Secretory IgA and intestinal epithelial cells at the interface between homeostasis regulation and protection against infection

RESUME DESTINE A UN LARGE PUBLICL'intestin est le siège d'intenses agressions de la part de l'ensemble des aliments ingérés, de bactéries agressives dites pathogènes mais également de bactéries dites commensales peuplant naturellement les surfaces intestinales muqueuses. Pour faire face, notre organisme arbore de nombreux niveaux de protections tant physiques, chimiques, mécaniques mais aussi immunitaires. La présence d'un type particulier de cellules, les cellules épithéliales (IEC) assurant une protection physique, ainsi que la production d'anticorps spécialisés par le système immunitaire appelés immunoglobulines sécrétoires A (SlgA) servent conjointement de première ligne de défense contre ces agressions externes. Néanmoins, comment le dialogue s'articule entre ces deux partenaires reste incomplet.Nous avons donc décidé de mimer ces interactions en modélisant les surfaces muqueuses par une monocouche de cellules différenciées en laboratoire. Des souches bactériennes isolées de l'intestin humain seules ou associées à des SlgA non-spécifiques ont été mises au contact de ce modèle cellulaire nous permettant de conclure quant à la présence effective d'une modulation du dialogue bactérie/lEC impliquant une activation de la réponse cellulaire vers un état de tolérance mutuelle. De façon surprenante, nous avons par ailleurs mis en évidence un type d'interaction nouveau entre ces anticorps et ces bactéries. Une étude biochimique nous a permis de détailler un nouveau rôle des SlgA médié par les sucres présents à leur surface dans le maintien d'une relation pacifique avec les commensaux perpétuellement présents, relations qualifiées d'homésostase intestinale.Le rôle protecteur des SlgA a par ailleurs été abordé pour avoir une meilleure appréhension de leur impact au niveau cellulaire lors d'infection par Shigella flexneri, bactérie causant la Shigellose, diarrhée sanglante responsable de la mort de plus d'un million de personnes chaque année. Basée sur le même modèle cellulaire, cette étude nous a permis de démontrer une nouvelle entrée de ce pathogène directement via les IEC. La présence d'anticorps spécifiques à la surface des bactéries restreint leur champs d'action contre les cibles intracellulaires identifiées que sont les filaments soutenant le squelette de la cellule, les fibres d'actine ainsi que les jonctions serrées, réseaux de protéines clés des interactions entre cellules. Cette ouverture au niveau cellulaire apporte un nouvel élan quant à la compréhension du rôle protecteur des SlgA lors d'attaques de l'intestin, protection semblant dépendante d'une agrégation des bactéries.Pour finir, nous avons mis en évidence la détection directe par les cellules de la présence d'anticorps libres dans l'intestin ajoutant une nouvelle réplique dans le dialogue complexe entre ces deux piliers de l'équilibre intestinal que sont les SlgA et les cellules épithéliales.RESUMELa muqueuse intestinale est dotée d'un réseau complexe de protections physico-chimiques, mécaniques ou immunologiques. Associées à un système immunitaire omniprésent, les cellules épithéliales intestinales {IEC) bordant la lumière intestinale ont la double tâche de protéger l'intérieur de l'organisme stérile contre l'invasion et la dissémination d'agents pathogènes, et de maintenir une relation pacifique avec la flore intestinale, rôles également joués par les immunoglobulines sécrétoires A (SlgA), anticorps les plus abondamment présents à la surface des muqueuses. Tant les IEC que les SlgA sont ainsi décrites comme convergeant vers le même objectif ; néanmoins, les rouages de leurs interactions restent largement inconnus.Pour répondre à cette question, des monocouches épithéliales reconstituées in vitro ont été incubées avec des souches commensales telles que des Lactobacillus ou des Bifodobacteria, seules ou complexées avec des SlgA non-spécifiques, nous permettant de décrypter l'influence des SlgA sur la détection des bactéries par les IEC, favorisant l'adhésion bactérienne et la cohésion cellulaire, augmentant l'activation de la voie NF-κΒ ainsi que la sécrétion de la cytokine thymic stromal lymphopoietin contrairement à celle de médiateurs pro-inflammatoires qui reste inchangée. Par ailleurs, une interaction Fab-indépendante est suggérée dans l'interaction SlgA/bactéries. Comme une interaction de faible affinité a été décrite comme prenant naturellement place au niveau de l'intestin, nous avons donc disséqué les mécanismes sous- jacents en utilisant un large spectre de bactérie associés à des protéines soit recombinantes soit isolées à partir de colostrum, mettant en évidence un rôle crucial des N-glycanes présents sur la pièce sécrétoire et soulignant une nouvelle propriété des SlgA dans l'homéostase intestinale.Intrinsèquement liés aux caractéristiques des SlgA, nous nous sommes également focalisés sur leur rôle protecteur lors d'infection par l'enteropathogène Shigella flexneri reproduites in vitro sur des monocouches polarisées. Nous avons tout d'abord démontré une nouvelle porte d'entrée pour ce pathogène directement via les IEC. L'agrégation des bactéries par les SlgA confère aux cellules une meilleure résistance à l'infection, retardant croissance bactérienne et entrée cellulaire, affectant par ailleurs leur capacité à cibler le cytosquelette et les jonctions serrées. La formation de tels cargos détectés de façon biaisée par les IEC apparaît comme une explication plausible au maintien de la cohésion cellulaire médiée par les SlgA.Enfin, le retrotransport des SlgA à travers les IEC a été abordé soulignant une participation active de ces cellules dans la détection de l'environnement extérieur, les impliquant possiblement dans l'activation d'un