Apports de la microscopie biphotonique intravitale pulmonaire à l'étude de la physiopathologie de la maladie du charbon

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires.Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.

International audience: Dynamics of the lung immune system at a microscopic level are largely unknown because of inefficient methods to rid chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process

La nouvelle version de MIMOSA, modèle de l'économie mondiale

MIMOSA, modèle macroéconomique de l’économie mondiale, construit en commun par le CEPII et l’OFCE, est maintenant réestimé. Le présent article en expose les principales caractéristiques ainsi que quelques propriétés variantielles. Les économies des six plus importants pays industrialisés sont décrites en détail par des modèles d’inspiration néo-keynésienne ; le découpage en cinq branches (trois pour les Etats-Unis et le Japon) permet d’isoler l’énergie, le secteur agricole, le secteur abrité, le secteur non-marchand et l’industrie où la fonction de production, de type « putty-clay », vise à assurer la cohérence des comportements d’emploi, d’investissement, d’accumulation de capacités de production. Ces modèles autorisent une analyse fine des conséquences des diverses mesures de politiques budgétaires ou monétaires. Douze zones, décrites plus sommairement, regroupent le reste du monde : le modèle intègre en particulier les contraintes de financement qui pèsent sur les possibilités d’importations des pays en développement. Les interdépendances commerciales sont décrites en quatre produits. L’article montre comment le modèle rend compte des effets différenciés sur l’économie considérée et sur l’économie mondiale d’une hausse des dépenses publiques survenant dans l’un des grands pays. Il étudie ensuite les conséquences d’une hausse mondiale des dépenses publiques, d’une hausse généralisée des taux d’intérêt, puis d’une dépréciation du dollar.Cet article a été écrit par l'équipe MIMOSA, commune au CEPII et à l'OFCE

Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models

PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model

Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org

Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms

The corporate centre in a financial conglomerate : governance under fundamental industry changes

