Secretory IgA: an actor of the interplay between the commensal flora and the intestinal immune system?

In the gastro-intestinal tract,Peyers patches have been describedas a major inductive site for mucosalsecretory IgA (SIgA) responses directedagainst pathogens. The classicalview is that SIgAserves as the firstline of defense against microorganismsby agglutining potential invadersand faciliting their clearance byperistaltic and mucociliary movements,a mechanism called immuneexclusion. Our laboratory has shownthat SIgA is not only able to be"retrotransported" into Peyers patchesvia the associated M cells, but also todeliver sizeable cargos in the form ofSIgA-based immune complexes, resultingin the onset of non-inflammatorytype of responses. Such a novelfunction raises the question of thepossible role of mucosal SIgA in theinterplay with commensal bacteriaand the contribution of the antibody inbacterial homeostasis. To address thisquestion, Lactobacillus rhamnosus(LPR) was administered into a mouseligated loop comprising a Peyerspatch, in association or not with SIgA.The fate of fluorescently labelled bacteriawas followed by laser scanningconfocal microscopy at different incubationtimes. After 2 hours of incubationin the loop, LPR bacteria arefound more abundantly in thesubepithelial dome (SED) regionwhen they are coated with SIgA thanLPR administered alone despite theyare absent from neighboring villi.Herein, it is shown that this mechanismof entry involves M cells inPeyers pathes. After their sampling byM cells, bacteria are engulfed by thedendritic cells of the subjacent SEDregion. Interestingly, LPR bacteriaare found coated by the endogenousnatural SIgA present in mice intestinalsecretions, confirming the requirementof SIgA for this type of entry.The subsequent effect on the maturationof dendritic cells after interactionwith LPR was investigated in vitroin presence or not of SIgA by measuringthe expression of CD40, CD80and CD86 surface markers with flowcytometry analyses. Results show thatDCs respond in the same way in presenceof SIgA than with LPR bacteriaalone, indicating that SIgA does notmodulate the interaction betweenDCs and bacteria in this context. Thiswork gives new evidences about theinvolvement of SIgA in the mechanismby which the intestinal immunesystem permanently checks the contentof the intestine.

