Feedback regulation of Gab2-dependent signaling by the Ras/MAPK pathway

La voie de signalisation des Récepteurs Tyrosine Kinase (RTK) occupe un rôle central dans la régulation de la croissance cellulaire, la prolifération, la différentiation et la motilité. Une régulation anormale des RTKs mène à plusieurs maladies humaines telles que le cancer du sein, la seconde cause de mortalité chez les femmes à cause de l’amplification et la mutation fréquente de la protéine tyrosine kinase HER2 (ERBB2). Grb2-associated binder (Gab) 2 est une protéine adaptatrice qui agit en aval de plusieurs RTKs, y compris HER2, pour réguler de multiples voies de signalisation. En réponse à la stimulation par de nombreux facteurs de croissances et cytokines, Gab2 est recruté à la membrane plasmique où il potentialise l’activation des voies de signalisation Ras/mitogen-activated protein kinase (MAPK) et PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B). En plus d’occuper un rôle essentiel durant le développement du système hématopoïétique, Gab2 est souvent amplifié dans les cancers, notamment le cancer du sein et les mélanomes. Cependant, les mécanismes moléculaires qui régulent le fonctionnement de Gab2 sont peu connus. Il est établi que lors de l’activation des RTKs, Gab2 est phosphorylé au niveau de plusieurs résidus Tyrosine, menant à l’association de différentes protéines comme p85 et Shp2. En plus de la phosphorylation en Tyrosine, notre laboratoire ainsi que d’autres groupes de recherche avons identifié que Gab2 est aussi phosphorylé au niveau de résidus Ser/Thr suite à l’activation de la voie de signalisation MAPK. Cependant, la régulation des fonctions de Gab2 par ces modifications post-traductionnelles est encore peu connue. Dans le but de comprendre comment Gab2 est régulé par la voie de signalisation MAPK, nous avons utilisé différentes approches. Dans la première partie de ma thèse, nous avons déterminé un nouveau mécanisme démontrant que la voie de signalisation Ras/MAPK, par le biais des protéines kinases RSK (p90 ribosomal S6 kinase), phosphoryle Gab2. Ce phénomène se produit à la fois in vivo et in vitro au niveau de trois résidus Ser/Thr conservés. Des mutations au niveau de ces sites de phosphorylation entrainent le recrutement de Shp2 menant à l’augmentation de la motilité cellulaire, ce qui suggère que les protéines RSK restreignent les fonctions dépendantes de Gab2. Ce phénomène est le résultat de la participation de RSK dans la boucle de rétroaction négative de la voie de signalisation MAPK. Dans la seconde partie de ma thèse, nous avons démontré que les protéines ERK1/2 phosphorylent Gab2 au niveau de plusieurs résidus pS/T-P à la fois in vitro et in vivo, entrainant l’inhibition du recrutement de p85. De plus, nous avons établi pour la première fois que Gab2 interagit physiquement avec ERK1/2 dans des cellules lors de l’activation de la voie de signalisation MAPK. Par ailleurs, nous avons montré un nouveau domaine d’attache du module ERK1/2 sur Gab2. Des mutations sur les résidus essentiels de ce domaine d’attache n’entrainent pas seulement la dissociation de ERK1/2 avec Gab2, mais diminuent également la phosphorylation dépendante de ERK1/2 sur Gab2. Ainsi, nos données montrent que la voie de signalisation MAPK régule les fonctions de la protéine Gab2 par le biais des kinases RSK et ERK1/2. Cette thèse élabore par ailleurs un schéma complet des fonctions de Gab2 dépendantes de la voie de signalisation MAPK dans le développement de nombreux cancers.