état muqueux stable.Conjointement, ces résultats indiquent que les SlgA représentent l'un des éléments-clés à la surface de la muqueuse et soulignent la complexité du dialogue établi avec l'épithélium en vue du maintien d'un fragile équilibre intestinal.ABSTRACTThe intestinal mucosa is endowed with a complex protective network melting physiochemical, mechanical and immunological features. Beyond the ubiquitous intestinal immune system, intestinal epithelial cells (IEC) lying the mucosal surfaces have also the dual task to protect the sterile core against invasion and dissemination of pathogens, and maintain a peaceful relationship with commensal microorganisms, aims also achieved by the presence of high amounts of secretory immunoglobulins A (SlgA), the most abundant immunoglobulin present at mucosal surfaces. Both IEC and SlgA are thus described to converge toward the same goal but how their interplay is orchestrated is largely unknown.To address this question, in vitro reconstituted IEC monolayers were first apically incubated with commensal bacteria such as Lactobacillus or Bifodobacteria strains either alone or in complexes with non-specific SlgA. Favoring the bacterial adhesion and cellular cohesion, SlgA impacts on the cellular sensing of bacteria, increasing NF-κΒ activation, and leading to cytokine releases restricted to the thymic stromal lymphopoietin and unaffected expression of pro-inflammatory mediators. Of main interest, bacterial recognition by SlgA suggested a Fab-independent interaction. As this low affinity, called natural coating occurs in the intestine, we further dissected the underlying mechanisms using a larger spectrum of commensal strains associated with recombinant as well as colostrum-derived proteins and pinpointed a crucial role of N-glycans of the secretory component, emphasizing an underestimated role of carbohydrates and another properties of SlgA in mediating intestinal homeostasis.As mucosal protection is also anchored in SlgA and IEC features, we focused on the cellular role of SlgA. Using IEC apical infection by the enteropathogen Shigella flexneri, we have first demonstrated a new gate of entry for this pathogen directly via IEC. Specific SlgA bacterial aggregation conferred to the cells a better resistance to infection, delaying bacterial growth and cellular entry, affecting their ability to damage both the cytoskeleton and the tight junctions. Formation of such big cargos differentially detected by IEC appears as a plausible explanation sustaining at the cellular level the antibody-mediated mucosal protection.Finally, SlgA retrotransport across IEC has been tackled stressing an active IEC sensing of the external environment possibly involved in the steady-state mucosal activation.All together, these results indicate that SlgA represents one of the pivotal elements at mucosal surfaces highlighting the complexity of the dialogue established with the epithelium sustaining the fragile intestinal balance.The Intestinal mucosa is endowed with a complex protective network melting physiochemical, mechanical and immunological features. Beyond the ubiquitous intestinal immune system, intestinal epithelial cells (IEC) lying the mucosal surfaces have also the dual task to protect the sterile core against invasion and dissemination of pathogens, and maintain a peaceful relationship with commensal microorganisms, aims also achieved by the presence of high amounts of secretory immunoglobulins A (SlgA), the most abundant immunoglobulin present at mucosal surfaces. Both IEC and SlgA are thus described to converge toward the same goal but how their interplay is orchestrated is largely unknown.To address this question, in vitro reconstituted IEC monolayers were first apically incubated with commensal bacteria such as Lactobacillus or Bifodobacteria strains either alone or in complexes with non-specific SlgA. Favoring the bacterial adhesion and cellular cohesion, SlgA impacts on the cellular sensing of bacteria, increasing NF-κΒ activation, and leading to cytokine releases restricted to the thymic stromal lymphopoietin and unaffected expression of pro-inflammatory mediators. Of main interest, bacterial recognition by SlgA suggested a Fab-independent interaction. As this low affinity, called natural coating occurs in the intestine, we further dissected the underlying mechanisms using a larger spectrum of commensal strains associated with recombinant as well as colostrum-derived proteins and pinpointed a crucial role of N-glycans of the secretory component, emphasizing an underestimated role of carbohydrates and another properties of SlgA in mediating intestinal homeostasis.As mucosal protection is also anchored in SlgA and IEC features, we focused on the cellular role of SlgA. Using IEC apical infection by the enteropathogen Shigella flexneri, we have first demonstrated a new gate of entry for this pathogen directly via IEC. Specific SlgA bacterial aggregation conferred to the cells a better resistance to infection, delaying bacterial growth and cellular entry, affecting their ability to damage both the cytoskeleton and the tight junctions. Formation of such big cargos differentially detected by IEC appears as a plausible explanation sustaining at the cellular level the antibody-mediated mucosal protection.Finally, SlgA retrotransport across IEC has been tackled stressing an active IEC sensing of the external environment possibly involved in the steady-state mucosal activation.All together, these results indicate that SlgA represents one of the pivotal elements at mucosal surfaces highlighting the complexity of the dialogue established with the epithelium sustaining the fragile intestinal balance.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Isolation of cardiovascular precursor cells from the human fetal heart.

Weakening of cardiac function in patients with heart failure results from a loss of cardiomyocytes in the damaged heart. Cell replacement therapies as a way to induce myocardial regeneration in humans could represent attractive alternatives to classical drug-based approaches. However, a suitable source of precursor cells, which could produce a functional myocardium after transplantation, remains to be identified. In the present study, we isolated cardiovascular precursor cells from ventricles of human fetal hearts at 12 weeks of gestation. These cells expressed Nkx2.5 but not late cardiac markers such as α-actinin and troponin I. In addition, proliferating cells expressed the mesenchymal stem cell markers CD73, CD90, and CD105. Evidence for functional cardiogenic differentiation in vitro was demonstrated by the upregulation of cardiac gene expression as well as the appearance of cells with organized sarcomeric structures. Importantly, differentiated cells presented spontaneous and triggered calcium signals. Differentiation into smooth muscle cells was also detected. In contrast, precursor cells did not produce endothelial cells. The engraftment and differentiation capacity of green fluorescent protein (GFP)-labeled cardiac precursor cells were then tested in vivo after transfer into the heart of immunodeficient severe combined immunodeficient mice. Engrafted human cells were readily detected in the mouse myocardium. These cells retained their cardiac commitment and differentiated into α-actinin-positive cardiomyocytes. Expression of connexin-43 at the interface between GFP-labeled and endogenous cardiomyocytes indicated that precursor-derived cells connected to the mouse myocardium. Together, these results suggest that human ventricular nonmyocyte cells isolated from fetal hearts represent a suitable source of precursors for cell replacement therapies.

Three essays on major trends in a slow clockspeed industry : the case of industrial automation