Tapping the innovation potential of corporate engagement with stakeholders

Summary This dissertation explores how stakeholder dialogue influences corporate processes, and speculates about the potential of this phenomenon - particularly with actors, like non-governmental organizations (NGOs) and other representatives of civil society, which have received growing attention against a backdrop of increasing globalisation and which have often been cast in an adversarial light by firms - as a source of teaming and a spark for innovation in the firm. The study is set within the context of the introduction of genetically-modified organisms (GMOs) in Europe. Its significance lies in the fact that scientific developments and new technologies are being generated at an unprecedented rate in an era where civil society is becoming more informed, more reflexive, and more active in facilitating or blocking such new developments, which could have the potential to trigger widespread changes in economies, attitudes, and lifestyles, and address global problems like poverty, hunger, climate change, and environmental degradation. In the 1990s, companies using biotechnology to develop and offer novel products began to experience increasing pressure from civil society to disclose information about the risks associated with the use of biotechnology and GMOs, in particular. Although no harmful effects for humans or the environment have been factually demonstrated even to date (2008), this technology remains highly-contested and its introduction in Europe catalysed major companies to invest significant financial and human resources in stakeholder dialogue. A relatively new phenomenon at the time, with little theoretical backing, dialogue was seen to reflect a move towards greater engagement with stakeholders, commonly defined as those "individuals or groups with which. business interacts who have a 'stake', or vested interest in the firm" (Carroll, 1993:22) with whom firms are seen to be inextricably embedded (Andriof & Waddock, 2002). Regarding the organisation of this dissertation, Chapter 1 (Introduction) describes the context of the study, elaborates its significance for academics and business practitioners as an empirical work embedded in a sector at the heart of the debate on corporate social responsibility (CSR). Chapter 2 (Literature Review) traces the roots and evolution of CSR, drawing on Stakeholder Theory, Institutional Theory, Resource Dependence Theory, and Organisational Learning to establish what has already been developed in the literature regarding the stakeholder concept, motivations for engagement with stakeholders, the corporate response to external constituencies, and outcomes for the firm in terms of organisational learning and change. I used this review of the literature to guide my inquiry and to develop the key constructs through which I viewed the empirical data that was gathered. In this respect, concepts related to how the firm views itself (as a victim, follower, leader), how stakeholders are viewed (as a source of pressure and/or threat; as an asset: current and future), corporate responses (in the form of buffering, bridging, boundary redefinition), and types of organisational teaming (single-loop, double-loop, triple-loop) and change (first order, second order, third order) were particularly important in building the key constructs of the conceptual model that emerged from the analysis of the data. Chapter 3 (Methodology) describes the methodology that was used to conduct the study, affirms the appropriateness of the case study method in addressing the research question, and describes the procedures for collecting and analysing the data. Data collection took place in two phases -extending from August 1999 to October 2000, and from May to December 2001, which functioned as `snapshots' in time of the three companies under study. The data was systematically analysed and coded using ATLAS/ti, a qualitative data analysis tool, which enabled me to sort, organise, and reduce the data into a manageable form. Chapter 4 (Data Analysis) contains the three cases that were developed (anonymised as Pioneer, Helvetica, and Viking). Each case is presented in its entirety (constituting a `within case' analysis), followed by a 'cross-case' analysis, backed up by extensive verbatim evidence. Chapter 5 presents the research findings, outlines the study's limitations, describes managerial implications, and offers suggestions for where more research could elaborate the conceptual model developed through this study, as well as suggestions for additional research in areas where managerial implications were outlined. References and Appendices are included at the end. This dissertation results in the construction and description of a conceptual model, grounded in the empirical data and tied to existing literature, which portrays a set of elements and relationships deemed important for understanding the impact of stakeholder engagement for firms in terms of organisational learning and change. This model suggests that corporate perceptions about the nature of stakeholder influence the perceived value of stakeholder contributions. When stakeholders are primarily viewed as a source of pressure or threat, firms tend to adopt a reactive/defensive posture in an effort to manage stakeholders and protect the firm from sources of outside pressure -behaviour consistent with Resource Dependence Theory, which suggests that firms try to get control over extemal threats by focussing on the relevant stakeholders on whom they depend for critical resources, and try to reverse the control potentially exerted by extemal constituencies by trying to influence and manipulate these valuable stakeholders. In situations where stakeholders are viewed as a current strategic asset, firms tend to adopt a