Tesi realitzada al Centre de Recerca en Agrigenòmica (CRAG) / La llum és un element essencial per les plantes, ja que d’ella en treuen l’energia necessària per créixer. És normal doncs que hagin adquirit mecanismes per percebre diferents paràmetres d’aquesta, relacionats tant amb la seva quantitat com amb la qualitat. Dels diferents receptors de llum o fotoreceptors, els fitocroms són els més estudiats. Probablement la resposta controlada pels fitocroms més important per a les plantes a la naturalesa és la síndrome de fugida de l’ombra (SAS, de l’anglès shade avoidance syndrome). Aquesta síndrome agrupa una sèrie de respostes que han adoptat les plantes amb flors per evitar l’ombra de la vegetació propera. Tals respostes, com l’allargament de l’hipocòtil, la disminució de l'expansió foliar o la floració primerenca, impliquen a la fi una disminució de la biomassa en general. L’estudi de la SAS, per tant, es perfila com de gran interès biotecnològic, i ha estat objecte de la manipulació genètica per tal de millorar el rendiment dels cultius. En la present tesi s’ha treballat amb dos factors prèviament identificats al laboratori i involucrats en les respostes de la SAS. 1. Estudi de la relació estructura-funció del factor regulador de la SAS ATHB4. D’una banda s’ha estudiat el factor de transcripció ATHB4, una proteïna de la família HD-Zip (de l’anglès homeodomain-leucine zipper) classe II. La sobre-expressió d’ATHB4 produeix plàntules més petites i fosques, amb hipocòtil lleugerament allargat i cotiledons estrets, i respostes molt menors a tractaments d’ombra. Mitjançant la sobre-expressió de diferents regions d’ATHB4 s’ha vist que proteïnes sense la regió Ct mantenen la mateixa activitat biològica que la sobre-expressió de la proteïna sencera, suggerint que la regió Ct no té rellevància en les respostes a ombra aplicada al laboratori. D’altra banda, línies sobre-expressant diverses parts d’ATHB4 sense la regió Nt, o amb mutacions en residus conservats d’aquesta, perden gran part del fenotip visible en la línia sobre-expressora d’ATHB4 sencera, suggerint que aquesta regió Nt és essencial per l’activitat d’ATHB4 en les respostes a ombra. A més, formes truncades d’ATHB4 que podrien tenir la capacitat d’unió al DNA afectada mantenen les mateixes respostes a ombra que la proteïna sencera, suggerint que ATHB4 podria actuar en ombra de manera independent a la unió al DNA. Conjuntament, els resultats fisiològics i moleculars de les diferents construccions analitzades en la present tesi, suggereixen que la regió Nt d’ATHB4 podria interaccionar amb altres proteïnes per tal de regular les respostes de la SAS. 2. Paper de les nucleoporines (NUPs) en la regulació de la SAS. En un escrutini genètic es va obtenir el mutant recessiu dracula2 (dra2) amb una resposta a ombra simulada atenuada. Aquesta mutació afecta un gen que codifica una possible NUP. L’al•lel obtingut de l’escrutini es va anomenar dra2-1. En la present tesi s’ha realitzat una caracterització general d’aquest mutant, i d’altres al•lels mutants del mateix gen per inserció de T-DNA, mitjançant quantificacions dels nivells de pigments (clorofil•les i carotenoides), respostes a hormones (BRs, GAs i auxines) i respostes a ombra, tant fisiològiques com moleculars. S’ha vist que aquest mutant presenta alteracions en diverses respostes, inclosa l’exportació d’mRNAs del nucli cap al citoplasma. A més, altres NUPs també afecten les respostes de la SAS a nivell fisiològic, però dra2-1 és l’únic amb efectes moleculars. En conjunt, en la present tesi presentem les NUPs com a elements importants per les correctes respostes de la SAS, tenint DRA2 algun paper específic en la senyalització per llum. / Plants sense the presence of competing neighboring vegetation as a change in light quality: i.e. they sense the reduced ratio of red to far-red light. The responses to shade, generally referred to as the shade avoidance syndrome (SAS), involve various developmental changes intended to outgrow or outcompete the neighboring plants. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 2 (dra2) mutant, that showed a reduced avoidance of shade, affected a gene encoding a nucleoporine-like protein. Nucleoporins (NUPs) are the components of the nuclear pore complex (NPC), a structure that regulates the nucleocytoplasmic transport. Our work demonstrate that a proper nucleocytoplasmic transport is needed for SAS responses, as several plant NUP mutants display attenuated physiological shade responses. Despite the pleiotropic phenotype of the loss of function of specific NUPs, we found that dra2-1 has some specific and distinct molecular activity by reducing the expression of several shade molecular markers. In summary, we propose that DRA2 is a true NUP with a unique role regulating nucleocytoplasmic traffic of SAS related macromolecules. ATHB4, a transcription factor of the homeodomain-leucine-zipper class II (HD-Zip II) family, participates in integrating shade perception and hormone-mediated growth and, on the other hand, regulating leaf and cotyledon development. In this work we demonstrated that ATHB4 is a transcription factor that binds DNA and repress the expression of some members of its own family. By means of a structure-function approach, we go further in the study of the biological activity of ATHB4 in the shade-modulated hypocotyl elongation and control of gene expression in transgenic plants. We found that the HD and Zip domains, which hold the DNA-binding as a dimer, is not required for the role of ATHB4 in SAS responses. By contrast, the N-terminal region, which is conserved between most of the HD-Zip II members and contains a putative EAR domain, is essential for the proper SAS activity. Taking together our results indicate that ATHB4 plays two different roles, one in plant development and other in SAS responses, and that for the shade related activity is not working as a DNA-binding transcriptional repressor.