The motivation for this research initiated from the abrupt rise and fall of minicomputers which were initially used both for industrial automation and business applications due to their significantly lower cost than their predecessors, the mainframes. Later industrial automation developed its own vertically integrated hardware and software to address the application needs of uninterrupted operations, real-time control and resilience to harsh environmental conditions. This has led to the creation of an independent industry, namely industrial automation used in PLC, DCS, SCADA and robot control systems. This industry employs today over 200'000 people in a profitable slow clockspeed context in contrast to the two mainstream computing industries of information technology (IT) focused on business applications and telecommunications focused on communications networks and hand-held devices. Already in 1990s it was foreseen that IT and communication would merge into one Information and communication industry (ICT). The fundamental question of the thesis is: Could industrial automation leverage a common technology platform with the newly formed ICT industry? Computer systems dominated by complex instruction set computers (CISC) were challenged during 1990s with higher performance reduced instruction set computers (RISC). RISC started to evolve parallel to the constant advancement of Moore's law. These developments created the high performance and low energy consumption System-on-Chip architecture (SoC). Unlike to the CISC processors RISC processor architecture is a separate industry from the RISC chip manufacturing industry. It also has several hardware independent software platforms consisting of integrated operating system, development environment, user interface and application market which enables customers to have more choices due to hardware independent real time capable software applications. An architecture disruption merged and the smartphone and tablet market were formed with new rules and new key players in the ICT industry. Today there are more RISC computer systems running Linux (or other Unix variants) than any other computer system. The astonishing rise of SoC based technologies and related software platforms in smartphones created in unit terms the largest installed base ever seen in the history of computers and is now being further extended by tablets. An underlying additional element of this transition is the increasing role of open source technologies both in software and hardware. This has driven the microprocessor based personal computer industry with few dominating closed operating system platforms into a steep decline. A significant factor in this process has been the separation of processor architecture and processor chip production and operating systems and application development platforms merger into integrated software platforms with proprietary application markets. Furthermore the pay-by-click marketing has changed the way applications development is compensated: Three essays on major trends in a slow clockspeed industry: The case of industrial automation 2014 freeware, ad based or licensed - all at a lower price and used by a wider customer base than ever before. Moreover, the concept of software maintenance contract is very remote in the app world. However, as a slow clockspeed industry, industrial automation has remained intact during the disruptions based on SoC and related software platforms in the ICT industries. Industrial automation incumbents continue to supply systems based on vertically integrated systems consisting of proprietary software and proprietary mainly microprocessor based hardware. They enjoy admirable profitability levels on a very narrow customer base due to strong technology-enabled customer lock-in and customers' high risk leverage as their production is dependent on fault-free operation of the industrial automation systems. When will this balance of power be disrupted? The thesis suggests how industrial automation could join the mainstream ICT industry and create an information, communication and automation (ICAT) industry. Lately the Internet of Things (loT) and weightless networks, a new standard leveraging frequency channels earlier occupied by TV broadcasting, have gradually started to change the rigid world of Machine to Machine (M2M) interaction. It is foreseeable that enough momentum will be created that the industrial automation market will in due course face an architecture disruption empowered by these new trends. This thesis examines the current state of industrial automation subject to the competition between the incumbents firstly through a research on cost competitiveness efforts in captive outsourcing of engineering, research and development and secondly researching process re- engineering in the case of complex system global software support. Thirdly we investigate the industry actors', namely customers, incumbents and newcomers, views on the future direction of industrial automation and conclude with our assessments of the possible routes industrial automation could advance taking into account the looming rise of the Internet of Things (loT) and weightless networks. Industrial automation is an industry dominated by a handful of global players each of them focusing on maintaining their own proprietary solutions. The rise of de facto standards like IBM PC, Unix and Linux and SoC leveraged by IBM, Compaq, Dell, HP, ARM, Apple, Google, Samsung and others have created new markets of personal computers, smartphone and tablets and will eventually also impact industrial automation through game changing commoditization and related control point and business model changes. This trend will inevitably continue, but the transition to a commoditized industrial automation will not happen in the near future.

Human fetal bone cells in delivery systems for bone engineering.

The aim of this study was to culture human fetal bone cells (dedicated cell banks of fetal bone derived from 14 week gestation femurs) within both hyaluronic acid gel and collagen foam, to compare the biocompatibility of both matrices as potential delivery systems for bone engineering and particularly for oral application. Fetal bone cell banks were prepared from one organ donation and cells were cultured for up to 4 weeks within hyaluronic acid (Mesolis(®)) and collagen foams (TissueFleece(®)). Cell survival and differentiation were assessed by cell proliferation assays and histology of frozen sections stained with Giemsa, von Kossa and ALP at 1, 2 and 4 weeks of culture. Within both materials, fetal bone cells could proliferate in three-dimensional structure at ∼70% capacity compared to monolayer culture. In addition, these cells were positive for ALP and von Kossa staining, indicating cellular differentiation and matrix production. Collagen foam provides a better structure for fetal bone cell delivery if cavity filling is necessary and hydrogels would permit an injectable technique for difficult to treat areas. In all, there was high biocompatibility, cellular differentiation and matrix deposition seen in both matrices by fetal bone cells, allowing for easy cell delivery for bone stimulation in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

Direct numerical simulations of interface dynamics to link capillary pressure and total surface energy

The flow of two immiscible fluids through a porous medium depends on the complex interplay between gravity, capillarity, and viscous forces. The interaction between these forces and the geometry of the medium gives rise to a variety of complex flow regimes that are difficult to describe using continuum models. Although a number of pore-scale models have been employed, a careful investigation of the macroscopic effects of pore-scale processes requires methods based on conservation principles in order to reduce the number of modeling assumptions. In this work we perform direct numerical simulations of drainage by solving Navier-Stokes equations in the pore space and employing the Volume Of Fluid (VOF) method to track the evolution of the fluid-fluid interface. After demonstrating that the method is able to deal with large viscosity contrasts and model the transition from stable flow to viscous fingering, we focus on the macroscopic capillary pressure and we compare different definitions of this quantity under quasi-static and dynamic conditions. We show that the difference between the intrinsic phase-average pressures, which is commonly used as definition of Darcy-scale capillary pressure, is subject to several limitations and it is not accurate in presence of viscous effects or trapping. In contrast, a definition based on the variation of the total surface energy provides an accurate estimate of the macroscopic capillary pressure. This definition, which links the capillary pressure to its physical origin, allows a better separation of viscous effects and does not depend on the presence of trapped fluid clusters.

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Pore-scale modeling of two-phase flow instabilities in porous media

Les problèmes d'écoulements multiphasiques en média poreux sont d'un grand intérêt pour de nombreuses applications scientifiques et techniques ; comme la séquestration de C02, l'extraction de pétrole et la dépollution des aquifères. La complexité intrinsèque des systèmes multiphasiques et l'hétérogénéité des formations géologiques sur des échelles multiples représentent un challenge majeur pour comprendre et modéliser les déplacements immiscibles dans les milieux poreux. Les descriptions à l'échelle supérieure basées sur la généralisation de l'équation de Darcy sont largement utilisées, mais ces méthodes sont sujettes à limitations pour les écoulements présentant de l'hystérèse. Les avancées récentes en terme de performances computationnelles et le développement de méthodes précises pour caractériser l'espace interstitiel ainsi que la distribution des phases ont favorisé l'utilisation de modèles qui permettent une résolution fine à l'échelle du pore. Ces modèles offrent un aperçu des caractéristiques de l'écoulement qui ne peuvent pas être facilement observées en laboratoire et peuvent être utilisé pour expliquer la différence entre les processus physiques et les modèles à l'échelle macroscopique existants. L'objet premier de la thèse se porte sur la simulation numérique directe : les équations de Navier-Stokes sont résolues dans l'espace interstitiel et la méthode du volume de fluide (VOF) est employée pour suivre l'évolution de l'interface. Dans VOF, la distribution des phases est décrite par une fonction fluide pour l'ensemble du domaine et des conditions aux bords particulières permettent la prise en compte des propriétés de mouillage du milieu poreux. Dans la première partie de la thèse, nous simulons le drainage dans une cellule Hele-Shaw 2D avec des obstacles cylindriques. Nous montrons que l'approche proposée est applicable même pour des ratios de densité et de viscosité très importants et permet de modéliser la transition entre déplacement stable et digitation visqueuse. Nous intéressons ensuite à l'interprétation de la pression capillaire à l'échelle macroscopique. Nous montrons que les techniques basées sur la moyenne spatiale de la pression présentent plusieurs limitations et sont imprécises en présence d'effets visqueux et de piégeage. Au contraire, une définition basée sur l'énergie permet de séparer les contributions capillaires des effets visqueux. La seconde partie de la thèse est consacrée à l'investigation des effets d'inertie associés aux reconfigurations irréversibles du ménisque causé par l'interface des instabilités. Comme prototype pour ces phénomènes, nous étudions d'abord la dynamique d'un ménisque dans un pore angulaire. Nous montrons que, dans un réseau de pores cubiques, les sauts et reconfigurations sont si fréquents que les effets d'inertie mènent à différentes configurations des fluides. A cause de la non-linéarité du problème, la distribution des fluides influence le travail des forces de pression, qui, à son tour, provoque une chute de pression dans la loi de Darcy. Cela suggère que ces phénomènes devraient être pris en compte lorsque que l'on décrit l'écoulement multiphasique en média poreux à l'échelle macroscopique. La dernière partie de la thèse s'attache à démontrer la validité de notre approche par une comparaison avec des expériences en laboratoire : un drainage instable dans un milieu poreux quasi 2D (une cellule Hele-Shaw avec des obstacles cylindriques). Plusieurs simulations sont tournées sous différentes conditions aux bords et en utilisant différents modèles (modèle intégré 2D et modèle 3D) afin de comparer certaines quantités macroscopiques avec les observations au laboratoire correspondantes. Malgré le challenge de modéliser des déplacements instables, où, par définition, de petites perturbations peuvent grandir sans fin, notre approche numérique apporte de résultats satisfaisants pour tous les cas étudiés. - Problems involving multiphase flow in porous media are of great interest in many scientific and engineering applications including Carbon Capture and Storage, oil recovery and groundwater remediation. The intrinsic complexity of multiphase systems and the multi scale heterogeneity of geological formations represent the major challenges to understand and model immiscible displacement in porous media. Upscaled descriptions based on generalization of Darcy's law are widely used, but they are subject to several limitations for flow that exhibit hysteric and history- dependent behaviors. Recent advances in high performance computing and the development of accurate methods to characterize pore space and phase distribution have fostered the use of models that allow sub-pore resolution. These models provide an insight on flow characteristics that cannot be easily achieved by laboratory experiments and can be used to explain the gap between physical processes and existing macro-scale models. We focus on direct numerical simulations: we solve the Navier-Stokes equations for mass and momentum conservation in the pore space and employ the Volume Of Fluid (VOF) method to track the evolution of the interface. In the VOF the distribution of the phases is described by a fluid function (whole-domain formulation) and special boundary conditions account for the wetting properties of the porous medium. In the first part of this thesis we simulate drainage in a 2-D Hele-Shaw cell filled with cylindrical obstacles. We show that the proposed approach can handle very large density and viscosity ratios and it is able to model the transition from stable displacement to viscous fingering. We then focus on the interpretation of the macroscopic capillary pressure showing that pressure average techniques are subject to several limitations and they are not accurate in presence of viscous effects and trapping. On the contrary an energy-based definition allows separating viscous and capillary contributions. In the second part of the thesis we investigate inertia effects associated with abrupt and irreversible reconfigurations of the menisci caused by interface instabilities. As a prototype of these phenomena we first consider the dynamics of a meniscus in an angular pore. We show that in a network of cubic pores, jumps and reconfigurations are so frequent that inertia effects lead to different fluid configurations. Due to the non-linearity of the problem, the distribution of the fluids influences the work done by pressure forces, which is in turn related to the pressure drop in Darcy's law. This suggests that these phenomena should be taken into account when upscaling multiphase flow in porous media. The last part of the thesis is devoted to proving the accuracy of the numerical approach by validation with experiments of unstable primary drainage in a quasi-2D porous medium (i.e., Hele-Shaw cell filled with cylindrical obstacles). We perform simulations under different boundary conditions and using different models (2-D integrated and full 3-D) and we compare several macroscopic quantities with the corresponding experiment. Despite the intrinsic challenges of modeling unstable displacement, where by definition small perturbations can grow without bounds, the numerical method gives satisfactory results for all the cases studied.