proactive/offensive posture in an effort to tap stakeholder contributions and connect the organisation to its environment - behaviour consistent with Institutional Theory, which suggests that firms try to ensure the continuing license to operate by internalising external expectations. In instances where stakeholders are viewed as a source of future value, firms tend to adopt an interactive/innovative posture in an effort to reduce or widen the embedded system and bring stakeholders into systems of innovation and feedback -behaviour consistent with the literature on Organisational Learning, which suggests that firms can learn how to optimize their performance as they develop systems and structures that are more adaptable and responsive to change The conceptual model moreover suggests that the perceived value of stakeholder contribution drives corporate aims for engagement, which can be usefully categorised as dialogue intentions spanning a continuum running from low-level to high-level to very-high level. This study suggests that activities aimed at disarming critical stakeholders (`manipulation') providing guidance and correcting misinformation (`education'), being transparent about corporate activities and policies (`information'), alleviating stakeholder concerns (`placation'), and accessing stakeholder opinion ('consultation') represent low-level dialogue intentions and are experienced by stakeholders as asymmetrical, persuasive, compliance-gaining activities that are not in line with `true' dialogue. This study also finds evidence that activities aimed at redistributing power ('partnership'), involving stakeholders in internal corporate processes (`participation'), and demonstrating corporate responsibility (`stewardship') reflect high-level dialogue intentions. This study additionally finds evidence that building and sustaining high-quality, trusted relationships which can meaningfully influence organisational policies incline a firm towards the type of interactive, proactive processes that underpin the development of sustainable corporate strategies. Dialogue intentions are related to type of corporate response: low-level intentions can lead to buffering strategies; high-level intentions can underpin bridging strategies; very high-level intentions can incline a firm towards boundary redefinition. The nature of corporate response (which encapsulates a firm's posture towards stakeholders, demonstrated by the level of dialogue intention and the firm's strategy for dealing with stakeholders) favours the type of learning and change experienced by the organisation. This study indicates that buffering strategies, where the firm attempts to protect itself against external influences and cant' out its existing strategy, typically lead to single-loop learning, whereby the firm teams how to perform better within its existing paradigm and at most, improves the performance of the established system - an outcome associated with first-order change. Bridging responses, where the firm adapts organisational activities to meet external expectations, typically leads a firm to acquire new behavioural capacities characteristic of double-loop learning, whereby insights and understanding are uncovered that are fundamentally different from existing knowledge and where stakeholders are brought into problem-solving conversations that enable them to influence corporate decision-making to address shortcomings in the system - an outcome associated with second-order change. Boundary redefinition suggests that the firm engages in triple-loop learning, where the firm changes relations with stakeholders in profound ways, considers problems from a whole-system perspective, examining the deep structures that sustain the system, producing innovation to address chronic problems and develop new opportunities - an outcome associated with third-order change. This study supports earlier theoretical and empirical studies {e.g. Weick's (1979, 1985) work on self-enactment; Maitlis & Lawrence's (2007) and Maitlis' (2005) work and Weick et al's (2005) work on sensegiving and sensemaking in organisations; Brickson's (2005, 2007) and Scott & Lane's (2000) work on organisational identity orientation}, which indicate that corporate self-perception is a key underlying factor driving the dynamics of organisational teaming and change. Such theorizing has important implications for managerial practice; namely, that a company which perceives itself as a 'victim' may be highly inclined to view stakeholders as a source of negative influence, and would therefore be potentially unable to benefit from the positive influence of engagement. Such a selfperception can blind the firm from seeing stakeholders in a more positive, contributing light, which suggests that such firms may not be inclined to embrace external sources of innovation and teaming, as they are focussed on protecting the firm against disturbing environmental influences (through buffering), and remain more likely to perform better within an existing paradigm (single-loop teaming). By contrast, a company that perceives itself as a 'leader' may be highly inclined to view stakeholders as a source of positive influence. On the downside, such a firm might have difficulty distinguishing when stakeholder contributions are less pertinent as it is deliberately more open to elements in operating environment (including stakeholders) as potential sources of learning and change, as the firm is oriented towards creating space for fundamental change (through boundary redefinition), opening issues to entirely new ways of thinking and addressing issues from whole-system perspective. A significant implication of this study is that potentially only those companies who see themselves as a leader are ultimately able to tap the innovation potential of stakeholder dialogue.