La transició evolutiva que va donar lloc als animals o metazous és una de les grans qüestions de la biologia evolutiva. Els animals tenen un procés de desenvolupament molt complex, en el qual molts gens controlen que una cèl•lula es converteixi en un ens multicel•lular. Aquests són els gens del desenvolupament, comuns a animals tan llunyans com els corals i els humans. Anàlisis genòmics primerencs van establir que aquests gens eren únics dels animals, ja que no es trobaven en espècies unicel•lulars. Però recents anàlisis filogenètics basats en dades moleculars han mostrat que hi ha certs llinatges de protists unicel•lulars molt estretament emparentats amb els animals. La seqüenciació del genoma del coanoflagel•lat Monosiga brevicollis, pertanyent al grup taxonòmic més proper als animals, va reportar moltes sorpreses, com la presencia de Cadherines, proteïnes claus en l’adhesió cel•lular als animals. El nostre laboratori està involucrat en la iniciativa UNICORN, on s’han seqüenciat més genomes d’espècies també emparentades als animals. Entre aquestes, hi destaca l’ameba Capsaspora owczarzaki, membre dels filasteris, que filogenèticament cau com a grup germà de coanoflagel•lats i animals. El genoma d’aquesta espècie ens ha aportat moltes dades noves sobre l’evolució dels gens del desenvolupament. Gens involucrats en l’adhesió cel•lular, com son les MAGUK o les Integrines es troben al genoma d’aquesta espècie. També hem analitzat els gens importants en el procés de diferenciació cel•lular, ja que aquest és bàsic per a la morfogènesis i la formació de tipus cel•lulars específics de teixits. Entre aquests, els factors de transcripció en son un grup clau. El nostre anàlisis a C. owczarzaki ens ha revelat que molts dels factors de transcripció que es creien únics d’animals de fet tenen orígens molt més antics, ja presents als ancestres unicel•lulars. També hem trobat vies de senyalització claus del desenvolupament presents en el genomes dels holozous unicel•lulars, com serien les tirosina cinases i els receptors associats a proteïnes G (GPCR). Tot plegat hem descobert que la pre-història animal va ser molt més complexa de l’esperat a nivell de contingut genòmic, i que la transició a la multicelul•lartiat va implicar la co-opció de moltes d’aquestes eines ancestrals en noves funcions associades al desenvolupament i no només guany de gens nous. / The origin of multicellular organisms from unicellular species is one of the major transitions of the history of life. Although there have been many independent transitions to multicellularity, only some groups have become surprisingly diverse and complex. Metazoans are one of the most complex multicellular groups, and present an intricate process of embryogenesis, where they develop from a single cell to an adult composed by many different organs and cell types. The genes that govern embryogenesis, the developmental genes, were thought to be exclusive to metazoans, the very core of their multicellular lifestyle. Nevertheless, recent molecular phylogenies have situated some protistan groups sister group to metazoans, allowing to address the question on the evolutionary origins of the developmental toolkit. To reconstruct the evolutionary origin of the developmental toolkit, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis had been reported to date. In this thesis my colleagues and me have analysed the complete sequence of the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans’ unicellular ancestors showing that they had a richer repertoire of proteins than what was thought before. For example MAGUKs, proteins involved in cell-adhesion and signalling scaffolding, evolved prior to the origin of metazoans, as well as many other adhesion proteins, such as Cadherins or Integrins. Not only proteins involved in adhesion where present, also cell differentiation is a very basic process for multicellular metazoans, as it allows differential gene expression and diverse cell behaviours in different cell types with the same genotype. Major regulators in this process are Transcription Factors; most of them key genes in developmental biology. Surveying the genome of C. owczarzaki and other holozoans (unicellular relatives of metazoans) shows that many of the metazoan transcription factors have ancient origins, as they are present and active in today’s living protists. In a broader perspective, we have seen that the assembly of the regulatory toolkit in both plants and animals is very similar. Both lineages are the richest in number and diversity of transcription factors among eukaryotes, and both repertoires have evolved by a step-wise process, having a first step of diversification in the unicellular relatives and a second one at the root of the respective lineages. Finally, cell-cell communication is also one of the major hallmarks of multicellularity and development. Here we present our results in two major signalling pathways, Receptor Tyrosine Kinases and G Protein Coupled Receptors. We demonstrate that both receptors where diverse and precede metazoans. Though the receptor types are there, no orthologs can be found between metazoans and unicellular holozoans. Contrastingly, most of the cytoplasmic machinery involved in the signal transduction is widely conserved. This may mean that signalling systems involved in sensing the environment where co-opted into multicellular functions, internalizing the ligand-signal relationships and forming the major signalling pathways of development. In a genome-wide perspective, we also show that pre-metazoan genomes have many pre-adaptations regarding composition of protein domains. Therefore we propose that the co-option of pre-existing genes into new and more complex regulatory networks had a major role in the evolutionary transition from unicellular protists to metazoans.

Tesi realitzada a l'Institut de Recerca Biomèdica de Barcelona (IRBB) / The integration of nutrient status to metabolic homeostasis at the cellular and organismal level is a complex process performed in multicellular organisms, and the ability of an organism to respond to nutritional stress is critical for its survival. In the last few years, the tumour suppressor protein p53 has emerged as an important regulator of several metabolic pathways such as glycolysis, oxidative phosphorylation and autophagy, that triggers a cellular adaptive response to fluctuations in nutrient availability, a function that may contribute not only to tumour suppression activities of this molecule but also to its non-cancer-associated functions. A better understanding of the molecular mechanisms underlying nutritional stress-mediated p53 activation and the potential role of p53 in organismal homeostasis is important to improve our current knowledge about p53 biology and its role in disease. In this regard, Drosophila is a very attractive model system whereby the physiology and the molecular mechanism that control metabolic homeostasis display significant parallels with humans. The fat body (FB), a functional analogue of vertebrate liver and adipose tissue, functions as a key sensor that couples nutrient status and energy expenditure. In conditions of nutrient deprivation, the FB supplies energy to the rest of the body by mobilizing stored glycogen and triacylglycerides (TAGs) to circulation in the form of trehalose and diacylglycerides (DAGs), respectively. The Drosophila homologue of p53 (Dp53) shares significant amino acid identity with mammalian p53, including specific residues frequently associated with human cancer. Like it mammalian counterpart, Dp53 promotes apoptotic cell death and cell cycle arrest in response to several stresses. The results presented in this thesis reveal a fundamental role of Dp53 in the organismal adaptation to nutrient deprivation. The depletion of Dp53 activity levels specifically in FB cells accelerates the consumption of the main energy stores, reduces the levels of sugars in the animal, and compromises organismal survival upon fasting. We present evidence that Dp53 has an impact on energy balance independently on the regulation of the endocrine signaling network (adipokinetic hormone and insulin) involved in metabolic homeostasis and the metabolic enzymes involved in the mobilization of energy resources upon starvation. Interestingly, we unveil a cell autonomous role of Dp53 in modulating the metabolic changes of FB cells to nutrient deprivation. We also identified the molecular mechanism by which Dp53 is activated by nutrient conditions in FB cells. We show that microRNAs (miRNAs), an abundant class of endogenous non-coding RNAs measuring 22-23 nucleotides in lenght, regulate dp53 expression by targeting its 3’ UTR, and we identify miR-305 as a major regulatory element. Interestingly, two elements involved in the biogenesis of miRNAs, Drosha and Dicer, and one catalytic component of the RISC complex, Argonaute-1, are downregulated during starvation, thus alleviating miR-305-dependent targeting of the dp53-3’UTR and contributing to organismal resistance to starvation. These results open up new avenues towards the molecular understanding of p53 activation under metabolic stress and reveal the participation of p53 in nutrient sensing and metabolic adaptation at the organismal level. During this thesis we also analyzed the classical tumour supressor functions of Dp53 and showed that upon ionizing radiation (IR)-induced DNA damage, Dp53 only regulates the early apoptotic response and induces DNA repair. Moreover, we present evidence that the miRNA machinery targets Dp53 in epithelial cells, thus preventing induction of programmed cell death. However, Dp53 activation upon DNA damage does not rely on the alleviation of miR305-mediated repression of the dp53-3’UTR. As p53 is found in primitive organisms, and cancer is unlikely to have significantly influenced evolution, suppressing tumour formation was almost certainly not the original function of this gene. In the last few years, a link between p53 and metabolic homeostasis has been uncovered, and together with our results, we will like to speculate that regulation of cellular metabolism could be one of the primary functions of p53. / Los organismos han desarrollado a lo largo de la evolución una amplia gama de estrategias para mantener un estado homeostático interno a pesar de ciertas fluctuaciones ambientales tales como la disponibilidad de nutrientes. En los últimos años, p53 ha surgido como un importante regulador de varias rutas metabólicas, capaz de desencadenar una respuesta celular adaptativa a fluctuaciones en la disponibilidad de nutrientes, una función que puede contribuir no sólo a la capacidad supresora de tumores de p53, sino también a otras funciones más fisiológicas no asociadas con el cáncer. El homólogo de Drosophila de p53 (Dp53) comparte una identidad significativa en secuencia de aminoácidos con p53 de mamíferos, capaz de promover funciones similares en respuesta a varios tipos de estrés. Los resultados presentados en esta tesis revelan un papel fundamental de Dp53 en la adaptación del organismo a la privación de nutrientes. Dp53 se requiere específicamente en el ‘cuerpo graso’ de Drosophila, un análogo funcional del hígado y el tejido adiposo de los vertebrados. La reducción de los niveles de actividad de Dp53 específicamente en este tejido acelera el consumo de las principales reservas de energía, reduce los niveles de azúcares en el animal, y compromete la supervivencia del organismo en ayuno. El impacto de Dp53 en el balance de energía es independiente a la regulación de las hormonas involucradas en el mantenimiento de la homeostasis metabólica y de las enzimas metabólicas implicadas en la movilización de los recursos energéticos. Desvelamos un papel autónomo-celular de Dp53 en la modulación de los cambios metabólicos que padecen las células del ‘cuerpo graso’ ante la privación de nutrientes. También identificamos el mecanismo molecular por el cual Dp53 se activa en condiciones de escasez nutricional en las células del ‘cuerpo graso’. Demostramos que los microRNAs (miRNAs) son capaces de regular la expresión dp53 e identificamos miR-305 como principal regulador. En una situación de privación de nutrientes, los niveles de expresión de varios elementos implicados en la biogénesis de los miRNAs se ven altamente reducidos, lo cual conlleva a la liberación de la represión mediada por miR-305, activando así Dp53 y contribuyendo a la resistencia del organismo frente a una situación de carencia nutricional.