CXCR3 biased signaling, heteromerization and decoy properties

Le récepteur de chimiokine CXCR3 est un récepteur couplé à la protéine G (RCPG) exprimé, entre autre, sur les cellules T activées lors d’une réponse immune. CXCR3 est activé par trois ligands inductibles par l’interféron-γ (CXCL9, 10, 11) et, plus récemment, il a été découvert que CXCL4 liait CXCR3. Nous savons que CXCR3 joue un rôle dans la chimiotaxie des leucocytes, mais peu d’attention a été portée sur la signalisation biaisée induite par ces quatre ligands. Alors que l’homodimérisation entre récepteurs de chimiokine est un concept grandement observé, l’hétéromérisation entre deux récepteurs reste un domaine de recherche active. La signalisation biaisée et l’hétéromérisation ont été testées grâce à la technique de bioluminescene resonance energy transfer (BRET) dans des cellules HEK293E. Nous présentons une caractérisation pharmacologique des quatre ligands de CXCR3 et démontrons l’hétéromérisation de CXCR3 avec CXCR4 et avec CXCR7. Nos résultats suggèrent que les ligands de CXCR3 n’agissent pas de manière redondante.

Elucidation of the biological roles of Wnt5a signaling in follicle development

La santé folliculaire est déterminée par un nombre de facteurs endocriniens, paracrines et autocrines. Les gonadotrophines hypophysaires sont les principaux moteurs du développement du follicule, mais leurs actions sont modulées localement par les hormones et des facteurs de croissance. Les glycoprotéines de la famille des WNTs représentent une grande famille de molécules impliquées dans différentes voies de signalisation. Ils sont sécrétés dans le but de moduler et coordonner la réponse des follicules aux gonadotrophines, et leurs activités sont indispensables à la fonction ovarienne et à la fertilité féminine. Les WNTs sont généralement classés en fonction de la (des) voie(s) qu’ils activent. Le rôle des membres de la voie canonique WNT et de ses composants tels que CTNNB1, WNT4, WNT2, FZD1 et FZD4 est bien établi au cours du développement du follicule chez les rongeurs. Un rôle similaire des WNTs dans les espèces mono-ovulatoires demeure essentiellement inconnu. De plus, le rôle des WNT non canoniques dans l'ovaire de rongeurs est méconnu. Les objectifs de cette thèse sont (1) d'élucider la régulation hormonale de l'expression de WNT5A et le rôle physiologique de WNT5A dans les cellules de la granulosa bovine in vitro et (2) d'identifier les rôles physiologiques de WNT5A dans l'ovaire de souris par inactivation génique conditionnelle. Chacun de ces objectifs a mené à la publication d’un article à partir des résultats obtenus au cours de cette thèse. Dans le premier article, le rôle de WNT5A dans les cellules de la granulosa bovine a été étudié in vitro. Nous avons constaté que WNT5A est un régulateur négatif de la stéroïdogenèse stimulée par la FSH issue des cellules de la granulosa, et qu'il agit en supprimant l'activité de signalisation des WNTs canoniques tout en induisant la voie de signalisation MAPK8/JUN. le deuxième article, afin d’examiner le rôle de deux WNTs non-canoniques, WNT5A et WNT11, à différents stades de développement folliculaire, nous avons généré des modèles de souris knock-out conditionnels ciblant les cellules de la granulosa pour chacun de ces WNTs. Les résultats obtenus ont permis de mettre en évidence que WNT5A est nécessaire pour assurer la fertilité normale chez la femelle, le développement folliculaire et la stéroïdogenèse ovarienne. Il est aussi un antagoniste de la réponse aux gonadotrophines, agissant par l’intermédiaire de la suppression de la signalisation canonique des WNTs. Chez les souris knock-out pour WNT11, nous ne constatons aucun défaut important dans la fertilité des femelles. L’ensemble de notre travail met en évidence que WNT5A est essentiel pour le développement normal du follicule et qu’il agit pour inhiber la différenciation des cellules de la granulosa. En résumé, nous avons fourni une étude novatrice et approfondie, utilisant plusieurs modèles et techniques pour déterminer les mécanismes par lesquels WNT5A régule le développement des follicules.