In part 1, we discuss 1) the fundamental changes in the financial services industry, 2) financial conglomerate structures and 3) value-based-management. These are core components for understanding the challenges and intentions of corporate level management of financial conglomerates. The financial services industry, financial conglomeration and value orientation In the first chapter, we highlight the major trend of consolidation in the financial services industry. This trend is most visible in business publications, and interacts with other trends. Financial conglomerates grow due to the fact that size is believed to be the answer to many difficulties. Consolidation has an effect on the systemic risk of the financial services industry because consolidation changes the risks of individual institutions, which in turn can affect other institutions. If the risk of an individual financial institution increases, the probability that the institution will fail or become illiquid before settling some of its payment obligations also increases. This increasing risk of an individual institution exposes other institutions directly as payees, or indirectly through contributing to panic runs or securities markets problems. As consolidation continues and non-traditional financial service activities enter into financial service activities by financial services firms, the safety net expand s, thereby imposing additional costs on the financial system. Regulators keep a close watch on the trends in the financial services industry: their objective is to maintain microeconomic stability, protect investors and support financial services firms’ proper behaviour, efficiency and competition. In intervening in consolidation decisions, regulators try to limit the government’s liability and to prevent exploitation of the safety net. To achieve this, regulators are looking for new ways of supervision. New capital requirements for credit, market and operational risk (Basel II), mandatory subordinated debt, a better supervisory structure and guidelines for corporate governance are all under development. The expectation is that, after a period of focus on model-based risk management, regulators will now be increasingly focusing their attention at management processes. Consolidation is a worldwide phe nomenon, primarily taking place in, but not limited to, well-developed economies. Globalisation of markets has contributed to (crossborder) mergers and acquisitions. In the United States, between 1988 and 1997, there has been a decrease in the number of financial services firms by 26.8 percent. In Europe we recognise the same trend: in Germany only there already has been a decline in the number of financial services firms from 5,000 to 36,000 between 1990 and 1995. In Japan and Singapore we see the same trend of consolidation. Although consolidation is primarily a domestic process, we also see cross-border mergers and acquisitions. Various trends contribute to the consolidation drive. For one, technological progress is very important as technology erodes entry barriers; financial services firms face pressures from a wider and more diverse range of competitors. Although information technology is pivotal for the internal processes of the financial services firms, clientfocused technologies make the difference on the market. New technologies support the development of new financial products and these products create new challenges for financial services firms. These products increasingly are capital-market based instead of straightforward savings- or credit facilities. Well-developed economies tend to shift from a bank-based system towards a market-based system in which the majority of the financing need is provided for by the capital market, which requires a efficient distribution in terms of scale and reach. While regulators attempt to supervise the financial system, deregulation leads to increased competition with margins being driven down and requiring scale economies. This dynamic is complemented by the drive to eradicate excess capacity in the global financial services industry. At the same time, and perhaps resulting from these industry changes, the lines of the financial services industry are blurring in various parts of the world. Non- financial services firms are making inroads in the financial services industry. We call this nearbanking and it can be understood to be the activity where non- financial services firms diversify into financial services. It implies that sections of the financial services industry are indeed attractive and that non- financial services firms are of the opinion that the present level of competition does not rule out profitability. This movement also reveals that specific banking and finance skills, once exclusive to financial services firms, have disseminated to other industries. A specific form of near-banking is in-house banking: non- financial firms perform several financial activities for themselves, such as provision of capital, investor relations, short-term financing, banking and custody, credits and collections, portfolio management, and tax optimisation. A special case of near-banking is internet banking. Innovations in communication and information technology has led to the increased possibility of total process automation of searching, buying, selling, producing and distributing and introduces the notion of contract banking, in which a complete package of services for a client consists of the management of contracts for those products individually. Electronic banking operating costs are estimated to be only 25 to 30 percent of the costs of providing financial services through traditional branches. This reduction in cost implies that financial services firms would have to adapt their business model to remain competitive and profitable. At its core, a financial service is about controlling the flow of money and financial information. Due to capital investments, regulations and loose (client) relationships, the internet is not as powerful as expected at first; on the other hand, it will increasingly become a central component of the financial services business. Financial services firms are reacting by setting up separate internet divisions, partnering with technology-oriented firms, and forming alliances with firms in- and outside of the financial services industry. In the second chapter, we discuss financial conglomerate structures. The financial services industry is made up of many types of participants of which financial conglomerates are often the most visible. Financial conglomerates deal with the fundamental changes in the financial services industry in part by choosing an appropriate structure. There are four different prototypes of financial conglomerates recognised, which range from almost complete to very loose integration: the fully integrated, German, British and American variant. These variants find their origin in historical reasons and different legal systems. Financial conglomerates are formed when management feels that this structure is best suited to achieve maximum synergies: economies of scope and scale in production and sales of financial services. Management always intends to increase market capitalization, reduce risk or create access to new products or