La Leucemia Mieloide Aguda (LMA) es una enfermedad hematológica heterogénea, que se caracteriza por la acumulación de células inmaduras en médula ósea. Los pacientes diagnosticados de LMA tienen una supervivencia del 40%, aproximadamente. Existen factores que predicen el pronóstico de los pacientes, como la edad, la leucocitosis, las comorbilidades, la enfermedad residual mínima y la citogenética. Además y gracias a la aparición de las nuevas técnicas moleculares se han descrito un elevado número de marcadores genéticos. Algunos de ellos ya se utilizan en la práctica clínica, pero para la mayor parte se necesitan mas evidencias sobre su efecto pronóstico para poder incluirlos en el diagnóstico habitual. El objetivo de nuestro estudio fue analizar el impacto pronóstico de algunas de las alteraciones moleculares en pacientes con LMA " de novo". Estudiamos el efecto de las mutaciones en IDH1 e IDH2 en pacientes con cariotipo normal. En el mismo estudio, también se analizó la frecuencia y el valor pronóstico de las mutaciones en TET2. Posteriormente, estudiamos los pacientes diagnosticados de LMA con los reordenamientos RUNX1-RUNX1T1 y CBFB-MYH11 (LMA CBF). Se analizaron los factores clínico-biologicos para establecer un "score" pronóstico. También se analizó el valor de la sobre-expresión de dos marcadores moleculares, MN1 y BAALC, y de la determinación del número de copias de tránscrito tras la quimioterapia de inducción. Finalmente, y utilizando toda la serie de pacientes se estudió la utilidad clínica y pronóstica de la determianción de los niveles de expresión del gen WT1 al diagnóstico, tras la quimioterapia de inducció y de intensificación. También se estudió el valor pronóstico y la frecuencia de las mutaciones en WT1. La frecuencia de mutaciones de IDH1 e IDH2 en nuestra serie de pacientes fue del 22,5%. Observamos que aportaban información adicional en los pacientes con cariotipo normal, sobretodo en pacientes que presentavan los genes NPM1 o CEBPA mutados sin la presencia de la FLT3-ITD. En estos grupos, las mutaciones en IDH1 e IDH2 se asociaron a peor pronóstico. Las mutaciones en TET2 fueron muy poco frecuentes en nuestra serie (6,3%) y por ello no se pudo identificar su impacto pronóstico. En el grupo de pacientes con LMA reordenamientos de RUNX1-RUNX1T1 y CBFB-MYH11 la edad y el recuento leucocitario fueron parámetros que permitieron generar un "score" pronóstico. Así, los enfermos menores de 50 años y sin leucocitosis tuvieron una supervivencia mayor que los de edad avanzada y más de 20x10e9/l leucocitos. En el análisis de la sobre-expresión tanto de MN1 como de BAALC, observamos que los pacientes que presentavan el reordenamiento CBFB-MYH11 tenian una expresión de MN1 mas elevada que los pacientes RUNX1-RUNX1T1. Asimismo, la sobre-expresión de BAALC y MN1 al diagnóstico nos identifican un grupo de pacientes con peor pronóstico y un elevado número de copias de tránscrito tras la inducción también fue un factor de pronóstico adverso. En el ánalisis del gen WT1, el 10% de los pacientes presentaron el gen mutado, sin efecto pronóstico. La determinación de los niveles de expresión de WT1 al diagnóstico, tras la quimioterapia de inducción y la de consolidación tuvieron impacto pronóstico adverso. Por lo tanto, podemos concluir que las mutaciones en IDH1 e IDH2 identifican un grupo de pacientes con peor pronóstico. Las mutaciones en TET2 y WT1 son muy poco frecuentes en nuestra serie y no aportaron información predictiva de la evolución. El "score" generado en pacientes con LMA CBF, permite diferenciar grupos de pacientes con distinto pronóstico. La sobre-expresión de MN1 y BAALC nos ayudaria también a estratificar el riesgo en la LMA CBF, considerada clásicamente como de relativo buen pronóstico, así como también la determinación de número de tránscritos tras el tratamiento La determinación de la expresión de WT1 es una buena técnica para el análisis de la enfermedad residual mínima. Además, la determinación tras la quimioterapia de intensificación fue un factor pronóstico independiente.