Unveiling the components of the signaling pathway between endosomes and the phagocytic uptake machinery in Dictyostelium discoideum

The soil amoebae Dictyostelium discoideum take up particles from their environment in order to obtain nutrition. The particle transits through the cell within a phagosome that fuses with organelles of different molecular compositions, undergoing a gradual degradation by different sets of hydrolytic enzymes. Griffiths’ concept of “phagosome individuality” predicts signaling from phagosomes into the cytoplasm, which might regulate many aspects of cell physiology. The finding that Dictyostelium cells depleted of the lysozyme AlyA or over-expressing the esterase Gp70 exhibit increased uptake of food particles, led to the postulation of a signaling cascade between endocytic compartments and the cytoskeletal uptake machinery at the plasma membrane. Assuming that Gp70 acts downstream of AlyA, gene-expression profiling of both mutants revealed different and overlapping sets of misregulated genes that might participate in this signaling cascade. Based on these results, we analyzed the effects of the artificial misregulation of six candidate genes by over-expression or negative genetic interference, in order to reconstruct at least part of the signaling pathway. SSB420 and SSL793 were chosen as candidates for the first signaling step, as they were up-regulated in AlyA-null cells and remained unaltered in the Gp70 over-expressing cells. The over-expression of SSB420 enhanced phagocytosis and raised the expression levels of Gp70, supporting its involvement in the signaling pathway between AlyA and Gp70 as a positive regulator of phagocytosis. However, this was not the case of cells over-expressing SSL793, as this mutation had no effects on phagocytosis. For the signaling downstream of Gp70, we studied four commonly misregulated genes in AlyA-depleted and Gp70 over-expressing cells. The expression levels of SLB350, SSB389 and TipD were lower in both mutants and therefore these were assumed as possible candidates for the negative regulation of phagocytosis. Cells depleted of SLB350 exhibited an increased phagocytic activity and no effect on Gp70 expression, proving its participation in the signaling pathway downstream of Gp70. Unlike SLB350, the disruption of the genes coding for SSB389 and TipD had no effects on particle uptake, excluding them from the pathway. The fourth candidate was Yipf1, the only gene that was commonly up-regulated in both mutants. Yet, the artificial over-expression of this protein had no effects on phagocytosis, so this candidate is also not included in the signaling pathway. Furthermore, localizing the products of the candidate genes within the cell helped unveiling several cellular organelles that receive signals from the phagosome and transduce them towards the uptake machinery.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41Δ, sap190Δ, ppm1Δ, rrd1Δ) and U34 modification defects (elp3Δ, kti12Δ, urm1Δ, ncs2Δ) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3Δ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3Δ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3Δ, urm1Δ) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TORsensitive NCR branch via Gln3 misregulation.

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca2+ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca2+ baseline concentration and frequency of oscillating Ca2+ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca2+ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca2+ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K+ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K+ and Na+ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.

La atm??sfera

En esta unidad did??ctica se pretende que los alumnos/as de Educaci??n Secundaria, mediante la realizaci??n de actividades sencillas, razonamiento inductivo y lecturas de ampliaci??n aprendan, de forma significativa, aquellos conceptos f??sicos que les permitan dar una explicaci??n a los fen??menos metereol??gicos, familiarizarse con el trabajo cient??fico, as?? como iniciar la formaci??n de una actitud cr??tica para la conservaci??n del medio ambiente. Como actividad anexa aparece la interpretaci??n de los s??mbolos del mapa del tiempo.

Differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis in brown adipocytes

Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-¹³C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-¹³C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine’s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin).

CAC controlled services sharing a link with connectionless data services in an ATM network: a performance evaluation study

In the B-ISDN there is a provision for four classes of services, all of them supported by a single transport network (the ATM network). Three of these services, the connected oriented (CO) ones, permit connection access control (CAC) but the fourth, the connectionless oriented (CLO) one, does not. Therefore, when CLO service and CO services have to share the same ATM link, a conflict may arise. This is because a bandwidth allocation to obtain maximum statistical gain can damage the contracted ATM quality of service (QOS); and vice versa, in order to guarantee the contracted QOS, the statistical gain have to be sacrificed. The paper presents a performance evaluation study of the influence of the CLO service on a CO service (a circuit emulation service or a variable bit-rate service) when sharing the same link

