Tesis por compendio / [EN] One of the defining features of a eukaryotic cell is the presence of a nuclear envelope. This allowed the physical separation between nucleus and cytoplasm, although the presence of a variable number of openings called nuclear pore complexes (NPCs) allowed a constant flow of molecules and information between the two compartments. Certain molecules passively diffuse, but others need energy and specific interactions with transporters and components of the NPC to travel between through both compartments. The messenger RNAs (mRNAs) are among the molecules selectively exported from the nucleus to the cytoplasm. The physical separation between nucleus and cytoplasm isolates the processes of transcription and translation in eukaryotic cells, allowing the cell to select core transcripts competent for export and that will lead to a functional protein in the cytoplasm. The right transcript levels in a cell, depending on the nutritional requirements, reproductive or relationship with the environment is essential for life. To this end, mechanisms regulating transcription, processing, stability, degradation, export or translation of the transcripts, are physically and spatially highly coupled in order to finely regulate the transcript levels in the cell. In Saccharomyces cerevisiae, SUS1 codes for a small protein of 11 kDa highly conserved in all eukaryotes. SUS1 is part of the SAGA transcriptional co-activator, being a submodule component involved in chromatin remodeling. In addition, SUS1 is one of the components of the TREX2 complex, wich interacts with the nuclear pore in the periphery of the nucleus and it is involved in the export of messenger RNAs. The presence of SUS1 in both complexes allows the physical and spatial coupling phenomena of transcription and export of mRNAs. In addition, SUS1 has two introns which is an unusual fact for the S. cerevisiae genome. Unlike other fungi or metazoans, the percentage of genes with introns in S. cerevisiae is very low (5%) and only 10 genes have more than one intron interrupting its coding sequence. The unusual characteristics of SUS1, the role of Sus1 coordinating processes during mRNA biogenesis and its functional conservation in higher eukaryotes, led the research conducted in this dissertation. In this work we studied in detail the biogenesis of SUS1 transcripts. We have identified different factors, acting in cis and trans that are involved in regulating the expression of SUS1 and function of the protein it encodes. On the other hand, we have studied the genetic relationship of SUS1 with components of the 5 '- 3' cytoplasmic degradation machinery and expanded the knowledge about the role of SUS1 during the biogenesis of mRNAs, not only in the nucleus but also in the cytoplasm. / [ES] Una de las características que definen a una célula eucariota es la presencia de una envoltura nuclear. Aunque este hecho permite la separación física entre núcleo y citoplasma, la presencia de un número variable de aberturas, denominadas complejos del poro nuclear (NPCs), permiten un flujo constante de moléculas e información entre ambos compartimentos. Ciertas moléculas difunden de forma pasiva, pero otras necesitan energía e interacciones específicas a través de transportadores y componentes del NPC para transitar entre ambos compartimentos. Entre las moléculas selectivamente exportadas del núcleo al citoplasma se encuentran los RNAs mensajeros (mRNAs). La separación física del núcleo y del citoplasma en eucariotas, aísla los procesos de transcripción y traducción, permitiendo a la célula seleccionar en el núcleo los transcritos competentes para ser exportados y que darán lugar a una proteína funcional en el citoplasma. Adecuar los niveles de transcritos en una célula, en función de las necesidades nutritivas, reproductivas o de relación con el entorno, es esencial para la vida. Para ello, los mecanismos encargados de regular la transcripción, el procesamiento, la estabilidad, la degradación, el exporte o la traducción de los transcritos, se encuentran altamente acoplados física y espacialmente con el fin de regular finamente los niveles de mensajeros en la célula. En el núcleo de las células de la levadura Saccharomyces cerevisiae se encuentra Sus1, una proteína de 11 kDa altamente conservada en eucariotas. Sus1 forma parte del co-activador transcripcional SAGA, siendo componente de un submódulo implicado en la desubicuitinación de la histona H2B. Además, Sus1 es uno de los componentes del complejo TREX2, que interacciona con el poro nuclear en la periferia del nucleo y está implicado en el exporte de RNAs mensajeros y en estabilidad genómica. La presencia de Sus1 en ambos complejos permite el acoplamiento físico y espacial de los fenómenos de transcripción y exporte de mRNAs. Además, el gen SUS1 posee dos intrones, siendo este un evento muy inusual en el genoma de S. cerevisiae. A diferencia de otros hongos o metazoos, el porcentaje de genes con intrones en S. cerevisiae es muy reducido (5%) y solo 10 genes poseen más de un intrón interrumpiendo su secuencia codificante. Las características peculiares del gen SUS1, el papel de la proteína que codifica coordinando procesos durante la biogénesis del mRNA y su conservación funcional en eucariotas superiores, motivó las investigaciones llevadas a cabo en esta tesis doctoral. En este trabajo hemos estudiado en detalle la