Tesi realitzada a l'Institut de Biologia Molecular de Barcelona (IBMB-CSIC) / How a chemoattractant can act on a tissue to induce its migration towards that source? I have tried to answer this question in this research using as migratory tissue model the Drosophila respiratory organ (trachea) during its embryonic development and studying the role of the actin-bundling protein Fascin (called Singed in Drosophila). The tracheal system has been widely used as a model system for tubulogenesis. The formation of tubules is the main organisation of several organs in vertebrates and invertebrates that are essential for life. They allow the exchange of liquid, gas or nutrients to the corresponding target tissues. Some examples are kidney, mammary gland and vascular system in vertebrates, and in insects the salivary gland or the excretory system. The conserved FGF (Branchless -Bnl- in Drosophila) / FGFR (Breathless -Btl-) signalling pathway plays crucial roles in tracheal formation and, among other processes, it regulates branch migration through the induction of filopodia and motile properties in the leading cells. It has been largely unknown how this pathway triggers the actin-cytoskeleton reorganisation required for filopodia formation and guided migration. Fascins are an evolutionary conserved family of actin-crosslinking proteins responsible for the tight packing of parallel filaments of actin into bundles that compose several cortical cell protrusions. Filopodia are among those protrusions and they typically extend at the cell front of migratory cells to act as guiding sensors and mechanical devices to facilitate guided migration. Fascin is the primary actin crosslinker in filopodia and is essential for their formation. Fascin function has already been associated in different organisms and tissues with cell migration and adhesion. Interestingly, fascin upregulation in several carcinomas is thought to be a key factor in the metastatic process, making our study also relevant in the medical field. In Drosophila an only fascin homolog is encoded, singed (sn). Therefore I decided to test the possibility that Sn is required for tracheal migration, and the possible involvement of the Bnl/Btl pathway in its regulation. In this study I find that the tracheal chemoattractant FGF/Bnl and its pathway induce sn expression in the tracheal system, and that this regulation is functional. The functional analysis of Sn in the tissue lead to the conclusion that Sn is required for several processes mainly related to the leading cells, where Sn is highly accumulated. These cells pull the trailing cells, inducing the migration of the tissue. I specifically find that sn is required in the tissue for: timely and guided migration of the branches, fusion between branches, terminal cell specification, cell extension and terminal lumen guidance. At the cellular level, I find that Sn provides enough stiffness to the filopodia and that it controls the formation of the correct number of filopodia. The rigid filopodia have the necessary strength to push the organized cell front that extends towards the correct place. A molecular analysis shows that Sn regulates the reorganisation of the actin-cytoskeleton of the tracheal cells. These results indicate that Sn acts as a molecular link between the Bnl/Btl signalling pathway and the actin-cytoskeleton driving the migration of the tissue. The fact that Fascin is a target of the FGF/FGFR pathway and that this relationship has a role in cortical extensions and migration, give new insights into the function and regulation of the actin-bundling cytoskeleton during morphogenesis, and provides important information to further understand the involvement of Fascin in metastatic cells, which is relevant in the health field. Moreover I find that the phosphorylation state of Sn is important for the tissue-specific roles of Sn, similarly to bristles and neurons, but in contrast to plasmatocytes or nurse cells. In addition, in this dissertation I show the partnership of Sn with another actin-crosslinker, Forked (F). F is an actin-bundling protein (only similar to the vertebrate Espins located in the actin-bundles of microvilli and stereocilia) so far only reported to act in bristle formation in cooperation with Sn. In this study I show that F is additionally required during the tracheal system development. Specifically F is required for the same processes than Sn. sn and f genetically interact, and both proteins act in concert for the correct formation of tracheal filopodia. / El sistema respiratorio de Drosophila (llamado tráquea) sufre varios procesos morfogenéticos durante su desarrollo embrionario, para dar lugar a un ramificado y continuo árbol de tubos que facilita el intercambio de gases en cada tejido. Muchos de estos procesos se han conservado durante la evolución y son similares a los que tienen lugar durante la formación de otros tejidos de animales superiores compuestos también por tubos (como son el sistema vascular o el riñón de vertebrados). Uno de estos procesos es la migración. Durante el desarrollo traqueal, la cascada de señalización de FGF (Branchless -Bnl- in Drosophila) / FGFR (Breathless -Btl-), también conservada evolutivamente, juega un papel crucial en el control de la reorganización del citoesqueleto de actina, para formar los filopodios que llevan a la migración del tejido. Uno de los aspectos más desconocidos había sido la manera en la que el quemo-atrayente del tejido, Bnl, podía conducir a esta reorganización de la actina celular. Por ello, en este estudio se ha querido ahondar en el papel de la cascada de señalización de Btl/Bnl, poniendo especial énfasis en el papel del citoesqueleto de actina en la formación de este tejido. En vertebrados, la principal proteína de unión a actina encontrada en los filopodios es la Fascina. La Fascina pertenece a una familia de proteínas (llamadas Fascinas) también muy conservada evolutivamente, que organiza y empaqueta haces de actina. Esta proteína globular une fuertemente los filamentos de actina, de misma polaridad y colocados en paralelo, dándolos la estabilidad necesaria para formar diferentes tipos de protrusiones celulares. Muchas de estas protrusiones están principalmente relacionadas con la migración de varios tejidos y tipos celulares. De hecho en los últimos años la expresión anormalmente elevada de la fascina en adultos, se ha relacionado con varios carcinomas muy agresivos. Se cree que aquí, la Fascina podría jugar un papel crucial en la formación de los invadosomas, unas protrusiones esenciales para la invasión de otros tejidos. De ésta manera, la Fascina facilitaría el proceso metastático, principal razón de fallecimiento en pacientes de cáncer. En Drosophila tan sólo existe una proteína homóloga, llamada Singed (Sn) (nombre que se le dio porque las moscas mutantes para sn tienen sus quetas muy rizadas, como quemadas, significado de “singed”). Durante esta investigación, se han estudiado las funciones de Sn durante el desarrollo del sistema traqueal, desde el nivel tisular hasta el molecular, centrada en la migración traqueal y su relación con la cascada de señalización que la controla, Bnl/Btl. Debido a que muchas proteínas de unión a actina suelen actuar en cooperación, se estudiaron además posibles colaboradores de Sn. Esta investigación ha demostrado que sn se expresa en las células que lideran la migración del sistema traqueal durante su desarrollo (células de fusión y células teminales). Esta expresión está inducida por la cascada de señalización de Bnl/Btl a través de sus factores de transcripción positivo y negativo (Pointed y Anterior Open, respectivamente). Además, Sn es necesaria para varios procesos que tienen lugar en los dos tipos celulares cuyas propiedades migratorias, incluida la formación de filopodios, llevan a la migración del tejido. En concreto, se ha mostrado en este trabajo que Sn es importante para la fusión de las ramas traqueales, la especificación de las células terminales, la guiada formación del lumen en las células terminales, y la correcta forma de las células de fusión y terminal. Como se esperaba, Sn se requiere durante la migración del sistema traqueal, tanto para la velocidad como para la direccionalidad. Un estudio más profundo del papel molecular de la proteína evidencia que Sn es esencial para la formación de los filopodios (modificando su forma, estabilidad y número). Muy probablemente esto es debido a que Sn modifica el citoesqueleto de actina de las células traqueales. La falta de largos haces de actina en las células líder, cambiaría la reorganización de la actina del lamelipodio y filopodios, dando lugar a células muy irregulares que parecen extender y moverse con dificultad al sitio correcto. Mi hipótesis tras estos resultados y una extensa bibliografía es que los haces de actina y los filopodios traqueales son necesarios para la correcta señalización celular, la adhesión y la migración celular durante el desarrollo del sistema traqueal. Además este estudio ha demostrado que la fosforilación de Sn regula su función en los procesos anteriormente mencionados, como ya había sido demostrado en la formación de las quetas y ciertas neuronas, pero al contrario de lo publicado en plasmatocitos y células nodrizas. Por último, este trabajo ha relacionado por primera vez a la proteína de unión a actina llamada Forked (F) con la formación de una protrusión dinámica, como es el filopodio en las células traqueales. Aquí se ha probado que sn y f interaccionan genéticamente para el correcto desarrollo del sistema traqueal y que, de hecho, cooperan para la correcta formación de los filopodios. En resumen, este trabajo da por fin un nexo molecular entre la cascada de señalización de Bnl/Btl y el citoesqueleto de actina, y da una pista más sobre cómo las células son atraídas hacia sus quemo-atrayente para generar un tejido sano. Además, da nuevas pistas a los investigadores de oncología sobre la regulación del gen y cómo funciona a nivel molecular. Nuestros datos deberían permitir comprender mejor por qué razón las células cancerígenas aumentan la expresión de la fascina (y qué ventajas les proporciona a estas células), aumentando así las posibilidades de utilizar la Fascina como posible diana de terapias anticancerígenas. Esta investigación proporciona nueva información sobre la migración del sistema traqueal, la dinámica de las células que la lideran, y sobre el versátil papel que juega el citoesqueleto de actina en cada momento y tipo celular. Procesos celulares ya conocidos poseen ahora nuevos detalles moleculares.