Probing functional groups at the gas-aerosol interface using heterogeneous titration reactions: a tool for predicting aerosol health effects?

The complex chemical and physical nature of combustion and secondary organic aerosols (SOAs) in general precludes the complete characterization of both bulk and interfacial components. The bulk composition reveals the history of the growth process and therefore the source region, whereas the interface controls--to a large extent--the interaction with gases, biological membranes, and solid supports. We summarize the development of a soft interrogation technique, using heterogeneous chemistry, for the interfacial functional groups of selected probe gases [N(CH(3))(3), NH(2)OH, CF(3)COOH, HCl, O(3), NO(2)] of different reactivity. The technique reveals the identity and density of surface functional groups. Examples include acidic and basic sites, olefinic and polycyclic aromatic hydrocarbon (PAH) sites, and partially and completely oxidized surface sites. We report on the surface composition and oxidation states of laboratory-generated aerosols and of aerosols sampled in several bus depots. In the latter case, the biomarker 8-hydroxy-2'-deoxyguanosine, signaling oxidative stress caused by aerosol exposure, was isolated. The increase in biomarker levels over a working day is correlated with the surface density N(i)(O3) of olefinic and/or PAH sites obtained from O(3) uptakes as well as with the initial uptake coefficient, γ(0), of five probe gases used in the field. This correlation with γ(0) suggests the idea of competing pathways occurring at the interface of the aerosol particles between the generation of reactive oxygen species (ROS) responsible for oxidative stress and cellular antioxidants.