Ovarian Cancer is the 5th most common cause of cancer death in women and the second most common gynaecological cancer in the UK. Worldwide, around 152,000 women were estimated to have died from ovarian cancer in 2012. Survival rates for women with epithelial ovarian cancer have not significantly changed since platinum-based treatment was introduced over 30 years ago. This is particularly disconcerting considering the fact that there is a less than 5% five year survival rate for patients diagnosed with late stage high grade serous ovarian cancer. This thesis examines the role of BMP signalling in ovarian cancer using in vitro cancer cell models. It builds upon the initial published work by the Inman lab identifying autocrine BMP9 as a promoter of ovarian cancer cell proliferation. The findings of Chapters 3-5 provide strong evidence indicating BMP9 as a context specific modulator of ovarian cancer cell proliferation. This significantly builds upon on the sole pro-proliferative BMP9 growth response previously described. Responding cell lines were subjected to a microarray with and without BMP9 treatment In order to determine early BMP target genes which were subsequently transiently knocked down in order to determine their role in the aetiology of said growth phenotype. ID1 gene expression was found to significantly contribute to the BMP9 proproliferative phenotype. Moreover several other BMP genes identified significantly alter basal cell proliferation. It was subsequently determined that BMP9 implemented a cell growth phenotype by negating apoptosis. .Excitingly, preliminary evidence suggests a marked reduction in detectable levels of a recently described Bax isoform, Bax β that coincide with BMP9 addition and the resultant anti-apoptotic phenotype observed. This is very interesting as no prior evidence correlating the BMP family and Bax β currently exists. These findings provide an enhanced understanding of BMP9s contribution to ovarian cancer pathogenesis that may result in the development of effective and targeted therapeutic interventions upon further stratification of the contextuality of the BMP induced growth response.

<p>Nerve injury induces up-regulation of the chaperone protein clusterin in affected neurons and adjacent astrocytes but the functional significance of this response is unclear. We find that motor neuron survival is significantly greater in clusterin(+/+) compared to (-/-) mice. These results suggest that endogenous expression of clusterin is neuroprotective after nerve injury. However, motor neuron survival in clusterin overexpressing mice was not different from that in wildtype mice. In contrast, treatment of neuronal cultures with clusterin-TAT recombinant protein is neuroprotective, including a positive effect on neuronal network complexity.</p><p>Since extracellular clusterin complexes are endocytosed after binding to various receptors, we examined the expression of known clusterin binding receptors in the spinal cord. We find that megalin is expressed in the nuclei of two cell populations in the mouse spinal cord: i) oligodendrocytes in late postnatal and adult spinal cord white matter, and ii) transiently (E11-15) in a population of immature astrocytes in the dorsal spinal cord. We find no correlation between clusterin and megalin in the intact or injured spinal cord. However, intranuclear localization of megalin, suggesting signalling properties, is supported by the co-localization with γ-secretase, the enzyme responsible for endodomain cleavage of megalin. Megalin deficient mice display a pronounced deformation of the dorsal part of spinal cord, an almost complete absence of oligodendroglial progenitor cells, and a marked reduction in the population of mature astrocytes at later prenatal developmental stages.</p><p>Taken together, our findings indicate that megalin is a novel signalling molecule for distinct populations of glial cells in the pre- and postnatal spinal cord. The functional role(s) of megalin is unknown. However, its expression patterns and cellular localization suggest that megalin regulates differentiation of oligodendrocytes and astrocytes in the prenatal spinal cord, as well as the function of myelinating oligodendrocytes in the postnatal spinal cord.</p>

Nerve injury induces up-regulation of the chaperone protein clusterin in affected neurons and adjacent astrocytes but the functional significance of this response is unclear. We find that motor neuron survival is significantly greater in clusterin(+/+) compared to (-/-) mice. These results suggest that endogenous expression of clusterin is neuroprotective after nerve injury. However, motor neuron survival in clusterin overexpressing mice was not different from that in wildtype mice. In contrast, treatment of neuronal cultures with clusterin-TAT recombinant protein is neuroprotective, including a positive effect on neuronal network complexity. Since extracellular clusterin complexes are endocytosed after binding to various receptors, we examined the expression of known clusterin binding receptors in the spinal cord. We find that megalin is expressed in the nuclei of two cell populations in the mouse spinal cord: i) oligodendrocytes in late postnatal and adult spinal cord white matter, and ii) transiently (E11-15) in a population of immature astrocytes in the dorsal spinal cord. We find no correlation between clusterin and megalin in the intact or injured spinal cord. However, intranuclear localization of megalin, suggesting signalling properties, is supported by the co-localization with γ-secretase, the enzyme responsible for endodomain cleavage of megalin. Megalin deficient mice display a pronounced deformation of the dorsal part of spinal cord, an almost complete absence of oligodendroglial progenitor cells, and a marked reduction in the population of mature astrocytes at later prenatal developmental stages. Taken together, our findings indicate that megalin is a novel signalling molecule for distinct populations of glial cells in the pre- and postnatal spinal cord. The functional role(s) of megalin is unknown. However, its expression patterns and cellular localization suggest that megalin regulates differentiation of oligodendrocytes and astrocytes in the prenatal spinal cord, as well as the function of myelinating oligodendrocytes in the postnatal spinal cord.

No description available.

El adenocarcinoma ductal pancreático (PDAC) es la quinta causa de muerte por cáncer en los países desarrollados y uno de los tumores humanos más agresivos. A pesar del papel clave de la angiogénesis -la formación de nuevos vasos a partir de otros preexistentes- en la progresión y metástasis de muchos tumores, su papel en PDAC ha sido poco caracterizado. El activador tisular del plasminógeno (tPA), una proteína multifuncional que regula numerosas funciones celulares, ejerce efectos proangiogénicos en modelos in vivo de PDAC, aunque no se han analizado los mecanismos moleculares responsables de estos efectos. Esta tesis trata de identificar el papel de tPA en la angiogénesis del PDAC, así como de descubrir los factores responsables de la sobreexpresión de tPA en cáncer de páncreas. En primer lugar, hemos demostrado que los efectos pro-angiogénicos de tPA pueden ser tanto directos como indirectos. Por un lado, aunque tPA no cambia los niveles de moléculas pro-angiogénicas como VEGF, TGF-b, IL-1 o IL-8 producidas por las células tumorales o endoteliales, sí que induce la sobreexpresión y activación de MMP-9, una metaloproteasa implicada en promover angiogénesis, sugiriendo por tanto que esta proteína puede mediar de forma indirecta los efectos proangiogénicos de tPA. Por otro lado, hemos encontrado que tPA, de forma independiente de su actividad catalítica, promueve directamente la proliferación, migración y tubulogénesis de las células endoteliales. Estos efectos son mediados por la activación en estas células de las rutas de señalización ERK1/2, AKT y JNK. Además, mediante siRNA o inhibidores químicos, hemos encontrado que Annexina A2, Galectina-1 y EGFR son necesarios para la activación de la señalización inducida por tPA en células endoteliales. Finalmente, hemos visto que citoquinas inflamatorias e hipoxia, dos eventos asociados a PDAC y además inductores de angiogénesis, dan lugar a un fuerte incremento de los niveles de tPA en células tumorales pancreáticos. Todos estos datos apoyan un mecanismo de retroalimentación positiva entre estímulos proangiogénicos presentes en las células tumorales y el estroma, y el incremento de la molécula proangiogénica tPA. / Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer death in the developed countries and one of the most aggressive human tumors. Despite the key contribution of angiogenesis – the growth of new vessels from pre-existing ones- to the progression and spread of many cancers, its role in PDAC has been poorly characterized. Tissue plasminogen activator (tPA), a multifunctional protein regulating a broad range of cellular functions, has been reported to exert pro-angiogenic effects in in vivo models of PDAC, although the underlying molecular mechanism has not been analyzed. This work aims to elucidate the role of tPA in the angiogenesis of PDAC as well as to identify the factors responsible for tPA increase in pancreatic cancer. First, we demonstrated that tPA pro-angiogenic effects are both indirect and direct. On the one hand, tPA does not change the levels of the pro-angiogenic molecules VEGF, TGF-b, IL-1 or IL-8 produced by pancreatic tumoral cells or endothelial cells, but it is involved in MMP-9 –a potent stimulator of angiogenesis- upregulation and activation in pancreatic and endothelial cells, suggesting that this matrix metalloproteinase can indirectly mediate tPA angiogenic effects. On the other hand, we found that tPA, in a catalytic-independent way, directly promotes endothelial cell proliferation, migration and tubulogenesis. These direct effects of tPA are mediated by activation of ERK1/2, AKT and JNK signaling pathways in endothelial cells. In addition, using siRNA technology or chemical inhibitors, we found that AnnexinA2, Galectin-1 and EGFR are required for tPA-mediated signaling activation in endothelium. Finally, we found that inflammatory cytokines and hypoxia, two hallmarks of PDAC and also angiogenic stimuli, lead to a sharp increase in tPA levels in pancreatic tumoral cells. These data support a feed-back loop between proangiogenic stimuli present in both tumoral and stromal cells and the increase of the proangiogenic molecule tPA.

Cdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.

Highly-trained athletes are accustomed to varied and high-volume based exercise stimuli and eliciting adaptation in individuals already possessing the necessary physiology to compete at the highest level is difficult. Therefore, identifying novel, potent and time efficient methods of achieving cumulative training stress is a continual quest for coaches and exercise scientists. This thesis examined the acute and chronic effects of manipulating exercise and recovery during brief all-out sprint cycling on adaptive responses favouring enhanced endurance capacity. Chapter 3 highlighted that low-volume non-work matched all-out sprint cycling, whether it be interval- (4 x 30 s bouts) or continuous based (1 x 2 min bout) provides a similarly potent stimulus for the acute induction of cell signalling pathways and key growth factors associated with mitochondrial biogenesis and angiogenesis in trained individuals. In line with manipulating recovery and in attempting to identify a novel and potent exercise intervention capable of giving athletes more return on their training investment, Chapters 4-6 investigated the efficacy of combining sprint interval training with post-exercise blood flow restriction (BFR). Firstly, it was demonstrated that BFR potentiates HIF-1α mRNA expression in response to SIT, tentatively suggesting an enhanced stimulus for hypoxia- and/or metabolic-mediated cell signalling associated with mitochondrial biogenesis and angiogenesis over SIT alone. Secondly, four weeks of SIT combined with post-exercise BFR provides a greater training stimulus over SIT alone in trained individuals to enhance VO2max (4.7 v 1.1 % change) and MAP (3.8 v 0.2 % change), but not 15-km TT performance. Finally, in response to four weeks of SIT combined with post-exercise BFR, an international female track sprint cyclist increased her CP and W by 7 and 2 % and VO2max and absolute MAP by 3 and 4 %, respectively. Through a combination of an acute in vivo molecular experiment, a training study and an athlete case study, this thesis has introduced a potentially potent and novel training concept that appears capable of augmenting aerobic capacity.

Allosteric inhibition of kinases provides high selectivity and potency due to lower evolutionary pressure in conserving allosteric vs. orthosteric sites. The former are regions distinct from the kinase active site, yet, when perturbed through allosteric effectors, induce conformational and/or dynamical changes that in turn modulate kinase function. Protein kinases involved in cyclic nucleotide signalling are important targets for allosteric inhibition due to their association with diseases, from infections to Cushing’s syndrome. This dissertation specifically focuses on elucidating the molecular mechanism of allosteric inhibition in the cAMP-dependent protein kinase (PKA) and the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG), which are targets for a generalized tumor predisposition commonly referred to as Carney Complex and for malaria, respectively. In chapters 2 and 3, we focus on the agonism-antagonism switch (i.e. allosteric pluripotency) observed as the phosphorothioate analog of cAMP, Rp-cAMPS (Rp), binds to PKA. Utilizing Nuclear Magnetic Resonance (NMR), Molecular Dynamics (MD) simulations and Ensemble Allosteric Model (EAM), we determined that two highly homologous cAMP-binding domains respond differently to Rp, giving rise to a conformational ensemble that includes excited inhibition-competent states. The free energy difference between this state and the ground inhibition-incompetent state is tuned to be similar to the effective free energy of association of the regulatory (R) and catalytic (C) subunits, leading to allosteric pluripotency depending on conditions that perturb the R:C affinity. The general significance of these results is a re-definition of the concept of allosteric target to include not only the isolated allosteric receptor, but also its metabolic and proteomic sub-cellular environment. In chapter 4, we utilize a mutant that silences allosteric pluripotency to reveal that the agonism-antagonism switch of PKA not only arises from the mixed response of tandem domains, but also from the mixed response of allosteric regions within a single domain that mediates interactions with Rp. In chapter 5, the allosteric inhibition of PfPKG associated with malaria is induced through base-modified cGMP-analogs and the underlying inhibitory mechanism is determined. We show that, when bound to a PfPKG antagonist, the regulatory domain of PfPKG samples a mixed intermediate state distinct from the native inhibitory and active conformations. This mixed state stabilizes key cGMP-binding regions, while perturbing the regions critical for activation, and therefore it provides an avenue to preserve high affinity, while promoting significant inhibition. Overall, in this thesis, previously elusive mechanisms of allosteric inhibition were elucidated through the combination of NMR, MD, and EAM methods. Through this integrated approach, we have unveiled an emerging theme of inhibitory ‘mixed’ states, either within a single domain or between domains, which offer a simple but effective explanation for functional allostery in kinases. / Thesis / Candidate in Philosophy

[ES] Los procesos de comunicación celular permiten a las células desarrollar una acción coordinada durante la embriogénesis y asimilar de forma coherente las señales recibidas a través del entorno. Algunas de las moléculas señalizadoras más usadas en la clínica y la investigación son las citoquinas. Sin embargo, existe una tendencia creciente en el uso de otro tipo de moléculas, como los iones metálicos. Algunos iones como el calcio y el zinc actúan como segundos mensajeros intracelulares. Otros como el litio son capaces de inactivar proteínas quinasa alterando rutas de señalización. En el desarrollo de esta tesis doctoral, se ha estudiado el efecto del zinc en células musculares de ratón, el papel del zinc en la auto-renovación de células madre embrionarias (CMEs), y el papel del litio en la diferenciación de CMEs. El estudio del efecto del zinc sobre los mioblastos demostró que el zinc es capaz de estimular la diferenciación de los mioblastos. El análisis del zinc intracelular, en los diferentes estadios de diferenciación de las células musculares, demostró que los miotubos eran capaces de albergar mayor cantidad de zinc en su interior. Los resultados mostraron que la adición de zinc extracelular estimula la fosforilación y activación de la proteína quinasa Akt. También se ha visto que el transportador de zinc, Zip7, es crítico en el proceso de diferenciación celular mediado por el zinc, además, su activación incrementa la fosforilación de Akt. La inhibición de Zip7 mediante ARN interferente redujo la fosforilación de Akt y consecuentemente origino unos niveles menores de diferenciación de los mioblastos expuestos a zinc extracelular. Nuestros resultados demuestran que altas concentraciones de zinc extracelular producen un incremento en la diferenciación de los mioblastos debido a la activación de Akt mediada por Zip7. Para el segundo estudio, se analizó el efecto del zinc sobre las CMEs. Como control de mantenimiento de la pluripotencia se usó medio suplementado con factor inhibidor de leucemia (LIF). Se ha observado que la adición externa de concentraciones de zinc superiores a 100 µM produce un incremento inmediato de la concentración de zinc intracelular activando Akt. Los resultados demuestran que las células tratadas con altas concentraciones de zinc mantienen su capacidad de auto-renovación. Para demostrar que el efecto del zinc en CMEs está asociado a la activación de Akt mediada por Zip7, se inhibió la fosforilación de Akt y se silenció Zip7. Ambos abordajes dieron como resultado un incremento en la diferenciación de las células tratadas con zinc. Por otro lado, CMEs cultivadas durante 30 días en presencia de zinc fueron capaces de retener su pluripotencia, mientras que el control sin zinc presentaba rasgos claros de diferenciación celular. Por último, la combinación de LIF con zinc produjo un incremento importante del efecto del LIF en cuanto al mantenimiento de la capacidad de auto-renovación celular. Por último, se ha estudiado el efecto del litio en la diferenciación de las CMEs. El litio es un inhibidor de la glucógeno sintasa quinasa 3ß (GSK3ß). En términos de CMEs, GSK3ß activa los mecanismos de diferenciación. Los resultados obtenidos indican que altas concentraciones de litio (10 mM) son capaces de fosforilar e inhibir la proteína GSK3ß. Sin embargo, en lugar de mantener la pluripotencia, las células madre se diferenciaron hacia el linaje del mesodermo tras 3 días de cultivo. Después de un total de 6 días, las células tratadas con 10 mM de litio presentaron características de endotelio hemogénico. La inhibición de GSK3ß dio como resultado la activación de la proteína ß-catenina, cuya actividad transcripcional es necesaria para la hematogénesis embrionaria. La capacidad de las células endoteliales con potencial hemogénico obtenidas de derivar en células madre hematopoyéticas fue confirmada tras su maduración durante 11 día / [CA] Els processos de comunicació cel·lular permeten a les cèl·lules desenvolupar una acció coordinada durant la embriogènesis y assimilar de forma coherent als senyals rebudes a través de l'entorn. Algunes de les molècules senyalitzadores més usades en la clínica i la investigació són les citocines. No obstant, hi ha una tendència creixent en l'ús d'un altre tipus de molècules, com els ions metàl·lics. Alguns ions com el calci i el zinc són capaços de dur a terme funcions de missatger secundari. Altres com el liti són capaços d'inactivar proteïnes quinasa alterant rutes de senyalització. Durant el desenvolupament d'aquest treball de tesi doctoral, s'ha estudiat l'efecte del zinc sobre mioblasts de ratolí, el paper del zinc en l'auto-renovació de les cèl·lules mare embrionàries (CMEs), i el paper del liti sobre la diferenciació de les CMEs. L'estudi de l'efecte del zinc sobre els mioblasts ha demostrat que el zinc és capaç d'incrementar la diferenciació dels mioblasts. L'anàlisi del zinc intracel·lular ha demostrat que els mioblasts diferenciats eren capaços d'albergar major quantitat de zinc intracel·lular. Els resultats han mostrat que suplementar les cèl·lules amb zinc extracel·lular produïx una major fosforilació i activació de la proteïna quinasa Akt. D'altra banda, s'ha observat que el transportador de zinc Zip7 es crític per a la diferenciació cel·lular mediada pel zinc. S'ha demostrat que l'activació d'aquest transportador mitjançant zinc extracel·lular és capaç d'incrementar la fosforilació d'Akt. La inhibició d'aquest transportador mitjançant ARN interferent ha donat com a resultat una menor fosforilació d'Akt i una menor diferenciació dels mioblasts exposats a zinc. Aquests resultats demostren que altes concentracions de zinc extracel·lular produeixen un incrementar la diferenciació dels mioblasts a causa de l'activació d'Akt per mitja de Zip7. Per al segon estudi, s'ha analitzat l'efecte del zinc sobre les CMEs. Com a control de manteniment de la pluripotència es va usar medi suplementat amb factor inhibidor de leucèmia (LIF). S'ha observat que les concentracions extracel·lulars de zinc a partir de 100 µM produïxen un increment immediat de la concentració intracel·lular, produint l'activació d'Akt per mitja de Zip7. Les CMEs tractades amb altes concentracions de zinc mantenen l'auto-renovació. Per demostrar que aquest efecte està associat a l'activació d'Akt mediada per Zip7, es va inhibir la fosforilació d'Akt i es va silenciar el transportador Zip7. Tots dos abordatges han donat com a resultat un increment en la diferenciació de les CMEs tractades amb zinc. D'altra banda, les CMEs van ser capaços de retenir la seva pluripotència després de ser cultivades durant 30 dies en presència de zinc, mentre que el control sense zinc presentava trets clars de diferenciació cel·lular. Finalment, la combinació de LIF amb zinc ha produit un increment sinèrgic de l'efecte del LIF. Finalment, també s'ha estudiat l'efecte del liti en la diferenciació de les CMEs. El liti és un inhibidor de la glicogen sintasa quinasa 3 beta (GSK3ß). En termes de CMEs, aquesta proteïna activa els mecanismes de diferenciació. Els resultats obtinguts indiquen que altes concentracions de liti (10 mM) tenen la capacitat de fosforilar i inhibir la proteïna GSK3ß. No obstant això, en lloc de mantenir la pluripotència, les CMEs es van diferenciar cap al llinatge del mesoderma després de 3 dies. Després d'un total de 6 dies, les cèl·lules tractades amb 10 mM de liti presentaven característiques d'endoteli hemogénic. La fosforilació de GSK3ß va donar com a resultat l'activació de la proteïna ß-catenina, l'activitat trasncripcional d'aquesta proteïna és necessària per a la hematogénesis embrionària. La capacitat de les cèl·lules endotelials amb potencial hemogénic obtingudes de derivar en cèl·lules mare hematopoètiques va ser confirmada després de la / [EN] The cell signalling process allows cells to develop a coordinated action during embryogenesis and assimilate coherently the signals received through the environment. Some of the most currently used signalling molecules in clinics and research are growth factors and cytokines. However, there is a growing trend in the use of other types of molecules, such as metal ions. Some ions such as calcium and zinc are able to carry out secondary messenger functions, transmitting signals in cascade. Others ions, such as lithium, are capable to inactivate protein kinases altering signalling pathways. During the development of this doctoral thesis, we investigated the effect of zinc on mouse muscle cells (myoblasts), the role of zinc in embryonic stem cells (ESCs) self-renewal, and the role of lithium in the differentiation of ESCs. In the first chapter, we showed that zinc is able to increase the differentiation of myoblasts. The analysis of intracellular zinc indicated that the differentiated myoblasts were capable to harbour higher concentration of intracellular zinc than undifferentiated ones. Addition of high concentration of extracellular zinc increased protein kinase Akt phosphorylation and activation. Akt activity is critical for myoblasts differentiation and has been well studied by other authors. Our results indicated that zinc transporter Zip7 was critical for zinc-mediated cell differentiation. It was prior demonstrated that the activation of this transporter by extracellular zinc increased the phosphorylation of Akt. The inhibition of Zip7 by interfering RNA resulted in a lower phosphorylation of Akt and reduced differentiation of the myoblasts exposed to extracellular zinc. These results demonstrated that high concentration of extracellular zinc enhances the differentiation of myoblasts through activation of Akt mediated by Zip7. In the second chapter, we have analysed the effect of zinc on ESCs. Leukaemia inhibitory factor (LIF) was used as pluripotency sustaining factor. We observed that extracellular supplementation of 100 ¿M zinc produced an immediate increase of the intracellular concentration, which resulted in the activation of Akt mediated by Zip7 transporter. ESCs treated with high concentrations of zinc maintained self-renewal. The role of Akt on ESCs self-renewal has been well established in the literature. To demonstrate that this effect is associated with the activation of Akt mediated by Zip7, we inhibited Akt phosphorylation and silenced the expression of Zip7. Both approaches resulted in an increase in the differentiation levels of the ESCs treated with zinc. We further demonstrated that ESCs treated with zinc during 30 days were able to retain their pluripotency, while the control condition cultured 30 days without zinc presented evident traits of spontaneous cellular differentiation. Finally, the combination of LIF with zinc produced a synergistic-like increase in the effect of LIF on ESCs self-renewal. Finally, we addressed the effect of lithium on the differentiation of ESCs. Lithium is an inhibitor of glycogen synthase kinase 3 beta (GSK3ß). In terms of ESCs, GSK3ß activates differentiation mechanisms. Our results indicated that high concentration of lithium (10 mM) was able to phosphorylate and strongly inhibit the activity of GSK3ß. However, instead of maintaining pluripotency, ESCs differentiated into the mesoderm lineage after 3 days of culture. After a total of 6 days, ESCs treated with 10 mM lithium showed haemogenic endothelium characteristics, expressing CD31, Sca-1 and CD31/Sca-1 positive cells. The phosphorylation of GSK3ß resulted in the activation of the ß-catenin protein, whose transcriptional activity is necessary for embryonic hematogenesis. The ability of endothelial cells with hemogenic potential obtained from lithium-treated ESCs to derive into hematopoietic stem cells was confirmed after maturation of these cells, resulting in rounded cell aggregates positive for Sox17. / Ministerio de Ciencia, Innovación y Universidades a través de la beca BES-2013-064052 y los proyectos MAT2012-38359-C03-01 y MAT2015-69315-C3-1-R. / Mnatsakanyan Movsesyan, H. (2019). ROLE OF IONS IN STEM CELLS SIGNALLING [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123063

Les histidine kinases (HKs) représentent une vaste famille de protéines impliquées dans la perception des signaux environnementaux chez les bactéries, les champignons et les plantes. Ces protéines joueraient notamment un rôle majeur dans l’adaptation aux stresses, mais aussi dans la virulence de nombreux micro-organismes procaryotes et eucaryotes. Si les HKs sont à présent bien connues chez les bactéries et les plantes, tant sur un plan structural que fonctionnel, les connaissances concernant ces protéines chez les autres clades de l’arbre du vivant demeurent plus que fragmentaires. C’est ainsi que le premier objectif de ce travail a consisté en l’exploration in silico de la structure et de la distribution des HKs chez les organismes eucaryotes dans le cadre de plusieurs études bioinformatiques : i) chez les champignons inférieurs, ii)chez les levures bourgeonnantes et enfin iii) à travers l’ensemble des super-groupes eucaryotes. Les HKs n’étant pas retrouvées chez les mammifères, elles suscitent depuis quelques années une attention particulière de la communauté scientifique en tant que nouvelles cibles pour le développement d’antimicrobiens. C’est précisément dans ce contexte que la partie expérimentale de ce projet a été initiée au sein du GEIHP. Cette équipe porte en effet ses efforts sur le filamenteux multi-résistant Scedosporium apiospermum qui se situe au second rang parmi les moisissures capables de coloniser chroniquement les poumons des patients atteints de mucoviscidose. Ainsi, dans l’optique d’identifier de nouvelles cibles thérapeutiques du champignon, la seconde partie de ce projet s’est focalisée sur la caractérisation fonctionnelle des HKs chez Scedosporium apiospermum. En parallèle, cette étude nous a également amenés à développer de nouveaux outils moléculaires adaptés à S.apiospermum en vue de futures études d’imageries de fluorescence et de bioluminescence. / Histidine kinases (HKs) represent a broad family of proteins involved in the perception of environmental signals in bacteria, fungi and plants.These proteins play a major role in stress adaptation, but also in the virulence of many prokaryotic and eukaryotic microorganisms. Although HKs are now well known in bacteria and plants, both structurally and functionally, knowledge about these proteins in other clades of the living tree remains more than fragmentary. Thus the first objective of this work was the in silico exploration of the structure and distribution of HKs in eukaryotic organisms through several bioinformatics studies : i) in the lower fungi, ii)in budding yeasts, and finally iii) across all eukaryotic supergroups. Since HKs are not found in mammals, they have been attracting attention in recent years from the scientific community as new targets for the development of antimicrobials. It is precisely in this context that the experimental part of this project was initiated in the GHEIHP. This team is focusing on the multi-resistant filamentous Scedosporiumapiospermum, which ranks second among the molds capable of chronycally conolizing the lungs of cysticfibrosis patients. Thus, in order to identify new therapeutic targets of the fungus, the second part of this project focused on the functional characterization of HKs in S. apiospermum. In parallel, this study also led us to develop new molecular tools adapted to S. apiospermum for future studies of fluorescence or bioluminescence imaging.