Amb la finalitat de garantir la seguretat dels consumidors i del medi ambient, a la UE els aliments modificats genèticament (MG) estan sotmesos a un rigorós procés d'autorització que inclou: caracterització molecular del transgèn i anàlisis comparatives a nivell nutricional i agronòmic. L'ús de tècniques de profiling per avaluar la possible existència d'efectes no esperats derivats de la inserció i/o expressió del transgèn s'ha proposat com a una eina complementària per l'avaluació de la seguretat alimentària. L'objectiu de la present tesis és avaluar la variabilitat associada a la inserció i expressió de transgens en plantes, utilitzant com a exemple el blat de moro MON810. Es pretén complementar les aproximacions ja existents basades en l'estudi de paràmetres concrets mitjançant les tècniques de profiling. Des del punt de vista transcriptòmic i proteòmic, les varietats de blat de moro MON810 semblen ser substancialment equivalents a les seves varietats comparables no-MG. En conseqüència, és possible la producció de plantes MG amb el mínim d'efectes no esperats. / To ensure the safety of consumers and the environment, genetically modified (GM) food and feed are submitted to strict legislation. The EU establishes an authorisation procedure requiring: molecular characterisation of the transgene and compositional and agronomic comparative analysis. The use of profiling approaches to evaluate the possible occurrence of unintended effects derived from the insertion and/or expression of the transgene has been proposed as complementary tool for safety assessment. The objective of this thesis was to evaluate the variability associated to the insertion and expression of transgenes in plants, using as example MON810 maize. We aim to complement the existing targeted approaches by providing more unbiased information on the basis of profiling techniques. From the transcriptomics and proteomics perspectives, MON810 maize varieties seem to be substantially equivalent to their non-GM comparators. Thus, the production of GM plants with minimal unexpected effects is possible.

Es presenta una breu síntesi dels resultats i les conclusions de cadascun dels treballs en què es divideix la tesi. 1. Quantitat de DNA. S'han aportat dades inèdites en 57 espècies i 133 poblacions del gènere Artemisia i gèneres afins i també s'ha cobert el 20% del gènere Tripleurospermum. Com a resultats remarcables, hi ha correlació positiva entre la quantitat de DNA, el nivell de ploïdia i el nombre de cromosomes i negativa entre la quantitat de DNA per genoma haploide i el nivell de ploïdia. En Artemisia aquest paràmetre varia significativament depenent del subgènere, essent el subgènere Tridentatae el que té una quantitat de DNA més elevada per genoma haploide i el subgènere Dracunculus el que la té més baixa. A més, l'heterogeneïtat en valors C dins d'un subgènere és reflex del suport que presenta en la filogènia. Es posa de manifest, doncs, el valor d'aquest paràmetre en l'avaluació d'espècies o grups d'emplaçament problemàtic. S'ha aprofundit l'estudi de quantitat de DNA en el subgènere Tridentatae, essent aquest el més ben cobert. Se n'ha analitzat la variació en un context filogenètic i considerant dades d'hàbitat, ecologia o morfologia. Es presenta la hipòtesi que les artemísies endèmiques nord-americanes mostren un contínuum d'una estratègia vital (tipus r, creixement ràpid, cicles vitals curts i elevada producció de llavors) a una altra (tipus K, creixement lent, cicles llargs i producció de llavors baixa), essent aquest procés acompanyat d'un augment considerable de la mida del genoma. 2. Cariologia i citogenètica clàssica. S'ha confirmat l'existència de dos nombres cromosòmics bàsics en Artemisia i s'ha vist que moltes de les espècies que poblen hàbitats extrems són poliploides, la qual cosa dóna suport a les hipòtesis que connecten tolerància ecològica amb poliploïdia. 3. Citogenètica molecular: hibridació in situ fluorescent (FISH) i bandatge amb fluorocroms en espècies del subgènere Tridentatae (Artemisia). El cariotip típic del grup consta de cromosomes metacèntrics i submetacèntrics, és molt simètric i homogeni i té tres loci positius per a cromomicina i per als dos tipus de DNA ribosòmic, 5S i 18S-5.8S-26S sempre en posicions telomèriques. S'ha demostrat que la seqüència telomèrica tipus Arabidopsis (TTTAGGG)n és present en aquest grup. Un dels resultats més interessants és la confirmació de la colocalització d'ambdues regions del DNA ribosòmic, que s'estén a tots els loci cromosòmics marcats; semblaria doncs que ambdós tipus de DNA ribosòmic no existeixen independentment en Artemisia, sinó que han evolucionat de manera concertada.4. Estudis de sistemàtica molecular en el subgènere Tridentatae d'Artemisia. Aquesta part del treball pretén aconseguir un marc filogenètic que reflecteixi la història evolutiva del grup. S'han seqüenciat quatre regions del DNA, dues de nuclears (ITS i ETS) i dues de cloroplàstiques (trnS-trnfM i trnC-trnS). Els arbres filogenètics generats a partir de l'ITS i l'ETS per una banda i d'ambdues regions cloroplàstiques per l'altra han produït filogènies incongruents. Les reconstruccions proporcionades per les regions nuclears estan d'acord amb els caràcters morfològics o altres estudis moleculars previs, mentre que la filogènia procedent de les regions cloroplàstiques no hi concorda. La regió ITS defineix clarament el que considerem el grup Tridentatae sensu stricto (97% provabilitat posterior). A més, l'ETS coincideix amb l'ITS a treure de Tridentatae s. s. espècies conflictives però clàssicament considerades com a membres d'aquest subgènere, com A. bigelovii, A. pygmaea, i A. rigida. Com a nou resultat, afegim que Sphaeromeria, gènere estretament relacionat amb el grup Tridentatae, queda immers dins del gènere Artemisia i és polifilètic i, per tant, un grup artificial. Les incongruències trobades fan pensar que la història evolutiva del grup Tridentatae és més semblant a una xarxa que no pas a un arbre filogenètic. Aquest treball il·lustra els problemes que poden aparèixer en intentar abordar la reconstrucció filogenètica de tàxons tan estretament relacionats i d'evolució relativament recent. / An approach to the study of the evolution and systematics in Artemisia and close genera in the scope of molecular cytogenetics and phylogeneticsBriefly, a synthesis of the main results and conclusions of this PhD thesis is presented:1. Genome size. New data for 57 species and 133 populations from genera Artemisia and allies is provided as well as for almost 20% of the genus Tripleurospermum. A positive relationship between genome size, ploidy level and chromosome number, and a negative one between monoploid genome size and ploidy have been found. In Artemisia, genome size varies significantly depending on the subgenus. The heterogeneity of C-value within a subgenus also reflects its support in the phylogeny. Additionally, genome sizes in the Tridentatae have been completely assessed, and its variation analyzed in a phylogenetic context, considering also habitat, ecological and morphological data. The North American endemic Artemisia seem to present a continuum from an "r" life strategy to a "K" life strategy, being this process accompanied by a considerable increase in genome size.2/3. Classical and molecular (FISH and chromosome banding) cytogenetics. The existence of two basic chromosome numbers is showed once more, as it is the prevalence of polyploidy. The typical Tridentatae karyotype consists of mostly metacentric and submetacentric chromosomes, with the same three positive loci for cromomycin and for both (5S and 18S-5.8S-26S) ribosomal DNA regions. The presence of the Arabidopsis-type telomeric repeat is reported for the first time in the genus. One of the most interesting outcomes is that both rDNA regions appear always co-localized; it would mean that concerted evolution has acted to homogenize this unusual rDNA repeat. 4. Molecular systematics in the Tridentatae. Four DNA regions (two nuclear, ITS and ETS, and two chloroplastic, trnS-trnfM and trnC-trnS) have been sequenced. The phylogenetic trees obtained from the nuclear data and from the chloroplast data produce inconsistent results. The phylogeny provided by the nuclear regions parallels to some extent results of previous works. ITS region clearly defines the Tridentatae sensu stricto, and coincides with ETS in placing out of this group some classically conflictual species. The genus Sphaeromeria, closely linked to the Tridentatae, appears as polyphyletic, hence artificial. The incongruousness found suggests that the relationships between these taxa are best explained as networks rather than as trees.