Rhythmic growth explained by coincidence between internal and external cues.

Most organisms use circadian oscillators to coordinate physiological and developmental processes such as growth with predictable daily environmental changes like sunrise and sunset. The importance of such coordination is highlighted by studies showing that circadian dysfunction causes reduced fitness in bacteria and plants, as well as sleep and psychological disorders in humans. Plant cell growth requires energy and water-factors that oscillate owing to diurnal environmental changes. Indeed, two important factors controlling stem growth are the internal circadian oscillator and external light levels. However, most circadian studies have been performed in constant conditions, precluding mechanistic study of interactions between the clock and diurnal variation in the environment. Studies of stem elongation in diurnal conditions have revealed complex growth patterns, but no mechanism has been described. Here we show that the growth phase of Arabidopsis seedlings in diurnal light conditions is shifted 8-12 h relative to plants in continuous light, and we describe a mechanism underlying this environmental response. We find that the clock regulates transcript levels of two basic helix-loop-helix genes, phytochrome-interacting factor 4 (PIF4) and PIF5, whereas light regulates their protein abundance. These genes function as positive growth regulators; the coincidence of high transcript levels (by the clock) and protein accumulation (in the dark) allows them to promote plant growth at the end of the night. Thus, these two genes integrate clock and light signalling, and their coordinated regulation explains the observed diurnal growth rhythms. This interaction may serve as a paradigm for understanding how endogenous and environmental signals cooperate to control other processes.

Influence of the implanted pulse generator as reference electrode in finite element model of monopolar deep brain stimulation.

Electrical deep brain stimulation (DBS) is an efficient method to treat movement disorders. Many models of DBS, based mostly on finite elements, have recently been proposed to better understand the interaction between the electrical stimulation and the brain tissues. In monopolar DBS, clinically widely used, the implanted pulse generator (IPG) is used as reference electrode (RE). In this paper, the influence of the RE model of monopolar DBS is investigated. For that purpose, a finite element model of the full electric loop including the head, the neck and the superior chest is used. Head, neck and superior chest are made of simple structures such as parallelepipeds and cylinders. The tissues surrounding the electrode are accurately modelled from data provided by the diffusion tensor magnetic resonance imaging (DT-MRI). Three different configurations of RE are compared with a commonly used model of reduced size. The electrical impedance seen by the DBS system and the potential distribution are computed for each model. Moreover, axons are modelled to compute the area of tissue activated by stimulation. Results show that these indicators are influenced by the surface and position of the RE. The use of a RE model corresponding to the implanted device rather than the usually simplified model leads to an increase of the system impedance (+48%) and a reduction of the area of activated tissue (-15%).

The role of MIC-1/GDF15 cytokine in glioblastoma

AbstractDespite advances in diagnosis and treatment made over the past two decades, high-gradeprimary brain tumors remain incurable neoplasms. Glioblastoma (GBM) represents the mostmalignant stage of astrocytic brain tumors. Identification of diagnostic and prognostic markers ineasily accessible biological material, such as plasma or cerebro-spinal fluid (CSF), would greatlyfacilitate the management of GBM patients. Elucidation of the molecular mechanisms that underlie thefunction of the factors implicated in GBM development would pave the way towards their potentialutility in cancer-targeting therapy.MIC-1/GDF15 (Macrophage Inhibitory Cytokine-1/ Growth Differentiation Factor 15), asecreted protein of the TGF-β superfamily, emerged as a candidate marker exhibiting increasingmRNA expression during astrocytoma malignant progression. However, injection of MIC-1/GDF15over-expressing GBM cell lines into nude mice has been previously shown to completely abolish theinherent tumorigenicity.In this study, determination of MIC-1/GDF15 protein levels in the CSF of a cohort of 94patients with intracranial tumors including astrocytomas (grades II, III and IV), meningioma, andmetastasis revealed significantly increased concentrations in GBM patients as compared to controlcohort of patients treated for non-neoplastic diseases. However, MIC-1/GDF15 levels were notelevated in the matching plasma samples from these patients. Most interestingly, GBM patients withthe increased concentrations of MIC-1/GDF15 in the CSF had worse outcome.In GBM tissue, it was found that the expression of MIC-1/GDF15 gene is low. Promotermethylation of the gene may partially explain the overall low expression levels. Investigation of thecellular origin of MIC-1/GDF15 expression in GBM tissue led to the MIC-1/GDF15 protein detectionin a subpopulation of the tumor infiltrating macrophages. These findings substantiated the workinghypothesis of MIC-1/GDF15 as harboring tumor-suppressive properties in GBM. Analysis of thesignaling pathway mediated by MIC-1/GDF15 in GBM highlighted the potential role of TGF-β signaltransduction. However, the lack of the functional response to the presence of MIC-1/GDF15 in-vitrosuggested operation of a paracrine loop for suppression of tumor formation which is evident solely invivo.In conclusion, MIC-1/GDF15 protein measured in the CSF may have diagnostic andprognostic values in patients with intracranial tumors. Molecular studies collectively proposeimplication of the tumor-host interactions in mediating the MIC-1/GDF15 tumor-suppressing activityduring GBM development.RésuméMalgré les progrès durant ces deux dernières décennies dans le diagnostique et le traitementdes tumeurs du cerveau primaires, ces néoplasmes restent incurables. Le glioblastome représente laforme la plus maligne des tumeurs astrocytiques du cerveau (astrocytomes). Pour le diagnostic et lepronostic, l'identification de marqueurs présents dans des substances facilement accessibles comme leplasma où le liquide céphalorachidien (LCR) faciliterait beaucoup la prise en charge des patients. Lacompréhension des mécanismes moléculaires de facteurs impliqués dans le développement du GBMpourrait ouvrir la voie vers l'utilisation de ces mécanismes dans des thérapies ciblées.MIC-1/GDF15 (Macrophage Inhibitory Cytokine-1/ Growth Differentiation Factor 15), uneprotéine secrétée qui appartient à la superfamille TGF-β, s'est révélé être un marqueur candidat, dontl'expression d'ARN messager augmente pendant la progression des astrocytomes malins. Cependant,une précedente étude montre que l'injection des lignées cellulaires de GBM fortement productrices deMIC-1/GDF15 dans des souris immunodéprimées abolit la tumorigénicité.Dans cette étude, les mesures dans une cohorte de 94 patients atteints de tumeursintracrâniennes comprenant des astrocytomes (grades II, III et IV), méningiomes et métastases,présentent des augmentations significatives des niveaux protéiques de MIC-1/GDF15 dans le LCRdes patients atteints de GBM par rapport aux patients traités pour des maladies non cancéreuses.Cependant, les niveaux de MIC-1/GDF15 n'étaient pas spécialement élevés dans le plasma. De plus,les patients atteints d'un GBM avec des niveaux élevés de MIC-1/GDF15 dans le LCR ont survécumoins longtemps. Dans les tissus de glioblastome, on observe que le gène MIC-1/GDF15 est peuexprimé. La méthylation du promoteur explique partiellement le faible niveau d'expression du gène.La recherche l'origine cellulaire de l'expression de MIC-1/GDF15, a permis de découvrir la présencede protéines MIC-1/GDF15 dans une sous-population de macrophages qui infiltrent les tumeurs. Cetteobservation supporte l'hypothèse que MIC-1/GDF15 présentait des propriétés de suppression destumeurs de type GBM. Des études sur les voies de signalisation régulées par MIC-1/GDF15 dans lesGBMs ont souligné l'importance de la voie de transduction du signal TGF-β. Cependant, l'absence deréponse fonctionnelle à MIC-1/GDF15 in vitro suggère fortement l'activité d'une boucle paracrinepour la répression de la formation de tumeur, qui n'est observé que in vivo.En conclusion, la protéine MIC-1/GDF15 mesurée dans le LCR pourrait avoir une valeur pourle diagnostic et le pronostic chez les patients atteints de GBM. Les études moléculaires suggèrent unepossible implication de l'interaction hôte-tumeur dans l'activité anti-tumorale de MIC-1/GDF15 sur leGBM.

Molecular characterization of a human matrix attachment region epigenetic regulator.

Matrix attachment regions (MAR) generally act as epigenetic regulatory sequences that increase gene expression, and they were proposed to partition chromosomes into loop-forming domains. However, their molecular mode of action remains poorly understood. Here, we assessed the possible contribution of the AT-rich core and adjacent transcription factor binding motifs to the transcription augmenting and anti-silencing effects of human MAR 1-68. Either flanking sequences together with the AT-rich core were required to obtain the full MAR effects. Shortened MAR derivatives retaining full MAR activity were constructed from combinations of the AT-rich sequence and multimerized transcription factor binding motifs, implying that both transcription factors and the AT-rich microsatellite sequence are required to mediate the MAR effect. Genomic analysis indicated that MAR AT-rich cores may be depleted of histones and enriched in RNA polymerase II, providing a molecular interpretation of their chromatin domain insulator and transcriptional augmentation activities.

Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers.

In shade-intolerant plants such as Arabidopsis, a reduction in the red/far-red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non-DNA-binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G-boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non-DNA-binding heterodimers. This negative feedback loop is important to limit the response of plants to shade.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

Personalized drug administrations using Support Vector Machine

The decision-making process regarding drug dose, regularly used in everyday medical practice, is critical to patients' health and recovery. It is a challenging process, especially for a drug with narrow therapeutic ranges, in which a medical doctor decides the quantity (dose amount) and frequency (dose interval) on the basis of a set of available patient features and doctor's clinical experience (a priori adaptation). Computer support in drug dose administration makes the prescription procedure faster, more accurate, objective, and less expensive, with a tendency to reduce the number of invasive procedures. This paper presents an advanced integrated Drug Administration Decision Support System (DADSS) to help clinicians/patients with the dose computing. Based on a support vector machine (SVM) algorithm, enhanced with the random sample consensus technique, this system is able to predict the drug concentration values and computes the ideal dose amount and dose interval for a new patient. With an extension to combine the SVM method and the explicit analytical model, the advanced integrated DADSS system is able to compute drug concentration-to-time curves for a patient under different conditions. A feedback loop is enabled to update the curve with a new measured concentration value to make it more personalized (a posteriori adaptation).

Hes1 is a critical but context-dependent mediator of canonical Notch signaling in lymphocyte development and transformation.

Although canonical Notch signaling regulates multiple hematopoietic lineage decisions including T cell and marginal zone B cell fate specification, the downstream molecular mediators of Notch function are largely unknown. We showed here that conditional inactivation of Hes1, a well-characterized Notch target gene, in adult murine bone marrow (BM) cells severely impaired T cell development without affecting other Notch-dependent hematopoietic lineages such as marginal zone B cells. Competitive mixed BM chimeras, intrathymic transfer experiments, and in vitro culture of BM progenitors on Delta-like-expressing stromal cells further demonstrated that Hes1 is required for T cell lineage commitment, but dispensable for Notch-dependent thymocyte maturation through and beyond the beta selection checkpoint. Furthermore, our data strongly suggest that Hes1 is essential for the development and maintenance of Notch-induced T cell acute lymphoblastic leukemia. Collectively, our studies identify Hes1 as a critical but context-dependent mediator of canonical Notch signaling in the hematopoietic system.

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

Rapamycin-mediated FOXO1 inactivation reduces the anticancer efficacy of rapamycin.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors such as rapamycin have shown modest effects in cancer therapy due in part to the removal of a negative feedback loop leading to the activation of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway. In this report, we have investigated the role of FOXO1, a downstream substrate of the PI3K/Akt pathway in the anticancer efficacy of rapamycin. MATERIALS AND METHODS: Colon cancer cells were treated with rapamycin and FOXO1 phosphorylation was determined by Western blot. Colon cancer cells transfected with a constitutively active mutant of FOXO1 or a control plasmid were treated with rapamycin and the antiproliferative efficacy of rapamycin was monitored. RESULTS: Rapamycin induced the phosphorylation of FOXO1 as well as its translocation from the nucleus to the cytoplasm, leading to FOXO1 inactivation. The expression of an active mutant of FOXO1 in colon cancer cells potentiated the antiproliferative efficacy of rapamycin in vitro and its antitumor efficacy in vivo. CONCLUSION: Taken together these results show that rapamycin-induced FOXO1 inactivation reduces the antitumor efficacy of rapamycin.

Capsid modified replicating adenovirus to selectively target colon cancer

Résumé: Pratiquement tous les cancers du colon contiennent des mutations dans la voie de signalisation de Wnt qui active constitutivement cette voie. Cette activation mène à la stabilisation de la β-catenine. La β-catenin est transportée dans le noyau ou elle active des gènes cible en interagissant avec le facteur de transcription de TCF/LEF. Des adénovirus qui peuvent sélectivement se répliquer dans les cellules tumorales sont les agents qui peuvent permettre la déstruction de la tumeur mais pas le tissu normal. In vitro, les adénovirus avec des sites d'attachement du facteur de transcription TCF dans les promoteurs de l'adénovirus montrent une sélectivité et une activité dans une large sélection de lignées cellulaires de cancer du colon. Au contraire, in vivo, quand les adénovirus modifiés sont injectés dans la circulation, ils sont moins efficaces à cause de leur fixation par le foie et à cause de l'absence d'expression du récepteur du Coxsackie-Adénovirus (CAR). Le but de ma thèse était de modifier la protéine principale de capside de l'adénovirus, fibre, pour augmenter l'infection des tumeurs du cancer du colon. La fibre de l'adénovirus est responsable de l'attachement aux cellules et de l'entrée virale. J'ai inséré un peptide RGD dans la boucle HI de la fibre qui dirige sélectivement le virus aux récepteurs des integrines. Les integrines sont surexprimées par les cellules du cancer du colon et l'endothélium des vesseaux de la tumeur. Le virus re-ciblé, vKH6, a montré une activité accrue dans toutes les lignées cellulaires de cancer du colon, tandis que la sélectivité était maintenue. In vivo, vKH6 était supérieur au virus avec une capside de type sauvage en retardant la croissance de la tumeur. Le virus s'est répliqué plus vite et dispersé graduellement dans la tumeur. Cet effet a été montré par hybridation in situ et par PCR quantitative. Cependant, la monothérapie avec le virus n'a pu retarder la croissance des cellules tumorales SW620 greffées que de 2 semaines, mais à cause des régions non infectées la tumeur n'a pas pu être éliminée. Bien que la combinaison avec les chimiothérapies conventionnelles soit d'intérêt potentiel, presque toutes interfèrent avec la réplication virale. Les drogues antiangiogéniques sont des agents anti-tumoraux efficaces et prometteurs. Ces drogues n'interfèrent pas avec le cycle de vie de l'adénovirus. RAD001 est un dérivé de la rapamycine et il inhibe mTOR, une protéine kinase de la voie de PI3K. RAD001 empêche la croissance des cellules et il a aussi des effets anti-angiogénique et immunosuppressifs. RAD001 in vitro n'affecte pas l'expression des gènes viraux et la production virale. La combinaison de VKH6 et RAD001 in vivo a un effet additif en retardant la croissance de la tumeur. Des nouveaux peptides plus efficaces dans le ciblage de l'adénovirus sont nécessaires pour augmenter l'infection des tumeurs. J'ai créé un système de recombinaison qui permettra la sélection de nouveaux peptides dans le contexte du génome de l'adénovirus. Summary Virtually all colon cancers have mutations in the Wnt signalling pathway which result in the constitutive activation of the pathway. This activation leads to stabilization of β-catenin. β-catenin enters the nucleus and activates its target genes through interaction with the TCF transcription factor. Selectively replicating adenoviruses are promising novel agents that can destroy the tumour but not the surrounding normal tissue. In vitro, adenoviruses with TCF binding sites in the early viral promoters show selectivity and activity in a broad panel of viruses but in vivo they are less effective due to the lack of expression of the Coxsackie-Adenovirus receptor (CAR). The aim of my thesis was to modify the major capsid protein of the adenovirus, fibre, to increase the infection of colon tumours. Fibre of adenovirus is responsible for the binding to cells and for the viral uptake. I inserted an RGD binding peptide into the HI loop of fibre that selectively targets the virus to integrins that are overexpressed on tumour cells and on tumour endothelium. The retargeted virus, vKH6, showed increased activity in all colon cancer cell lines while selectivity was maintained. In vivo, vKH6 is superior to a matched virus with a wild type capsid in delaying tumour growth. vKH6 replicates and gradually spreads within the tumour as shown by in situ hybridization and Q-PCR. The virus alone can delay the growth of SW620 xenografts by 2 weeks but due to uninfected tumour regions the tumour cannot be cured. Although combination with conventional chemotherapeutics is of potential interest, almost all of them interfere with the viral replication. Growing evidence supports that anti-angiogenic drugs are effective and promising anti-tumour agents. These drugs interfere less with the viral life cycle. RAD001 is a rapamycin derivative and it blocks mTOR, a protein kinase in the PI3K pathway. RAD001 inhibits cell growth and has strong anti-angiogenic and immunosuppressive effects. RAD001 in vitro does not affect viral gene expression and viral burst size. In vivo vKH6 and RAD001 have an additive effect in delaying tumour growth, but tumour growth is still not completely inhibited. To further increase tumour infection new tumour specific targeting peptides are needed. I created an adenovirus display library that will allow the selection of targeting peptides. This system may also facilitate the production of fibre modified viruses.

Génération d'une souris dépourvue du gène VIT32 de manière conditionnelle: étude du phénotype rénal chez les souris dépourvues de RGS2

Résumé Dans le rein, la vasopressine possède un rôle essentiel dans la régulation fine du transport d'eau et participe au contrôle de la réabsorption du sodium. Cette action est conduite par l'activation du récepteur à la vasopressine V2R situé dans l'anse de Henle, dans le tubule connecteur et dans le canal collecteur du néphron des rongeurs et conduit à la formation d'AMPc entraînant un mécanisme d'action caractérisé par deux phases distinctes. Le premier effet de la vasopressine est non génomique et a lieu rapidement après l'activation du récepteur, la deuxième phase est plus tardive et possède la caractéristique de moduler la transcription d'un réseau de gènes. Parmi ces gènes, plusieurs sont directement impliqués dans le transport d'eau et de sodium, comme l'Aqp2 et 3, ENaC et la Na,K-ATPase. L'identification des effets de la voie de signalisation de la vasopressine représente un point crucial pour la compréhension des mécanismes moléculaires de la réabsorption de l'eau et du sodium dans le néphron. L'analyse en série de l'expression de gènes (SAGE) réalisée en 2001 dans notre laboratoire a permis de caractériser le transcriptome dépendant de la vasopressine dans la lignée cellulaire mpkCCDc14,a dérivée du canal collecteur cortical (CCD) de souris. Deux des transcrits induits par la vasopressine (VIT) ont fait l'objet des études de ce travail de thèse. Le premier est VIT32 (Vasopressin induced transcript 32) qui code pour une protéine ne possédant aucune homologie avec des domaines protéiques dont la fonction est connue. Dans le système d'expression de l'ovocyte de Xenopus laevis, VIT32 induit la maturation des ovocytes et diminue le courant sensible à l'amiloride de manière dépendante de la voie des MAPK. Dans les mpkCCDc14, l'inhibition de la voie des MAPK diminue le courant sodique en diminuant l'activité de la Na,K-ATPase, mais sans modifier le courant d'ENaC. Ainsi la voie de signalisation des MAPK peut avoir des cibles différentes suivant le système dans lequel elle est étudiée. C'est pourquoi nous avons décidé de poursuivre l'étude de VIT32 dans un contexte physiologique en créant une souris dépourvue du gène codant pour VIT32 de manière conditionnelle (conditional knockout). La première partie de cette thèse a donc consisté à générer cette souris. Le deuxième transcrit induit par la vasopressine qui a été étudié dans cette thèse est RGS2 (Regulator of G protein Signaling 2). In vitro, il a été montré que RGS2 inhibe des voies de signalisation dépendantes de récepteurs couplés à des protéines Gq et Gs. Dans notre étude, nous avons montré que dans le néphron de rein de souris, RGS2 est colocalisé avec V2R. In vivo, la vasopressine sécrétée lors d'une restriction en eau imposée à des souris augmente l'expression de RGS2. De plus, l'accumulation d'AMPc engendrée par l'action de la vasopressine sur les canaux collecteurs est significativement plus grande chez les souris dépourvues de RGS2 (rgs2 -/-). Cette induction de la signalisation de la vasopressine est corrélée à une augmentation de la réabsorption d'eau chez les souris rgs2 -/-. Ainsi RGS2 serait impliqué dans le rétrocontrôle négatif de la voie de signalisation de la vasopressine. Abstract In the kidney, vasopressin plays a key role in the control of water balance and participates in salt reabsorption. These actions are induced by the activation of V2 vasopressin receptor (V2R) located in the loop of Henle, in the connecting tubule and in the collecting duct leading to an increase in intracellular cAMP levels. The V2R-mediated vasopressin action elicits a rapid, non-genomic effect, during which water and salt reabsorption is rapidly increased and a late or genomic effect characterised by the long-term regulation of water and salt reabsorption through the transcriptional activation of a gene network that includes Aqp2, Aqp3, ENaC and Na,K-ATPase. Serial analysis of gene expression (SAGE) performed in 2001 in our laboratory characterised the vasopressin induced transcripts (VIT) in the mpkCCDc14 cell line. Two of them are studied in this thesis. The first one is VIT32 (Vasopressin induced transcript 32) that encodes a protein that has no homology with any protein domain of known function. In the Xenopus laevis oocyte, VIT32 induces oocyte maturation and downregulates the ENaC amiloride sensitive current via the activation of the MAPK pathway. In mpkCCDc14 cell line, the MAPK pathway inhibition leads to a decrease of Na,K-ATPase activity without affecting ENaC current. Therefore, the MAPK pathway can act on different targets depending on the cellular context. Thus, we decided to investigate the function of VIT32 in its physiological environment by performing a conditional knockout mouse of VIT32. The first part of this thesis consisted in generating this mouse. The second studied vasopressin induced transcript is RGS2 (Regulator of G protein Signaling 2). In vitro, RGS2 has been shown to inhibit Gq and Gs protein-coupled receptor pathway. In our study we show that RGS2 is co-localized with V2R in the mouse nephron. In vivo, vasopressin secreted during water restriction up-regulates RGS2 expression. Moreover, vasopressin-dependant accumulation of CAMP is significantly increased in the cortical collecting duct of RGS2 knockout mice. This increase is correlated with an increase in water reabsorption. RGS2 could be involved in the negative feedback regulation of V2R signalling. Résumé tout public Le corps humain est composé d'environ 60% d'eau répartie à l'intérieur et à l'extérieur des cellules de notre organisme. Les cellules, unités fondamentales du vivant, puisent l'oxygène et les nutriments indispensables à leur fonctionnement dans le liquide extracellulaire. La composition du milieu doit être constante, car les variations peuvent perturber considérablement et parfois fatalement la fonction des cellules. Ainsi les organismes pluricellulaires ont développé des mécanismes permettant de contrôler la constance du milieu extracellulaire afin de maintenir l'état d'équilibre nommé homéostasie. Le rein joue un rôle majeur dans cette homéostasie grâce à sa capacité de réabsorber l'eau et les solutés en fonction des besoins de l'organisme. Cette fonction du rein est régulée par différentes hormones comme la vasopressine, qui permet de contrôler la réabsorption fine de l'eau et des solutés. Dans leurs membranes, les cellules possèdent des récepteurs leur permettant de répondre aux signaux extracellulaires comme le sont entre autres les hormones. Ainsi les cellules sensibles à la vasopressine possèdent un récepteur nommé V2R qui permet d'intégrer les signaux de la vasopressine en déclenchant tout une cascade d'événements conduisant à une modification de l'expression de certaines protéines impliquées directement ou non dans la réabsorption de l'eau et des solutés. Une étude précédente élaborée au sein de notre laboratoire a permis de répertorier les protéines dont l'expression est augmentée par de la vasopressine. Deux de ces protéines ont fait l'objet des études de cette thèse. La première protéine induite par la vasopressine est VIT32 (Vasopressin induced transcript 32). Cette protéine est entre autres impliquée dans la réabsorption du sodium, mais la fonction précise de VIT32 dans ce transport n'a pas pu être déterminée. Une des approches possibles pour l'étude de la fonction d'une protéine est de supprimer son expression chez la souris et d'étudier les conséquences de son absence. Ces souris sont appelées des souris knockout, puisque la protéine en question ne peut plus agir. La première partie de cette thèse a donc consisté à générer une souris dépourvue du gène de VIT32. La deuxième protéine étudiée est RGS2 (Regulator of G protein Signaling 2). Cette protéine inhibe certaines voies de signalisation activées par différentes hormones. Dans cette partie du travail de thèse, nous avons pu mettre en évidence que RGS2 agit comme un inhibiteur de la voie de signalisation de la vasopressine. En modifiant cette signalisation, RGS2 serait donc un médiateur du contrôle de la réabsorption d'eau dans les cellules du rein sensibles à la vasopressine.