Multicast algorithms evaluation using an adaptative routing in ATM networks

This paper proposes a multicast implementation based on adaptive routing with anticipated calculation. Three different cost measures for a point-to-multipoint connection: bandwidth cost, connection establishment cost and switching cost can be considered. The application of the method based on pre-evaluated routing tables makes possible the reduction of bandwidth cost and connection establishment cost individually

An ATM distributed simulator for network management research

Due to the high cost of a large ATM network working up to full strength to apply our ideas about network management, i.e., dynamic virtual path (VP) management and fault restoration, we developed a distributed simulation platform for performing our experiments. This platform also had to be capable of other sorts of tests, such as connection admission control (CAC) algorithms, routing algorithms, and accounting and charging methods. The platform was posed as a very simple, event-oriented and scalable simulation. The main goal was the simulation of a working ATM backbone network with a potentially large number of nodes (hundreds). As research into control algorithms and low-level, or rather cell-level methods, was beyond the scope of this study, the simulation took place at a connection level, i.e., there was no real traffic of cells. The simulated network behaved like a real network accepting and rejecting SNMP ones, or experimental tools using the API node

Functional studies of the deubiquitinating enzymes USP19, USP4 and UCH-L1

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis.

Congestion probability routing in virtual path ATM network

This dissertation focuses on the problem of providing mechanisms for routing point to point and multipoint connections in ATM networks. In general the notion of multipoint connection refers to connections that involve a group of users with more than two members. The main objective of this dissertation is to contribute to design efficient routing protocols with alterative routes in fully connected VP-based ATM Networks for call establishment of point to point and multipoint VC connections. An efficient route should be computed during this connection establishment phase.

Enhanced convolution approach for CAC in ATM networks, an analytical study and implementation

The characteristics of service independence and flexibility of ATM networks make the control problems of such networks very critical. One of the main challenges in ATM networks is to design traffic control mechanisms that enable both economically efficient use of the network resources and desired quality of service to higher layer applications. Window flow control mechanisms of traditional packet switched networks are not well suited to real time services, at the speeds envisaged for the future networks. In this work, the utilisation of the Probability of Congestion (PC) as a bandwidth decision parameter is presented. The validity of PC utilisation is compared with QOS parameters in buffer-less environments when only the cell loss ratio (CLR) parameter is relevant. The convolution algorithm is a good solution for CAC in ATM networks with small buffers. If the source characteristics are known, the actual CLR can be very well estimated. Furthermore, this estimation is always conservative, allowing the retention of the network performance guarantees. Several experiments have been carried out and investigated to explain the deviation between the proposed method and the simulation. Time parameters for burst length and different buffer sizes have been considered. Experiments to confine the limits of the burst length with respect to the buffer size conclude that a minimum buffer size is necessary to achieve adequate cell contention. Note that propagation delay is a no dismiss limit for long distance and interactive communications, then small buffer must be used in order to minimise delay. Under previous premises, the convolution approach is the most accurate method used in bandwidth allocation. This method gives enough accuracy in both homogeneous and heterogeneous networks. But, the convolution approach has a considerable computation cost and a high number of accumulated calculations. To overcome this drawbacks, a new method of evaluation is analysed: the Enhanced Convolution Approach (ECA). In ECA, traffic is grouped in classes of identical parameters. By using the multinomial distribution function instead of the formula-based convolution, a partial state corresponding to each class of traffic is obtained. Finally, the global state probabilities are evaluated by multi-convolution of the partial results. This method avoids accumulated calculations and saves storage requirements, specially in complex scenarios. Sorting is the dominant factor for the formula-based convolution, whereas cost evaluation is the dominant factor for the enhanced convolution. A set of cut-off mechanisms are introduced to reduce the complexity of the ECA evaluation. The ECA also computes the CLR for each j-class of traffic (CLRj), an expression for the CLRj evaluation is also presented. We can conclude that by combining the ECA method with cut-off mechanisms, utilisation of ECA in real-time CAC environments as a single level scheme is always possible.