The general aim of the thesis was to improve knowledge of the ecology and taxonomy of estuarine benthic diatoms in a highly stratified Mediterranean estuary, in order to evaluate their potential use as bioindicators of this ecosystem. To achieve that, diatom community composition was described and the factors affecting its composition and distribution were elucidated. Once the main anthropogenic pressures in the Ebro Estuary had been established, the response of diatoms to these pressures was tested through: i) the evaluation of existing diatom indices, and ii) the identification of groups of species indicative of potentially altered environmental conditions. Field studies were supported with experimental studies to test the response of species to the main environmental gradient in the Ebro Estuary (i.e. salinity). Several taxonomical aspects of estuarine diatoms were studied in detail to aid in the understanding and interpretation of ecological results. First of all, the morphological variability and ecophysiological response of selected species to salinity was documented. The morphology of the valves of several small and morphologically similar diatom species from the genus Nitzschia (one of the most abundant in the Ebro Estuary) was compared and studied in detail. Finally, the thesis focused on a species complex, i.e. Nitzschia inconspicua, to analyse the morphological, genetic, reproductive and ecophysiological variability of a taxonomically difficult but ecologically important species of both freshwater and transitional waters. / L’objectiu general de la tesi és avançar en el coneixement de l’ecologia i la taxonomia de les diatomees bentòniques d’un estuari Mediterrani altament estratificat (l’estuari de l’Ebre), per tal d’avaluar el seu ús potencial com a indicadors biològics d’aquest ecosistema. Per a assolir-ho, es va estudiar la composició de les espècies de diatomees bentòniques i es van determinar els factors que afecten l’estructura de la comunitat. Un cop detectades les principals pressions antropogèniques a l’estuari de l’Ebre, la resposta de les diatomees a aquestes pressions i el seu paper com a bioindicadors es va testar mitjançant: i) l’avaluació dels índexs de diatomees existents, i ii) la identificació de grups d’espècies indicadores de condicions ambientals potencialment alterades. Els estudis de camp es van complementar amb estudis experimentals per tal de testar la resposta de les espècies al principal gradient ambiental a l’estuari de l’Ebre (és a dir, la salinitat). Es van estudiar detalladament diversos aspectes taxonòmics per tal d’ajudar a la comprensió i interpretació dels resultats ecològics. En primer lloc, es va documentar la variabilitat morfològica i la resposta ecofisiològica de diverses espècies a la salinitat. Seguidament, es va comparar la morfologia de les valves d’algunes espècies petites i morfològicament molt similars del gènere Nitzschia (un dels més abundants a l’estuari de l’Ebre). Finalment, la tesi es va centrar en l’espècie Nitzschia inconspicua per tal d’analitzar la variabilitat morfològica, genètica, reproductiva i ecofisiològica d’una espècie taxonòmicament complexa però ecològicament important, tant en ecosistemes d’aigua dolça com en aigües de transició.

Tesi realitzada al Centre de Recerca en Agrigenòmica (CRAG) / The soil-borne plant pathogen Ralstonia solanacearum is the causing agent of bacterial wilt, a devastating disease of wide geographical distribution and host range with enormous economic impact worldwide. To cause disease, R. solanacearum injects a suite of type Ill effector (T3E) proteins into its hosts, from which only a few have been assigned a function, as it is the case for the T3E repertoires of other bacterial pathogens. Using yeast as a model system, we show that expression of the R. solanacearum awr type Ill effector family caused growth inhibition to different extents, and AWR5 displayed the most dramatic effect on yeast cells. Production of the full-length AWR5 protein in yeast targeted a cellular process leading to a growth inhibition and reduced cell size, but not involving an evident cell cycle arrest or cell death. Furthermore, we demonstrate that AWR5 is an inhibitor of TOR (target of rapamycin), a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of awr5 in yeast caused autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or by nitrogen starvation. The observation that deletion of two components of the PP2A heterotrimeric forms -CDC55 and TPD3- and mutation in SCH9 abolished the dramatic growth defect of cells expressing awr5 indicates AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A and Sch9. We speculate targeting of TOR pathways might be conserved among AWR2, AWR4 and AWR5, since their expression in yeast leads to similar downstream transcriptional responses. Also, we present evidence in planta that AWR5 T3E caused a reduction in TOR-regulated plant nitrate reductase activity and that the bacterial growth inhibition caused by delivery of AWR5 into host cells was mediated by TOR. Our work demonstrates that yeast is a powerful model for T3E function discovery and reveals not only a new mode of action for T3Es but also a new virulence target that might be conserved across kingdoms. / El patogen vegetal Ralstonia solanacearum és l'agent causant del marciment bacterià, una malaltia devastadora amb una àmplia distribució geogràfica i un extens ventall d'hostes que té un enorme impacte econòmic a nivell mundial. Per tal de causar malaltia, R. solanacearum injecta un conjunt de proteïnes efectores de tipus III (T3Es) als seus hostes. Tant a R. solanacearum com a altres patògens bacterians, pocs T3Es han sigut caracteritzats funcionalment. Utilitzant el llevat com a sistema model, demostrem que l'expressió de la família de T3Es awr provoca una inhibició del creixement de les cèl•lules del llevat en diferents graus segons l'efector, éssent AWR5 el que mostra l'efecte més dramàtic. L'expressió de la proteïna AWR5 en llevat resulta en la inhibició del creixement i la reducció de la mida de la cèl•lula, sense aturar el cicle cel•lular o la mort cel•lular. A més, hem demostrat que l'AWR5 és un inhibidor de la via TOR (target of rapamycin), un regulador central en eucariotes que integra els estímuls cel•lulars i prioritza creixement o resposta a estrés. L'expressió heteròloga d'awr5 en llevat causa una inducció de l'autofàgia acoblada a canvis transcriptòmics massius, que recorden a la inhibició de la via TOR per rapamicina o per manca de nitrogen. La fosfatasa PP2A i la kinasa SCH9 són dos components essencials de la via TOR. L'observació que la deleció de dos components de la forma heterotrimèrica PP2A -CDC55 i TPD3- i la mutació en SCH9 aboleixen els defectes de creixement en cèl•lules que expressen awr5 indiquen que AWR5 podria exercir la seva funció directa o indirectament mitjançant la inhibició de la via TOR, aigües amunt de PP2A i Sch9. En base als perfils transcripcionals d'AWR2 i AWR4, similars als d'AWR5, especulem que aquests efectors actuarien de forma similar, inhibint la via. També presentem evidència que l'AWR5 té un efecte sobre la via TOR in planta, ja que que aquest efector causa una reducció en l'activitat nitrat reductasa, regulada per la via TOR. A més a més, la inhibició del creixement bacterià causada pel lliurament d'AWR5 en les cèl.lules hostes també està mediada per TOR. El nostre treball demostra que el llevat és un model molt efectiu per descobir noves funcions dels T3Es i revela no només un nou mode d'acció per T3Es sinó també una nova diana de virulència que podria estar conservada entre els regnes biològics.

Tesi realitzada al Centre de Supercomputació de Barcelona (BSC-CNS) / Se ha demostrado en un número de estudios de metagenómica que la adición y la pérdida de genes específicos ha permitido a los microbiomas adaptarse a condiciones ambientales típicas de los entornos. Pero, aún no se conoce mucho acerca de como la regulación de la expresión génica contribuye a la adaptación. Aquí hemos caracterizado y analizado los metaregulomas de tres ambientes diferentes (muestra tomada de una Mina Acidificada en Iron Mountain, California; muestra de restos de ballenas en sedimentos colectados cerca de la península Antártica y en la costa oeste de Estados Unidos y suelo de granja colectado en Minessota, USA), así también hemos evaluado su impacto en la adaptación a condiciones físico-químicas variables. Para ello, se ha desarrollado un protocolo computacional para extraer las regiones reguladoras y sus correspondientes sitios de unión a factores de transcripción. Tomando la densidad de sitios de unión por promotor como una medida del potencial y la complejidad de la regulación de genes; encontramos que ésta se mantiene constante en los tres nichos analizados; a pesar de sus diferentes condiciones físico-químicas y la composición de especies. Sin embargo, se encontró que cada entorno distribuye su potencial de regulación diferente a través de su espacio funcional. Entre las funciones con mayor potencial regulador en cada nicho, se observó un importante enriquecimiento en los procesos relacionados con la detección y amortiguación de factores dinámicos en cada entorno, como por ejemplo, la disponibilidad de cofactores en alta mar; de oligosacáridos en el suelo dedicado a agricultura y la regulación del pH en la antigua mina dedicada a extracción de hierro. Unido a este estudio también evaluamos la capacidad de regulación de cepas de E. coli con diferente biolocalización. Todos los resultados de estos estudios ponen en relieve el impacto del potencial de regulación de genes en la adaptación de las bacterias a los diferentes hábitats. La adaptación ocurre a través de la distribución de su potencial de regulación; entre las funciones específicas con redes de factores de transcripción complejas, observamos alta prevalencia de genes que respondían a factores ambientales críticos para el crecimiento de cualquier comunidad microbiana. / The incapacity to culture over 90% of the species of microorganisms and the impossibility to reproduce the interaction among species in controlled conditions have limited the knowledge about microorganism communities in their natural habitats. In this sense, metagenomics studies have brought some light about, (1) natural microorganism communities compositions, (2) non-cultivable species (Allen & Banfield, 2005) and (3) the identification of functional fingerprints related to specific habitats (Tringe et al., 2005; Gianoulis et al., 2009). More recently single cell genomics analysis allows for the first time the sequencing of the complete genomes of environment-isolated bacterial cells collected directly from their natural habitats. Both approaches generate a massive amount of sequence information of communities and organisms living under different physicochemical conditions. All this allows, for the first time, to search for the molecular and genetic basis of adaptation through the comparison and the study of genomes of different species sharing the same environment, and of similar species living in different conditions. It has been shown in a number of metagenomic studies that the addition and removal of particular molecular functions have allowed microbiomes to adapt to specific environmental conditions by losing and gaining specific genes. But little is known about to which level the regulation of the expression of these genes contributes to adaptation. To cover this limitation, my PhD thesis aims to the study whether and how the environment shapes the regulatory regions of organisms to allow adaptation to variable external factors. This is done through the identification, analysis and comparison of regulatory regions (promoters) of Bacteria and Archaea species sharing the same environment, and of the same (or similar) species living under different physicochemical conditions. Our general approach is based on the analysis of proximal regulatory regions from free living (metagenomic) and laboratory prokaryotic organisms, as to their regulatory potential, i.e. their density of transcription factors binding sites (TFBSs). More specifically, we want to answer the following questions: (1) Do prokaryotic communities from different environments distribute and organize their regulatory potential differently? (2) Can these differences be explained and correlated with variable physico-chemical factors of the environment they live in? (3) Does the same bacteria (or clade) distribute its regulatory potential differently in environments with distinct external conditions? Below we explain the work and the results obtained in the last four years of my PhD thesis. The work is divided in two main blocks. In the first one, we investigate how prokaryotes in different environments distribute their regulatory potential and how this is correlated with variable external factors of each of the niches. This part is finished and the manuscript has been already sent for publication. The second block aims at answering the same main question, but now, by comparing the distributions of the regulatory potential through the same species inhabit different environments I. REGULATORY POTENTIAL VARIABILITY AT THE COMMUNITY LEVEL. 1. Selection of regulatory regions through metagenomics sequencing data. The first step of this project consisted in the identification of putative promoter using metagenomic sequencing reads. The pipeline we developed could be applied to Sanger sequencing reads as well as to partial genomes assembling from shorter reads obtained by Illumina or 454 sequencing. All the promoter data sets we have identified using this protocol in three metagenomic samples (Acid Mine (AM), Whale Falls (WhF), Waseca Soil (WS) will be publicly available in due time. These data sets have been carefully curated at different levels, not only to ensure their reliability by comparing with known sets of TFBS. 2. TFBS occurrences per promoter as a way to estimate regulatory potential. When the expression of a gene is regulated by a high number of transcriptions factors (TFs) makes the gene response more versatile in order to sense environmental physicochemical parameters specific of the habitat. In addition, we suspect that the species able to moderate their transcription requirements to face dynamic environmental factors will have more chance to be selected to growth under these conditions. What is the same, we expect that the habitat shapes the regulatory regions positively selecting TFBSs upstream of genes, which are on charge of sensing key environmental factors strongly related to microorganism survival rates. Based on this hypothesis we aimed to the identification of putative TFBSs in prokaryotic promoters as a way to estimate the regulatory potential of a gene. First, we applied a de novo method (Li et al, 2002) suitable to new sequencing genomes without any previous information about their TFBSs and also useful in the comparison of communities with high diversity, to avoid biases towards well known or abundant species. This method is based on the searching for overrepresented palindromic sequences, preferred DNA structures for TFs binding in prokaryotes (Rodionov, 2007), in the set of promoters we previously selected. Furthermore, in order to evaluate the presence of false positive, we performed different quantitative and qualitative comparisons with available independent data and methodology. From a quantitative point of view, we (1) first observed that the global average of 10 TFBS per promoter (with 0 as minimum and 25 as maximum values) that we identify from all three environments is in agreement with previous estimates obtained with different bacterial species and methodologies (Liu et al, 2008) (Sun et al, 2007). (2) We also evaluated the performance of our methodology by comparing our results with those obtained with an independent method, MotifClick, which predicts cis-regulatory regions using a graph-based polynomial-time algorithm (Zhang et al, 2011). After running both predictors over intergenic E. Coli regions, we observed that the densities of TFBS resulting from one or the other strategy showed high correlation values (rho= 0.52, p-value < 2.2 x 10-6 ). (3) Furthermore, we also assessed the biological significance of our predictions by carrying out a randomization test consisting in applying the same prediction pipeline to our collection of promoters with their nucleotide sequence completely shuffled, i.e. with no biological information. From a qualitative point of view, we (1) screened for coincidences between our predicted TFBSs and those reported in the Regprecise database (Novichkov et al, 2010), which consist on manually curated site reconstructions in various bacteria genomes. We observed that our approach has been able to find at least one site described among all possible binding sequences for all TFs described in Regprecise. This overlap involves 28% of our predictions. (2) In addition, we also searched for a particular type of false predictions, which consist on regulatory palindromic repeats with no binding potential, named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). The results that we obtained on our promoters using the CRISPRFinder web tool (Grissa et al, 2007) showed a negligible amount of these regions among our TFBS predictions, i.e. less than 1% of the promoters that were removed from the analysis. In summary, all these evaluation tests suggest that our TFBS prediction method is both, quantitatively and qualitatively reliable, as they show a small fraction of false positives. But, most importantly, these are not expected to affect our final conclusions because they derive from comparisons within and between environments and do not rely on absolute TFBSs counts. 