markets. Synergies, from the firm’s perspective, can be reached in improved client management, when distribution channels are used for more products, simultaneous marketing of products, use of information for different products and the reduction of portfolio risk. From the client’s perspective, synergies may exist in the procurement and use of a complete financial services package instead of individual products: costs for searching, information, monitoring and transaction may be lower. As switching costs can be higher with a complete services package, clients may be less likely to switch financial services provider. There are also diseconomies of scope, which may jeopardize the advantages of the financial conglomerate structure. We recognize regulation and compliance costs of a large bureaucracy, complexity of managing several businesses, lack of client trust in the use of information on them and decrease of transparency as a result of reduction in market discipline, as potential issues. The advantages of financial conglomeration have not (yet) been proven in practice and good management seems to be paramount to achieving these advantages. As we see in the third chapter, in dealing with environmental turbulence and the optimisation of the structure, financial conglomerates focus on the creation of shareholder value in all their activities. Shareholder value creation can be used as a yardstick to measure how well the financial conglomerate is doing; there is a zone between bankruptcy and absolute value creation leadership, which implies there is space to give other interests a higher priority when necessary. Functional excellence is not the only pre-condition for success, increasingly a client focus, reducing xinefficiency, and company repositioning in the (financial services) industry, e.g. through alliances, becomes important. There are various methodologies to measure shareholder value creation, which in essence is about achieving a return on capital higher than its cost. Shareholder value creation requires a management approach to support this. Corporate aspirations, analytical techniques and management processes should be aligned to assist the financial conglomerate maximise its value by focusing decision making on the key drivers of value: this is called value-based management. Dynamic value drivers should be developed down to the level of detail that aligns the value driver with the decision variables directly under the control of line management. Six characteristics determine to what extent a financial conglomerate is valueoriented: performance drive, value-base, low cost, self-reinforcing processes, two-way communications, and bottom-up and top-down management (dual control approach). Implementing value-based management in financial conglomerates follows the lines of the triad of value-based bank management: value-oriented business philosophy, value-oriented growth policy and value-oriented risk policy. The triad focuses on profitability, goal-oriented management, supported by planning, decision-making, implementation and control, an appropriate organisational and operational structure for the institutionalised controlling cycle, and a management information system reporting transparently on transaction level. The company organisation for valuebased management is the profit centre. Profit centres are organisational units, which are responsible for their own results and are able to decide independently from each other. Profit centres increase revenue and cost transparency, enable faster and a more flexible approaches to (market) changes, improve staff motivation, enhance earnings and value orientation, and allow for objective performance evaluation of management. Profit centres, therefore, improve the management of the financial conglomerate as a whole. The profit centre concept can be implemented when various requirements have been met. These requirements include: decentralisation of management structures, operational independence, correct allocation of costs and revenues and creation of technical accounting and settlement units in accordance with the areas of responsibility. Adjacent to the profit centre concept is lean banking, which focuses on the optimisation of operational processes through well-known concepts from industrial management, including continuous improvement, quality management and customer-focused production. Measuring the results of value-based management is of utmost importance on both corporate and divisional levels, as well as within divisions. Value controlling measures the gap between the market value of the firm and its potential, and supports divisions with the implementation of value-enhancing strategies. This implies that the value controlling system has to be integrated in the corporate philosophy, be accepted by users, adapt flexibly to changes, and have a good cost-benefit relationship. Instruments for value-controlling include the Market Rate Method, which attempts to isolate the revenue contribution made by each individual transaction, Activity Based Costing, which allocates costs more precise to activities than other methods, and Process-oriented Standard Direct Cost Accounting, which uses planned or standard costs for references to the future. The non-financial dimension is assessed with the Balanced Scorecard, which, in addition to the financial view, takes on the following views: customer perspective, internal business process perspective, and organisational learning and growth perspective. The Balanced Scorecard can be seen as a system of (partly) operational measures (i.e. ratios), which are connected to each other via cause-and-effect relationships. One important determinant of shareholder value creation is lowering risk as this results in a lower firm cost of capital. Financial conglomerates, though, accept risk to generate profits. Profitability (volatility) concerns and the financial conglomerate’s ability to carry risk must be taken into account. For financial conglomerates there are five risk categories: systemic risk, company-specific strategic risks, market risk, credit risk, and operational risk. A risk matrix, formed by risk categories affecting the individual business areas, is the core of a risk control system. Allocating internal (risk) capital to the organisational units and activities is common to both value-based management and asset and liability management. The concept of value at risk (VaR) is currently widely used in the financial services industry to ascertain and value the market risks inherent in financial positions. In general, the VaR is treated as a measure of the maximum possible change in the value of a portfolio of financial instruments within a given likelihood and for a specified period and is intended to quantify the potential losses inherent in that portfolio. Credit risk, at a minimum, is the risk of loss due to borrower defaults and is attributed to all units with borrower or counterparty exposure. Credit risk management has evolved from credit scoring of individual borrows to sophisticated aggregate models of borrowers’ default probabilities and the extent of asset recovery. Operational risk is the potential for any disruption in the financial conglomerate’s (operational) processes and includes reputational risk, legal