biogénesis de los transcritos de SUS1. Se han identificado diferentes factores, tanto en cis como en trans, implicados en la regulación de la expresión de SUS1 y en la función de la proteína que codifica. Por otro lado, hemos estudiado la relación genética de SUS1 con componentes de la maquinaria de degradación citoplasmática 5'-3' y hemos ampliado los conocimientos respecto al papel de Sus1 durante la biogénesis de los mRNAs, no solo en el núcleo sino también en el citoplasma. / [CA] Una de les característiques que definixen a una cèl·lula eucariota és la presència d'un embolcall nuclear. Este fet permet la separació física entre nucli i citoplasma, encara que la presència d'un nombre variable d'obertures, denominades complexos del porus nuclear (NPCs), permet un flux constant de molècules i informació entre ambdós compartiments. Certes molècules difonen de forma passiva, però altres necessiten energia i interaccions específiques amb transportadors i components del NPC per a transitar entre ambdós compartiments. Entre les molècules selectivament exportades del nucli al citoplasma es troben els RNAs missatgers (mRNAs). La separació física del nucli i del citoplasma aïlla els processos de transcripció i traducció en eucariòtes, permetent a la cèl·lula seleccionar en el nucli els transcrits competents per a ser exportats i que donaran lloc a una proteïna funcional en el citoplasma. Adequar els nivells de transcrits en una cèl·lula, en funció de les necessitats nutritives, reproductives o de relació amb l'entorn, és essencial per a la vida. Per a això, els mecanismes encarregats de regular la transcripció, el processament, l'estabilitat, la degradació, l'exportació o la traducció dels transcrits, es troben altament acoblats física i espacialment amb el fi de regular finament els nivells de missatgers en la cèl·lula. En el nucli de les cèl·lules del rent Saccharomyces cerevisiae es troba Sus1, una xicoteta proteïna d'11 kDa altament conservada en eucariotes. Sus1 forma part del coactivador transcripcional SAGA, sent component d'un submòdul implicat en la modificació de histones. A més, Sus1 és un dels components del complex TREX2, que interacciona amb l'embolall nuclear a la perriferia del nucli i està implicat en l'export de RNAs missatgers. La presència de Sus1 en ambdós complexos permet l'adaptament físic i espacial dels fenòmens de transcripció i exportació de mRNAs. A més, el gen SUS1 posseïx dos introns i este fet és inusual en el genoma de S. cerevisiae. A diferència d'altres fongs o metazous, el percentatge de gens amb introns en S. cerevisiae és molt reduït (5%) i només 10 gens posseïxen més d'un intró interrompent la seua seqüència codificant. Les característiques peculiars del gen SUS1, el paper de la proteïna que codifica coordinant processos durant la biogènesi del mRNA i la seua conservació funcional en eucariotes superiors, va motivar les investigacions dutes a terme en esta tesi doctoral. En este treball hem estudiat en detall la biogènesi dels transcrits de SUS1. S'han identificat diferents factors, tant en cis com en trans, implicats en la regulació de l'expressió de SUS1 i en la funció de la proteïna que codifica. D'altra banda, hem estudiat la relació genètica de SUS1 amb components de la maquinària de degradació citoplasmática 5'-3' i hem ampliat els coneixements respecte al paper de Sus1 durant la biogènesi dels mRNAs, no sols en el nucli sinó també al citoplasma. / Cuenca Bono, B. (2016). Identificación y caracterización de elementos reguladores de la expresión de SUS1 y nuevas funciones celulares para la proteina Sus1 en Saccharomyces cerevisiae [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62327 / Compendio

The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 protein, all are associated with ribonucleoprotein (RNP) complexes. Understanding the role of G-patch proteins in modulating the DExD/H box protein Prp43 biological function was the motivation of this thesis. The G-patch domain has been proposed as a protein-protein or a protein-RNA interaction module for RNP proteins. This study found that the three Prp43-associated G-patch domains interact with Prp43 in a yeast 2 hybrid (Y2H) assay but differ in apparent relative affinities. Using a systemic Y2H analysis, I identified the conserved Winged-helix (WH) domain in Prp43 as a major binding site for G-patch motif. Intriguingly, removal of the non-essential N-terminal domain (NTD) of Prp43 (amino acids 2-94), greatly improves G-patch binding, suggesting that the NTD may play a role in modulating enzyme activity by the G-patch effectors. I identify a second site within the Pxr1 that strongly binds Prp43 but, unlike the G-patch, is dispensable for Pxr1 function in vivo. By constructing chimeric proteins, I demonstrated that individual G-patch peptides differ in the ability to reconstitute Spp382 and Pxr1 function in support of pre-mRNA splicing and rRNA biogenesis, respectively. Through amino acid sequence comparisons and selective mutagenesis I identified several residues within the G-patch motif critical for Prp43-stimulated pre-mRNA splicing without greatly altering its ability to bind Prp43. These data lead me to propose that the G-patch motif is not a simple Prp43 binding interface but may contribute more directly to substrate selection or Prp43 enzyme activation in the biologically distinct pre-mRNA splicing and rRNA processing pathways.

Le mode vie autotrophique des plantes repose entièrement sur l’intégrité du chloroplaste et notamment l’étape de la biogénèse. La transcription des gènes chloroplastiques, assurée par une PEP (ARN polymérase encodée par le chloroplaste) et deux NEPs (ARN polymérase encodée par le noyau), est l’une des étapes primordiales dans le développement d’un chloroplaste photosynthétique. On distingue trois classes de gènes chloroplastiques : les gènes de classe I, transcrit par la PEP exclusivement; les gènes de classe II, transcrits par la PEP ou les NEPs; et les gènes de classe III, transcrits exclusivement par les NEPs. Pour assurer sa fonction, la PEP doit être associée à des facteurs sigmas. L’un de ceux-ci, la protéine SIG6, est un facteur sigma général et, associé à la PEP, assure la transcription de l’ensemble des gènes de classe I et II lors du développement du chloroplaste photosynthétique. Ainsi, le mutant sig6 présente un phénotype de cotylédons pâles, associé à un retard de biogénèse chloroplastique, ainsi qu’une diminution de la transcription des gènes de classe I, provoquant la diminution de la quantité de protéines de classe I. Dans le laboratoire, nous étudions les deux protéines WHIRLY chloroplastiques (WHY1 et WHY3) pour leur rôle dans le maintien de la stabilité génomique chloroplastique. Toutefois, peu de choses sont encore connues sur leur rôle potentiel dans la transcription ou la biogénèse chloroplastique. Par exemple, lorsque l’on tente de purifier la PEP, on obtient un gros complexe transcriptionnel nommé PTAC (Plastid Transcriptionally Active Chromosome) dans lequel sont retrouvées les deux protéines WHIRLY, suggérant qu’elles pourraient être impliquées dans la transcription chloroplastique. De plus, un possible rôle dans la biogénèse chloroplastique leur a été prêté, notamment chez le maïs. Dans cette étude, nous avons donc cherché à vérifier l’implication des protéines WHIRLY dans la biogénèse chloroplastique par une approche génétique de croisements entre les mutants sig6 et why1why3. Pour cela, nous avons isolé des doubles mutants sig6why1 et sig6why3, ainsi qu’un triple mutant sig6why1why3. À l’aide d’une caractérisation phénotypique et de la quantification de quelques protéines chloroplastiques, nous avons remarqué que la perte d’un des WHIRLY permet de complémenter le phénotype de cotylédons pâles du mutant sig6 et favorise l’expression normale de protéines en principe sous-exprimées dans le mutant sig6. Toutefois, la perte des deux WHIRLY ne permet pas de compenser le phénotype de cotylédons pâles et provoque l’apparition d’un phénotype persistant associé à une expression anormale des protéines chloroplastiques. Ces résultats ne peuvent être expliqués par le rôle des WHIRLY dans le maintien de la stabilité génomique chloroplastique étant donné que le triple mutant sig6why1why3 présente moins de réarrangements que le double mutant why1why3. Finalement, nous montrons que les effets de la perte d’un WHIRLY sur le mutant sig6 peuvent être mimés par l’utilisation de la rifampicine, une drogue inhibant l’ARN polymérase chloroplastique de type bactérienne (PEP). Ensemble, ces résultats démontrent donc l’implication des protéines WHIRLY chloroplastiques dans la biogénèse chloroplastique en association avec la protéine SIG6. Nous proposons un modèle selon lequel les deux protéines WHIRLY permettraient de favoriser l’activité de l’ARN polymérase de type bactérienne, notamment lors du développement du chloroplaste photosynthétique. En cas d’absence d’une des deux protéines, cette diminution partielle d’activité de la PEP favoriserait la mise en place d’un mécanisme de complémentation par le NEPs, permettant finalement de rétablir la biogénèse chloroplastique dans un mutant sig6. En l’absence des deux WHIRLY, le mécanisme de complémentation par les NEPs serait incapable de compenser la forte inhibition de la PEP, se traduisant par une aggravation du retard de développement du chloroplaste dans le mutant sig6. / The autotrophic lifestyle of plants relies entirely on the integrity of chloroplasts and particularly on their biogenesis. Chloroplast gene transcription, performed by a Plastid-Encoded Polymerase (PEP) and two Nuclear-Encoded Polymerases (NEPs), is one of the key steps during the development of photosynthetic chloroplast. There are 3 classes of genes, one transcribed by PEP alone (class I), one by both PEP and NEPs (class II), and the third by NEPs alone (class III). To carry out transcription, PEP associates with plastid sigma factors including the general sigma factor SIG6. sig6 mutants have a pale cotyledon phenotype, a severe decrease in class I gene transcription and a reduction in the level of class I proteins. In our laboratory, we study the role of the two plastid WIHRLY proteins (WHY1 and WHY3) in maintaining plastid genome stability. However, little is known about any role these proteins may play in transcription or chloroplast biogenesis. It seems likely they are involved in plastid gene transcription since they are found in the Plastid Transcriptionally Active Chromosome (PTAC). Moreover, they have been implicated in chloroplast biogenesis in maize. In this study, we verified the implication of these proteins in plastid biogenesis using a genetic approach in which we crossed a sig6 mutant with a why1why3 mutant. We isolated sig6why1 and sig6why3 double mutants and a sig6why1why3 triple mutant. Using a phenotypic characterisation and quantification of some plastid proteins, we show that loss of one of the two Why genes complements the sig6 pale cotyledon phenotype and allows a more normal pattern of expression of plastid proteins that are under-expressed in the sig6 mutant. However, we also show that loss of the two Why genes does not alleviate the sig6 phenotype. Moreover, the triple mutant shows a second pale phenotype on true leaves, and the plastid protein expression pattern is abnormal compared to either sig6 or wild type plants. Those results cannot be explained by the role of WHIRLY proteins in plastid genome stability since the triple mutant shows fewer plastid genome rearrangements than the why1why3 mutant. Finally, we show that inhibition of the PEP polymerase using rifampicin elicits the same complementation of the sig6 phenotype as the loss of one of the two WHIRLY. Together, these results show the implication of WHIRLY proteins in plastid biogenesis in association with SIG6. We propose a model in which WHIRLY act as activators of PEP activity, particularly during the chloroplast biogenesis. Therefore, the absence of one of the WHIRLY would cause a weak inhibition of PEP, facilitating the set-up of a rescue mechanism by NEPs and, consequently, allowing the complementation of plastid biogenesis in the sig6 mutant. However, the absence of the two WHIRLY proteins would cause a strong inhibition of PEP, and the inability of the rescue mechanism by NEPs to compensate for this strong inhibition, resulting in a more severe phenotype in the sig6 mutant.

La réponse stringente est un processus mis en place lors d'une carence nutritionnelle qui permet l'arrêt coordonné de la croissance. Cette réponse essentielle à la survie des bactéries est très conservée. Elle se caractérise par la production et l'accumulation de guanosine tretra- et pentaphosphate (ppGpp). Le ppGpp, en se fixant sur l'ARN polymérase modifie ses propriétés cinétiques et affecte ainsi de manière globale la transcription de très nombreux gènes. Principalement, l'accumulation de ppGpp inhibe la biosynthèse des ARNs stables (ARNr et ARNt) et en conséquence inhibe la biogenèse des ribosomes. Chez Escherichia coli, le niveau de ppGpp est régulé par les deux enzymes RelA et SpoT. Lors d'une carence en acides aminés, RelA fixée au ribosome détecte le blocage de la machinerie traductionnelle causée par la fixation d'un ARNt déacylé au site A du ribosome, et synthétise du ppGpp. SpoT quant à elle serait capable de détecter et de synthétiser le ppGpp en réponse à d'autres carences nutritionnelles notamment en source de carbone, mais les mécanismes et les signaux détectés sont inconnus. Il a été proposé que la protéine CgtA serait impliquée dans le contrôle de la réponse stringente, en interagissant avec SpoT au niveau des ribosomes. CgtA est une GTPase conservée et essentielle de la famille Obg, mais sa fonction précise est inconnue. Elle a été impliquée à la fois dans la maturation des ribosomes et dans la ségrégation des chromosomes et la division. Le gène cgtA est situé en aval des gènes rplU, rpmA, et yhbE codant respectivement pour les protéines L21 et L27 de la sous-unité 50S du ribosome et pour une protéine intégrale de membrane interne de fonction inconnue. / The stringent response is a physiological process that occurs when bacterial cells encounter nutritional stresses, and allowing coordinated growth arrest. This conserved response is characterized by the accumulation of tetra- and pentaphosphate guanosine (ppGpp). ppGpp bind to RNA polymerase and modifies its kinetic properties, thereby affecting the transcription of many genes. Prinicpaly, ppGpp accumulation inhibits stable RNAs (rRNA and tRNA) biosynthesis, which in consequence inhibits ribosome biogenesis. Escherichia coli contains two enzymes involved in ppGpp metabolism, RelA and SpoT. During amino acid starvation, RelA bound to ribosomes produces ppGpp in response to the presence of uncharged tRNA in the ribosomal A-site. In contrast, SpoT produces ppGpp in response to other types of nutrient limitations, such as carbon starvation, but the detected signals and mechanism involved are still unknown. It has been proposed that the CgtA protein is involved in the stringent response control by interacting with SpoT at the ribosome. CgtA is a conserved and essential small GTPase of the Obg family. CgtA has also been implicated in ribosome maturation, chromosome segregation and division, but its precise function remains unknown. The cgtA gene is located downstream of rplU, rpmA and yhbE genes coding respectively for L21 and L27 proteins of the 50S subunit of the ribosome, and an integral inner membrane protein of unknown function. This genetic proximity with rplU and rpmA genes is highly conserved in bacteria. My thesis work was therefore organized around three questions. First, understanding the role of CgtA in growth control and in the stringent response.

Cell replication and division are central to the proliferation of life, and have implications for normal growth and development as well as disease state. Assembly of a complete picture of the systems which control this process requires identification of individual genetic components, but the identity and complete sequence of events that trigger initiation of cell division, at a point called START in yeast, remain unknown. Here, we evaluated panels of non-essential single gene deletion strains and tested the effects of FDA-approved drugs on cell-cycle progression, using flow cytometry to detect altered DNA content. Previous studies relied mainly on cell size changes to systematically identify genes required for the timely completion of START. This analysis revealed that most gene deletions that altered cell-cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell-cycle control mechanisms. We found that cystathionine-beta-synthase (CBS) advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function which does not require that activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, it is generally not known whether the compounds tested alter the timing of particular cell-cycle transitions. Our approach revealed strong cell-cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell-cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation. Our studies not only transform our view of START, but also expand the repertoire of genetic and chemical means to modulate the eukaryotic cell cycle.

En este trabajo de tesis doctoral se caracteriza funcional y molecularmente el canal TRPV4 en varios modelos de células epiteliales respiratorias mostrando por primera vez la participación de este canal en la función osmoreguladora a nivel celular así como la identificación de nuevas variantes del canal. Se demuestra que la entrada de Ca2+ en respuesta a un hinchamiento hipotónico se produce a través del canal TRPV4 y es necesaria para una eficiente recuperación del volumen o RVD. Por su parte, las células epiteliales respiratorias con fenotipo de fibrosis quística no son capaces de reducir su volumen en un medio hipotónico a causa de una regulación defectuosa del canal, indicando, al mismo tiempo, que la regulación del TRPV4 por el estímulo hipotónico es dependiente de la CFTR.La caracterización de las variantes del canal TRPV4 demuestra que los dominios de ANK son determinantes moleculares claves en el proceso de oligomerización del canal. Al mismo tiempo este trabajo describe nuevos aspectos relacionados con la biogénesis del TRPV4 hasta ahora desconocidos: la oligomerización del canal tiene lugar en el RE, orgánulo donde es N-glicosilado de forma simple antes de ser transportado hacia el Golgi donde sus N-glicanos son madurados. / This thesis characterizes molecularly and funcionally the TRPV4 channel in various models of airway epithelial cells showing, for the first time, the involvement of this channel in an osmoregulatory cellular function as well as the isolation of new splice variants of this channel. It is demonstrated that the TRPV4 channel is the molecular Ca2+ pathway activated by hypotonic estimulus needed to trigger the RVD response. Furthermore, the cystic fibrosis airway epithelial cells showed an impaired RVD due to the misregulation of the TRPV4 channel, indicating that the regulation by the hypotonic stimulus is CFTR-dependent.The characterization of the new variants demonstrated that the ANK domains are key structural determinants in the oligomerization process of the TRPV4. This work also describes new aspects related to the biogenesis of this channel: oligomerization is achieved in the ER, where the TRPV4 is N-glycosilated and then transported to the Golgi where the glycans are matured.

<p>Nitric oxide (NO) is an endogenous gaseous signaling molecule produced by three NO synthase isoforms (NOS1, 2, 3) and important in host defense. The induction of NOS2 during bacterial sepsis is critical for pathogen clearance but its sustained activation has long been associated with increased mortality secondary to multiple organ dysfunction syndrome (MODS). High levels of NO produced by NOS2 incite intrinsic cellular dysfunction, in part by damaging macromolecules through nitration and/or nitrosylation. These include mitochondrial DNA (mtDNA) and enzymes of key mitochondrial pathways required for maintenance of normal O2 utilization and energy homeostasis. However, animal studies and clinical trials inhibiting NOS2 have demonstrated pronounced organ dysfunction and increased mortality in response to live bacterial infections, confirming that NOS2 confers pro-survival benefits. Of particular interest here, the constitutive NOS1 and NOS3 have been linked to the up-regulation of nuclear genes involved in mitochondrial biogenesis but no comparable role has been described for NOS2. <italic> Therefore, I hypothesized that NOS2 is indispensible for host protection but must be tightly regulated to ensure NO levels are high enough to activate mitochondrial and other pro-survival genes, but below the threshold for cellular damage.</italic></p><p>This hypothesis was explored with two major Aims. The <italic>first Aim</italic> was to define the role of NOS2 in the activation of mitochondrial biogenesis in the heart of <italic>E. coli</italic>-treated mice. The <italic>second</italic> was to investigate the ability of NOS2 to be transcriptionally regulated by an enzyme previously shown to induce mitochondrial biogenesis, heme oxygenase-1 (HO-1). This hypothesis was tested using an <italic>in vivo</italic> model of sublethal heat-killed <italic>E. coli</italic> (<italic>HkEC</italic>) peritonitis in C57B/L6 (Wt), NOS2-/-, and TLR4-/- mice. Additionally, <italic>in vitro</italic> systems of mouse AML-12 or Hepa 1-6 cells pretreated with HO-1 activators or <italic>Hmox1</italic> shRNA prior to inflammatory challenge with lipopolysaccharide (LPS) +/- tumor necrosis factor-&alpha; (TNF-&alpha;). For the first Aim, Wt, NOS2-/-, and TLR4-/- mice were treated with (<italic>HkEC</italic> and cardiac tissue analyzed for mitochondrial function, expression of nuclear and mitochondrial proteins needed for mitochondrial biogenesis, and histological expression of NOS2 and TLR4 relative to changes in mitochondrial mass. For the second Aim, Wt mice were pretreated with hemin or carbon monoxide (CO) to activate HO-1 prior to <italic>HkEC</italic>-peritonitis. Liver tissue in these animals was evaluated at four hours for HO-1 induction, <italic>Nos2</italic> mRNA expression, cytokine profiles, and nuclear factor (NF)-&kappa;B activation. Liver cell lines were pretreated with hemin, CO-releasing molecule (CORM), or bilirubin one hour before LPS exposure and the <italic>Nos2</italic> transcriptional response evaluated at two and 24 hours. The MTT assay was used to confirm that <italic>in vitro</italic> treatments were not lethal. </p><p>These studies demonstrated that <italic>HkEC</italic> induced mtDNA damage in the heart that was repaired in Wt mice but not in NOS2-deficient mice. In KO mice, sustained mtDNA damage was associated with the reduced expression of nuclear (NRF-1, PGC-1&alpha;) and mitochondrial (Tfam, Pol-&gamma;) proteins needed for mitochondrial biogenesis. The findings thus supported that NOS2 is required for mitochondrial biogenesis in the heart during Gram-negative challenge. Evaluation of the relationship between HO-1 and NOS2 in murine liver was more complex; HO-1 activation in <italic>HkEC</italic>-treated Wt mice attenuated 4-hour <italic>Nos2</italic> gene transcription. In liver cell lines, hemin, CORM, and bilirubin were unable to suppress <italic>Nos2</italic> expression at the time of maximal induction (2 hours). <italic>Nos2</italic> was, however, suppressed by 24 hours, suggesting that the regulatory impact of HO-1 induction was not engaged early enough to reduce <italic>Nos2</italic> transcription at 2 hours. It is concluded that NOS2 induction in bacterial sepsis optimizes the expression of the mitochondrial biogenesis transcriptional program, which subsequently can also be regulated by HO-1/CO in murine liver. This provides a potential new mechanism by which immune suppression and mitochondrial repair can occur in tandem during the acute inflammatory response.</p> / Dissertation

Le mode vie autotrophique des plantes repose entièrement sur l’intégrité du chloroplaste et notamment l’étape de la biogénèse. La transcription des gènes chloroplastiques, assurée par une PEP (ARN polymérase encodée par le chloroplaste) et deux NEPs (ARN polymérase encodée par le noyau), est l’une des étapes primordiales dans le développement d’un chloroplaste photosynthétique. On distingue trois classes de gènes chloroplastiques : les gènes de classe I, transcrit par la PEP exclusivement; les gènes de classe II, transcrits par la PEP ou les NEPs; et les gènes de classe III, transcrits exclusivement par les NEPs. Pour assurer sa fonction, la PEP doit être associée à des facteurs sigmas. L’un de ceux-ci, la protéine SIG6, est un facteur sigma général et, associé à la PEP, assure la transcription de l’ensemble des gènes de classe I et II lors du développement du chloroplaste photosynthétique. Ainsi, le mutant sig6 présente un phénotype de cotylédons pâles, associé à un retard de biogénèse chloroplastique, ainsi qu’une diminution de la transcription des gènes de classe I, provoquant la diminution de la quantité de protéines de classe I. Dans le laboratoire, nous étudions les deux protéines WHIRLY chloroplastiques (WHY1 et WHY3) pour leur rôle dans le maintien de la stabilité génomique chloroplastique. Toutefois, peu de choses sont encore connues sur leur rôle potentiel dans la transcription ou la biogénèse chloroplastique. Par exemple, lorsque l’on tente de purifier la PEP, on obtient un gros complexe transcriptionnel nommé PTAC (Plastid Transcriptionally Active Chromosome) dans lequel sont retrouvées les deux protéines WHIRLY, suggérant qu’elles pourraient être impliquées dans la transcription chloroplastique. De plus, un possible rôle dans la biogénèse chloroplastique leur a été prêté, notamment chez le maïs. Dans cette étude, nous avons donc cherché à vérifier l’implication des protéines WHIRLY dans la biogénèse chloroplastique par une approche génétique de croisements entre les mutants sig6 et why1why3. Pour cela, nous avons isolé des doubles mutants sig6why1 et sig6why3, ainsi qu’un triple mutant sig6why1why3. À l’aide d’une caractérisation phénotypique et de la quantification de quelques protéines chloroplastiques, nous avons remarqué que la perte d’un des WHIRLY permet de complémenter le phénotype de cotylédons pâles du mutant sig6 et favorise l’expression normale de protéines en principe sous-exprimées dans le mutant sig6. Toutefois, la perte des deux WHIRLY ne permet pas de compenser le phénotype de cotylédons pâles et provoque l’apparition d’un phénotype persistant associé à une expression anormale des protéines chloroplastiques. Ces résultats ne peuvent être expliqués par le rôle des WHIRLY dans le maintien de la stabilité génomique chloroplastique étant donné que le triple mutant sig6why1why3 présente moins de réarrangements que le double mutant why1why3. Finalement, nous montrons que les effets de la perte d’un WHIRLY sur le mutant sig6 peuvent être mimés par l’utilisation de la rifampicine, une drogue inhibant l’ARN polymérase chloroplastique de type bactérienne (PEP). Ensemble, ces résultats démontrent donc l’implication des protéines WHIRLY chloroplastiques dans la biogénèse chloroplastique en association avec la protéine SIG6. Nous proposons un modèle selon lequel les deux protéines WHIRLY permettraient de favoriser l’activité de l’ARN polymérase de type bactérienne, notamment lors du développement du chloroplaste photosynthétique. En cas d’absence d’une des deux protéines, cette diminution partielle d’activité de la PEP favoriserait la mise en place d’un mécanisme de complémentation par le NEPs, permettant finalement de rétablir la biogénèse chloroplastique dans un mutant sig6. En l’absence des deux WHIRLY, le mécanisme de complémentation par les NEPs serait incapable de compenser la forte inhibition de la PEP, se traduisant par une aggravation du retard de développement du chloroplaste dans le mutant sig6. / The autotrophic lifestyle of plants relies entirely on the integrity of chloroplasts and particularly on their biogenesis. Chloroplast gene transcription, performed by a Plastid-Encoded Polymerase (PEP) and two Nuclear-Encoded Polymerases (NEPs), is one of the key steps during the development of photosynthetic chloroplast. There are 3 classes of genes, one transcribed by PEP alone (class I), one by both PEP and NEPs (class II), and the third by NEPs alone (class III). To carry out transcription, PEP associates with plastid sigma factors including the general sigma factor SIG6. sig6 mutants have a pale cotyledon phenotype, a severe decrease in class I gene transcription and a reduction in the level of class I proteins. In our laboratory, we study the role of the two plastid WIHRLY proteins (WHY1 and WHY3) in maintaining plastid genome stability. However, little is known about any role these proteins may play in transcription or chloroplast biogenesis. It seems likely they are involved in plastid gene transcription since they are found in the Plastid Transcriptionally Active Chromosome (PTAC). Moreover, they have been implicated in chloroplast biogenesis in maize. In this study, we verified the implication of these proteins in plastid biogenesis using a genetic approach in which we crossed a sig6 mutant with a why1why3 mutant. We isolated sig6why1 and sig6why3 double mutants and a sig6why1why3 triple mutant. Using a phenotypic characterisation and quantification of some plastid proteins, we show that loss of one of the two Why genes complements the sig6 pale cotyledon phenotype and allows a more normal pattern of expression of plastid proteins that are under-expressed in the sig6 mutant. However, we also show that loss of the two Why genes does not alleviate the sig6 phenotype. Moreover, the triple mutant shows a second pale phenotype on true leaves, and the plastid protein expression pattern is abnormal compared to either sig6 or wild type plants. Those results cannot be explained by the role of WHIRLY proteins in plastid genome stability since the triple mutant shows fewer plastid genome rearrangements than the why1why3 mutant. Finally, we show that inhibition of the PEP polymerase using rifampicin elicits the same complementation of the sig6 phenotype as the loss of one of the two WHIRLY. Together, these results show the implication of WHIRLY proteins in plastid biogenesis in association with SIG6. We propose a model in which WHIRLY act as activators of PEP activity, particularly during the chloroplast biogenesis. Therefore, the absence of one of the WHIRLY would cause a weak inhibition of PEP, facilitating the set-up of a rescue mechanism by NEPs and, consequently, allowing the complementation of plastid biogenesis in the sig6 mutant. However, the absence of the two WHIRLY proteins would cause a strong inhibition of PEP, and the inability of the rescue mechanism by NEPs to compensate for this strong inhibition, resulting in a more severe phenotype in the sig6 mutant.

Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.

Helicobacter pylori est une bactérie qui colonise les cellules épithéliales gastriques humaines. Une des conséquences de cette infection est l'induction de cancers de l'estomac dans 1 à 3 % des cas, via l'injection d'une cytotoxine appelée CagA qui dérégule les voies de signalisation des cellules cibles. Cette injection, dont le mécanisme est encore inconnu, se fait grâce à un système de sécrétion de type IV (T4SS). Le pilus du cagT4SS est encore mal caractérisé. Les protéines CagI, CagL et CagH sont essentielles à la fonctionnalité du cagT4SS et à la biogenèse du pilus. De plus les trois protéines forment un sous-complexe dont les détails moléculaires n'ont pas encore été élucidés. Par conséquent mes études se sont focalisées sur ces trois protéines, leurs interactions et leur relation structure/fonction. J'ai mis en évidence que CagL interagissait directement avec CagI et CagH avec une affinité de l'ordre du micromolaire et que CagI et CagH n'interagissaient pas entre elles. La caractérisation de ces interactions a permis notamment d'identifier un complexe CagL-CagI. Afin de comprendre les détails structuraux de ce complexe, j'ai entrepris deux études structurales. La première consiste à déterminer les résidus de CagL impliqués dans l'interface d'interaction avec CagI par RMN. La seconde étude se focalise sur la détermination de la structure 3D du complexe CagI-CagL par microscopie électronique. Pour cela j'ai purifié le complexe CagI-CagL, monodisperse et stable en solution. Nous avons collecté des images du complexe par cryoEM et généré des classes 2D. Cette étude a permis pour la première fois de caractériser les interactions entre CagL-CagI-CagH et d'obtenir des informations structurales du complexe CagI-CagL / Helicobacter pylori is a bacterium that colonizes the human stomach in half of the world population. It is estimated that 20% and 3% of patients develop peptic ulcer and gastric cancer, respectively. For these reasons, H. pylori was identified as a group 1 carcinogen by the World Health Organization (WHO) in 1994. The most virulent strains of H. pylori carry a type IV secretion system (Cag-T4SS) responsible for the injection of the oncoprotein CagA into gastric epithelial cells. One remarkable feature of the cagT4SS is its external pilus which composition is not clear. CagL, CagH and CagI proteins are critical components of the Cag-T4SS because these proteins are necessary for CagA translocation and are involved in pilus formation. Moreover CagL forms a sub-assembly with CagI and CagH but the molecular details of the complex are still to be discovered. Our objective is to better understand the molecular basis of CagLIH complex by interaction and structural study. CagL interacts with CagI and CagH with a with Kds of 5 µM. CagI does not interact with CagH. The structural study of CagL/CagI complex has been investigated by a two-pronged approach. First I have purified the CagL/CagI complex and collected cyo-EM micrographs. In parallel I have collected NMR spectra of CagL in the presence of CagI and identify the changes in the spectra to determine the residues involved in the interaction. In this study we have, for the first time, characterize the CagL-CagI-CagH interactions and obtained structural informations of the CagI-CagL complex