3. Analysis of functional and taxonomical organization of regulatory potential. As the general regulatory potentials distributions (determined as is explained in section 2) per habitat in AM, WFS, WhF are similar among them (i.e., calculated average and standard deviation is about 10.4 (±3.49), 9.49 (±3.47) and 10.08 (±3.31) for Acid Mine Drainage, Waseca Farm Soil and Whale Falls samples, respectively), we further checked the occurrences of regulatory potential variability related to taxon and function within and among environments. a) Regulatory potential distribution per taxon. At this point we observed similar profiles for the majority of the species assigned by MEGAN (Huson et al., 2007) in AM, WFS, WhF (median of TFBSs/promoter ~ 9 -12), only a few exceptions were out of this range. b) Regulatory potentials distributions per function. The functional enrichment analysis was done by first ranking all promoters as to their number of predicted TFBSs. Then, we retained significant cases based on two criteria 1) functions whose p << 0.05 within environment and 2) functions with orthologous in the other three environments. Those selected groups were compared again, this time among environments. In contrast to the taxon analysis, we observed here functions with a significant higher regulatory potential comparing among habitats. We have found, for instance, highly regulatory potential in processes involved in the adaptation to variable environmental factors, such as cofactor availability in deep sea and fluctuations on the concentration of di-and-oligosacharides in soil. Another interesting observation is the variability on the regulatory potential of the orthologous genes depending on the environment and to this respect it is worth to highlight some examples listed below. -In Whale Falls Samples, beside the cofactor metabolism, we found high regulation in functions that are involved in the oxidative stress response, i.e., the hydrogen peroxide inducible gene activator and NnrS protein in response to NO and the non-specific DNA binding protein (Dps). This response is also needed in order to protect genomic DNA during prolonged non-growing phases, which are typical of oceanic environments (Storz & Imlay, 1999). -In Waseca Farm Soil carbohydrates metabolism related functions appear as highly regulated, more precisely di and oligosaccharides metabolism. This fact could be in concordance with the fluctuations in organic matter in the soil, such as, plant debris. -In Acid Mine, we identified high regulatory potential in the promoters of genes related to the Ton and Tol B transport systems, these genes are involved in avoiding toxicity by keeping metal homeostasis inside the cell (Osorio et al., 2008), in particular of iron. The high regulatory potential of the TonB-dependent receptor and the iron chelator utilization proteins (particularly in Leptospirilum group) compare to other orthologous found in Waseca Soil, for example, might provide homeostasis and, therefore, plasticity to acid mine bacteria living under variable ferric concentrations. In brief, through these analyses we have found specific functional enrichments among highly regulated functions in each of the metagenomes that point to possible interaction points between gene regulation and dynamic parameters of the niche. What is more, these results also highlight the impact of gene regulation in the adaptation of microbes to their habitat. Thus, with the outcomes explained above we found important clues aimed to solve two out of three main questions exposed at the beginning of this summary, both related to how is distributed the regulatory potential at the community level. II. REGULATORY POTENTIAL VARIABILITY IN THE SAME (OR HIGHLY RELATED) SPECIES LIVING IN DIFERENT HABITATS. The second block of my PhD aims to study the same general question on the relationship of gene regulation and adaptation, but now by studying the variability found on the same species living in different habitats. Here we present promising preliminary results that are currently being confirmed and expanded. We have analysed the regulatory potential of nine Escherichia Coli strains (downloaded from IMG database, http://img.jgi.doe.gov/). E. Coli is an excellent model to try our hypothesis at the species level, first because the regulatory network of several strains are well characterized and also because is a very versatile microorganism related to niche specificities. Thus, due to all the information available this species is a perfect candidate to study TFBSs abundances under different environmental conditions. 4. De novo TFBS predictions in different E. Coli strains. We first estimated the regulatory potential in several Escherichia Coli strains isolated from different sources, such as, human and animal gut (labeled as 2513237219, 2518645559, 2513237251, 2506520037), urinary bladder (2512047041, 2511231170, 2511231198), cerebrospinal fluid (2511231131) and a strain isolated for first time in 1977, which was later engineered for ethanol production (2513237200). This data was downloaded from IMG/JGI database (http://img.jgi.doe.gov/). After running two different approaches to predict TFBSs per promoter we observed a high correlation coefficient between both predictions for all the strains analyzed here except the engineered (2513237200). In spite of the great sequences identity among strains, top highly regulated functions are different among them. The most remarkably case is for colanic acid biosynthesis, in which the strain located in the gut present a complex regulation in some genes of this pathway, differently as occur in the strains located in the urinary tract and cerebrospinal fluid. However, for pyrimidine metabolism related genes we observed a similar behavior in the regulatory potential for the strains located in the gut and urinary tract. 5. Identification of TFBSs in different strains of E.Coli using homology mapping of experimentally validated binding sequence matrices. The second is an approach compatible with the analysis of regulatory regions in well-characterized species, such as E. Coli, because it relies on the TFBSs mapping using position specific sequencing matrices derived mostly from experimental information. More specifically, we predicted TFBSs through position specific sequencing matrices (PSSM) of TFs from RegulonDB (http://regulondb.ccg.unam.mx:80/index.html). We first calculated (with MATSCAN software, Blanco et al., 2006) the mapping score of known TFBS in nine Escherichia Coli strains isolated from different sources, as is explained in section 4 (http://img.jgi.doe.gov/). Afterwards, these isolated strains were clustered (using the TFBSs scores as input) by body location and also we observed a separation of these strains by the capacity to synthesize or not selenocysteine. We also analyzed the TFBSs abundances per TF in these E. Coli strains. After analyzing 86 TFs, we observed that the TFBSs distributions per TF have a great similarity among them, with a few exceptions. The outstanding differences between strains are given by the non- selenocysteine synthesizer and is related to FhlA. FhlA is a DNA-binding transcriptional activator; required for the induction of formate dehydrogenase H (FDH-H) expression. In addition, FDH-H enzyme contains selenium as selenocysteine incorporated cotranslationally. The dependence of this strain on external selenocysteine, increases the necessity of a major control on the FDH-H synthetic pathway. What is more, the maintenance of a FDH-H enzyme stock inside the cell is also critical under fermentative growth conditions, frequently observed in these host associated microorganisms. 6. Conclusions and perspectives. With this PhD thesis we intend to tackle some questions up to now unsolved, such as, 1) whether the environment influence the structure and complexity of regulatory regions (e.g. the regulation of gene expression) within the same microbial community, and how this affects the regulation of essential functions of the community in general, and 2) whether the core genome of different strains or highly related species, shows differences in terms of gene regulation, and how this correlates with media conditions (natural or not). For instance, we were able to find niche specific gene regulatory potentialities and also intraspecific plasticity of gene regulation. As far as we know this work constitutes the first study on how the regulatory regions of a gene are shaped by the environmental factors. Furthermore, this kind of analysis would have a wide applicability in biomedicine for instance in the design of in silico microbial communities for specific environments as therapeutic strategies. 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