enforcement of contracts and claims, possibly having a severe impact on the financial conglomerate’s perception in the market, share price devaluation and a loss of standing. Underlying causes of operational risk include complacency or a false sense of security, cost, as controlling operational risk can be seen as a new activity, difficulties in measuring operational risk, miscommunication when using jargon, over reliance on outside vendors, incompatible systems, decentralisation complicating oversight of operational risks, and organisation-specific factors. Four types of qualitative guidelines are particularly relevant: industry guidelines for good operations practices, guidelines for internal control, process and resource quality guidelines and regulatory capital requirements. Operational risk seems to be more in the management realm than in accounting. An overemphasis on the quantitative measurement of operational risks may be dangerous to firms without good management and staff or well-designed processes. Ultimately, the main defence against operational risks must be governance: management and staff who are knowledgeable about the risks and the good processes and systems that embody that knowledge. Discussion of the corporate centre Given that we now understand the major governance issues for financial conglomerates: fundamental changes in the financial services industry, financial conglomerate structures and value-based and (operational) risk management, we discuss the main governance body of financial conglomerates in part 2: the corporate centre. We focus on its nature, management and organisation, and economics In the first chapter, we discuss (de-) centralisation, roles and contribution of the corporate centre. There is an ongoing discussion on the level of decentralisation, i.e. what belongs to the corporate centre and what belongs to the divisions. On the continuum between centralisation and decentralisation, we distinguish between five statuses: core, policy, matrix, service, and autarchy. These statuses have different characteristics and are appropriate in situations of different complexity and environmental dynamics. Decentralisation without coordination leads to a lack of focus. Coordination is implemented with the use of special instruments, which manage the interdependencies between organisational units. In the ranking of increasing coordination, we recognise the following instruments: company (sub) culture, role standardisation, self-management, plans, programs, and personal instruction. In conjunction with issues of decentralisation and coordination is the issue of influence of the corporate centre. Influence can be seen as the result of decisions of design and the implemented level of decentralisation; influence tends to be highest in the general planning areas e.g. setting of budgets and financial targets, major capital investments, business strategy and new business creation. Influence tends to be lower in the functional areas e.g. human resources and marketing. Companies with greater diversity tend to have more decentralised decision-making. Striking the correct influence balance, i.e. fine tuning decentralisation and coordination proves to be difficult. Continuous tests are necessary to assess if the influence exercised is appropriate and effective. High levels of influence in combination with a very diverse portfolio could indicate either that the company is too centralised in its decisionmaking, or that the portfolio is insufficiently focused. On the other hand, low levels of influence in combination with a focused portfolio could indicate that the company is missing opportunities to add value. There are two different types of corporate centres: separate and embedded corporate centres. The latter type is integrated in the main division and is most common when the company comprises either a single major division together with a few minor divisions. If the minor divisions develop into more major operations, such companies tend to move to the separate corporate centre form. In this study, focusing on financial conglomerates, we concentrate on the separate corporate centre. Another typology for (parts of) the corporate centre is determined by the intensity of coordination; we distinguish between the operational, financial, and management holding. We recognise that companies can optimise their centralised services by placing them in divisions, which, depending on qualifications, regional or functional considerations, are the best location for those activities. From a strategy viewpoint, we can assume that the corporate centre, as instrument of corporate management, is primarily there to help develop and implement the overall corporate strategy. Hence, the aim is to combine the advantages of the flexibility and autonomy of the divisions with the unified strategic direction and management of the company as a whole. High performing firms do not distinguish themselves from the low performers by being more or less diversified or more or less decentralised: high performers have a better fit between their diversification strategy and the role their corporate centre performs, even in dynamic circumstances. There are three requirements for value creation by the corporate centre: 1) opportunity, 2) skills and resources, and 3) the degree of understanding by the corporate centre. Opportunities arise from an imperfect fit between divisions and the environment. The following five types of opportunities are distinguished: build, stretch, link, leverage and portfolio development. Synergy, the notion that the whole is larger than the sum of its parts, is hard to quantify and proves hard to reach in financial conglomerates. One way to realise the synergy potential is to identify affinities and critical interrelationships within the group, develop and analyse value chains per division and look for common characteristics, formulation of a strategy in coordination with corporate and division strategies with goals supporting the pursuit of interrelationships, and configuration of the synergy activities by e.g. centralisation of these activities on a separate location. In these steps, different instruments can be deployed: change of attitude of management, structuring collaboration, and supporting systems and procedures. Obstacles to achieving synergy include a classic, one-dimensional strategic planning approach, predominant vertical organisational boundaries, lack of motivation, and a lack of symmetric benefits in interrelationships. In the second chapter we focus on the management and organisation of the corporate centre. The form of the corporate centre is decreasingly determined by historical and cultural differences and increasingly by industry trends. Further, when a firm is active in a regulated industry, such as the financial services industry, the corporate centre has to take special influences into

Contribution of in vivo two-photon lung microscopy to the study of anthrax pathophysiology.

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires.Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections

Apports de la microscopie biphotonique intravitale pulmonaire à l'étude de la physiopathologie de la maladie du charbon

Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires