New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies.

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Role of myosin V in actin cable organization in fission yeast

Functional specialization is tightly linked to the ability of eukaryotic cells to acquire a particular shape. Cell morphogenesis, in turn, relies on the capacity to establish and maintain cell "polarity", which is achieved by orienting the trafficking of signaling molecules and organelles towards specific cellular locations and/or membrane domains. The "oriented" transport is based upon cytoskeletal polymers, microtubules and actin filaments, which serve as tracks for molecular motors. These latter generate motion that is translated either into pulling forces or directed transport. Fission yeast, a rod-like unicellular eukaryote, shapes itself by restricting growth at cell tips through the concerted activity of microtubules and actin cables. Microtubules, which assemble into 2-6 bundles and run parallel to the long axis of the cell, serve to orient growth to the tips. Growth is supported by the actin cytoskeleton, which provides tracks, the cables, for motor-based transport of secretory vesicles. The molecular motors, which bind cargos and deliver them to the tips along cables, are also known as type V myosins (hereafter indicated as myosin V). How the bundles of parallel actin filaments, i.e. the cables, extend from the tips through the cell and whether they serve any other purpose, besides providing tracks, is poorly understood. It is also unclear how the crosstalk between the two cytoskeletal systems is achieved. These are the basic questions I addressed during my PhD. The first part of the thesis work (Chapter two) suggests that the sole function of actin cables in polarized growth is to serve as tracks for motors. The data indicate that cells may have evolved two cytoskeletal systems to provide robustness to the polarization process but in principle a unique cytoskeleton might have been able to direct and support polarized growth. How actin cables are organized within the cell to optimize cargo transport is addressed later on (Chapter three). The major finding, based on the actin cable defect of cells lacking myosin Vs, is that actin filaments self-organize through the activity of the transport motors. In fact, by delivering cargos to cell tips and exerting physical pulling forces on actin filaments, Myosin Vs contribute not only to polarize cargo transport but also actin tracks. Among the cargos transported by Myosin V, which may be relevant to its function in organizing cables, there is likely the endoplasmic reticulum (ER). Actin cables, which run parallel to cortical ER, may serve as tracks for Myosin V. Myosin V-driven displacement, in turn, may account for the dynamic expansion and organization of ER during polarized growth as suggested in Chapter four. The last part of the work (Chapter five) highlights the existence of a crosstalk between actin and microtubules. In absence of myosin V, indeed, microtubules contribute to actin cable organization, likely playing a scaffolding/tethering function. Whether or not the kinesin 1, Klp3, plays any role in such process has to be demonstrated. In conclusion the work proposes a novel role for myosin Vs in actin organization, besides its transport function, and provides molecular tools to further dissect the role of this type of myosin in fission yeast. - La spécialisation fonctionnelle est étroitement connectée à la capacité des cellules eucaryotes d'acquérir une forme particulière. La morphogenèse cellulaire à son tour, est basée sur la capacité d'établir et de maintenir la polarité cellulaire, polarité réalisée en orientant le trafic des molécules signales et des organelles vers des zones cellulaires spécifiques. Ce transport directionnel dépend des polymères du cytosquelette, microtubules et microfilaments, qui servent comme des voies pour les moteurs moléculaires. Ces derniers engendrent du mouvement, traduit soit en force de traction soit en transport directionnel. La levure fissipare, un eucaryote unicellulaire en forme de bâtonnet, acquière sa forme en limitant sa croissance aux extrémités par l'action concertée des microtubules et de l'actine. Les microtubules, qui s'assemblent de façon antiparallèle et parcourent la cellule parallèlement à l'axe longitudinal, servent à orienter la croissance aux extrémités. Cette croissance est permise par le cytosquelette d'actine, fournissant des voies, les câbles, pour le transport actif des vésicules de sécrétion. Les moteurs moléculaires, responsables de ce transport actif sont aussi appelés myosines de type V (par la suite appelés myosines V). La manière dont ces câbles s'étendent depuis l'extrémité jusqu'à l'intérieur de la cellule est peu connue. De plus, on ignore également si ces câbles présentent une fonction autre que le transport. L'interaction entre les deux cytosquelettes est également obscure. Ce sont ces questions de base auxquelles j'ai tenté de répondre lors de ma thèse. La première partie de cette thèse (chapitre II) suggère que les câbles d'actine, pendant la croissance polarisée, fonctionnent uniquement comme des voies pour les moteurs moléculaires. Les données indiqueraient que les cellules ont fait évoluer deux systèmes de cytosquelette pour assurer plus de robustesse au processus de polarisation, bien que, comme nous le verrons, un système unique est suffisant. Au chapitre III, nous verrons comment les câbles d'actine sont organisés à l'intérieur de la cellule afin d'optimiser le transport des cargo. La découverte majeure, réalisée en observant des cellules dont la myosine V fait défaut, est que ces filaments d'actine s'auto organisent grâce au passage des moteurs moléculaires le long de ces voies. En réalité, en délivrant les cargos aux extrémités de la cellule et en exerçant des forces de traction sur les câbles, les myosines V contribuent non seulement à polariser le transport mais également à polariser les voies elles mêmes. Nous verrons également au chapitre IV, que parmi les cargos importants pour l'organisation des câbles, il y aurait le réticulum endoplasmique (RE). En effet, les câbles d'actine, qui s'étalent parallèlement au RE cortical, pourraient servir comme voie pour la myosine V. Cette dernière en retour pourrait être responsable de l'expansion dynamique et de l'organisation du RE pendant la croissance polarisée.

Leniency programs, antitrust enforcement and multimarket contact : three essays in industrial organization

The Layout of My Thesis This thesis contains three chapters in Industrial Organization that build on the work outlined above. The first two chapters combine leniency programs with multimarket contact and provide a thorough analysis of the potential effects of Amnesty Plus and Penalty Plus. The third chapter puts the whole discussion on leniency programs into perspective by examining other enforcement tools available to an antitrust authority. The main argument in that last chapter is that a specific instrument can only be as effective as the policy in which it is embedded. It is therefore important for an antitrust authority to know how it best accompanies the introduction or modification of a policy instrument that helps deterrence. INTRODUCTION Chapter 1 examines the efféct of Amnesty Plus and Penalty Plus on the incentives of firms to report cartel activities. The main question is whether the inclusion of these policies in a leniency program undermine the effectiveness of the latter by discouraging the firms to apply for amnesty. The model is static and focus on the ex post incentives of firms to desist from collusion. The results suggest that, because Amnesty Plus and Penalty Plus encourage the reporting of a second cartel after a first detection, a firm, anticipating this, may be reluctant to seek leniency and to report in the first place. However, the effect may also go in the opposite direction, and Amnesty Plus and Penalty Plus may encourage the simultaneous reporting of two cartels. Chapter 2 takes this idea further to the stage of cartel formation. This chapter provides a complete characterization of the potential anticompetitive and procompetitive effects of Amnesty Plus in a infinitely repeated game framework when the firms use their multimarket contact to harshen punishment. I suggest a clear-cut policy rule that prevents potential adverse effects and thereby show that, if policy makers follow this rule, a leniency program with Amnesty Plus performs better than one without. Chapter 3 characterizes the socially optimal enforcement effort of an antitrust authority and shows how this effort changes with the introduction or modification of specific policy instruments. The intuition is that the policy instrument may increase the marginal benefit of conducting investigations. If this effect is strong enough, a more rigorous detection policy becomes socially desirable.

Ant genomics sheds light on the molecular regulation of social organization.

Ants are powerful model systems for the study of cooperation and sociality. In this review, we discuss how recent advances in ant genomics have contributed to our understanding of the evolution and organization of insect societies at the molecular level.

Temporal and spatial appearance of the membrane cytoskeleton and perineuronal nets in the rat neocortex.

Parvalbumin-immunoreactive interneurons are surrounded by perineuronal nets, containing molecules of the extracellular matrix (e.g. tenascin-R). Furthermore, they seem to have a special cytoskeleton composed of, among others, ankyrinR and beta Rspectrin. In the present developmental study we showed that the intracellular markers parvalbumin, ankyrinR and beta Rspectrin as well as Vicia Villosa agglutinin, an extracellular marker for perineuronal nets, appeared in the second postnatal week. In the third postnatal week, ankyrinR and beta R spectrin were present in the parvalbumin-positive interneurons. Tenascin-R appeared in a similar topographic distribution as the intracellular markers. The adult pattern was established upon the end of the fourth postnatal week. Our results indicate that cytoskeletal maturity maybe a prerequisite for the organization of perineuronal nets of extracellular matrix.

Public particiation and the legitimacy of intergovernmental decision-making : a focus on the World Trade Organization

The legitimacy of the WTO's decision-making process has always been questioned, and many have advocated public participation mechanisms as a remedy. The present study considers the limits and potential of these mechanisms by advancing a conceptual framework, which distinguishes the four 'implementation parameters' of public participation: the goal, the object, the modalities, and the actors. It addresses the issue of legitimacy by considering to what extent, and by virtue of which legal developments, one can see implementing the democratic principle as a goal for public participation in the context of the WTO. By analyzing the institutional structure of the WTO and its different types of decisions, it then outlines how this goal should influence the object and modalities of public participation, which decision-making procedures should be opened to public participation, and how the mechanisms should be implemented in practice. Finally, it suggests speciflrc amendments to existing WTO affangements on public participation

The Hindu Model of Social Organization and the bhakti Movement : the Example of Vallabha's sampradāya

(Résumé de l'ouvrage) This volume addresses research topics within the field of Bhakti literature, the devotional poetry and other compositions of devotional character in the earlier literature of the modern South Asian languages. Its papers range from the roots of the Bhakti tradition in the early history of Krsna to its modern adaptations in nineteenth- and twentieth-century culture. Geographically, they span Bengal to Sind, Panjab to Maharashtra. Contemporary study of the modern Indian languages has broadened the scope of scholarship to consider today's Hindu attitudes, and those of a mixed society, against the background of ancient culture. Here, materials in six modern Asian languages are discussed: Bengali, Gujarati, Hindi in its main literary forms, Marathi, Panjabi and Sindhi; with assessment also of material in Sanskrit, Arabic and Chinese. In addition to studies of literary (and orally transmitted) works in the Krsna or Rama traditions, and of Sufi compositions and their interpretation, there are papers on the early history of sacred sites, the emergence of the religion of Rama, later religious formulations throughout the subcontinent, and the interaction of the Islamic and the Hindu.

Physical activity in 22 African countries: results from the World Health Organization STEPwise approach to chronic disease risk factor surveillance.

BACKGROUND: Baseline physical activity data are needed to effectively plan programs and policies to prevent noncommunicable diseases, but for many African countries these data are lacking. PURPOSE: To describe and compare levels and patterns of physical activity among adults across 22 African countries. METHODS: Data from 57,038 individuals from 22 countries (11 national and 11 subnational samples) that participated in the STEPwise approach to chronic disease risk factor surveillance (2003-2009) were analyzed in 2010. The validated Global Physical Activity Questionnaire (GPAQ) was used to assess days and duration of physical activity at work, for transport, and during leisure time in a typical week. RESULTS: Overall, 83.8% of men and 75.7% of women met WHO physical activity recommendations (at least 150 minutes of moderate activity per week or equivalent). Country prevalence ranged from 46.8% (Mali) to 96.0% (Mozambique). Physical activity, both at work and for transport, including walking, had large contributions to overall physical activity, while physical activity during leisure time was rare in the analyzed countries. CONCLUSIONS: Physical activity levels varied greatly across African countries and population subgroups. Leisure time activity was consistently low. These data will be useful to inform policymakers and to guide interventions to promote physical activity.

Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

High bi-atrial organization in patients with long-standing persistent atrial fibrillation terminated within the left atrium

Sustained atrial fibrillation (AF) is maintained by sites displaying high dominant frequency (DF). In patients (pts) with long-standing persistent AF (LS-pAF), their spatial distribution and the presence of a left-to-right atrial DF gradient remain poorly known. We hypothesized that the pre-ablation bi-atrial frequency characteristics of LS-pAF pts terminated within the left atrium (LT) are different from that of non terminated (NT) ones. Methods: 23 consecutive pts (59±7y, LS-pAF duration 19±12m) underwent stepwise catheter ablation (step-CA) consisting in pulmonary veins isolation, left atrial (LA) defragmentation, and right atrial (RA) ablations for non terminated AF. A quadripolar catheter (CAT) was placed into the RA appendage (RAA), a decapolar CAT into the coronary sinus (CS) and a duodecapolar CAT into the LA divided into 8 segments. For each segment, 20-sec of bipolar recording was acquired. The DF was defined as the largest peak in the power spectrum (3-15 Hz). The inter-atrial DF gradient was defined as the DF difference between LA and RA appendages. Results: LS-pAF was terminated in 83% (19/23) of the pts: 17 LT, 2 during RA ablation and 4 NT. The figure shows that before ablation bi-atrial DF values of LT pts are significantly lower than that of NT pts for each LA segment as well as for the RAA (p < 0.05). No significant LA-to-RA DF gradient was observed both for LT (0.3±0.5 Hz, p=ns) and NT (0.5±0.03 Hz, p=ns) pts. No significant difference in DF values was observed between LA segments. Conclusions: The lower DF of LT pts is suggestive of a higher organization within both atria compared to NT pts. Our findings suggest that low bi-atrial DF values, but not inter-atrial DF gradient, might be of interest for selecting LS-pAF candidates for sinus rhythm restoration by step-CA.

Early functional differentiation in the chick embryonic disc: interaction between mechanical activity and extracellular matrix

The mechanical behaviour of ectodermal cells in the area opaca and the supracellular organization of fibronectin in the adjacent extracellular matrix were studied in whole chick blastoderms developing in vitro. The pattern of spontaneous mechanical activity and its modification by immunoglobulins against fibronectin were determined using a real-time image-analysis system. The pattern of fibronectin was studied using immunocytochemical techniques. It was found that the ectodermal cells in the area opaca actively develop a radially oriented contraction, which leads to a distension of the area pellucida from which the embryo develops. Abnormally increased tension resulted in perturbations of gastrulation and neurulation. An optimized mechanical equilibrium within the blastoderm seems to be necessary for normal development. Anti-fibronectin antibodies applied to the basal side of the blastoderm led rapidly and reversibly to an increase of tension in the contracted cells. This observation indicates that modifications of the extracellular matrix can be transmitted to cytoskeletal elements within adjacent cells. The extracellular matrix of the area opaca contains fibronectin arranged in radially oriented fibrils. This orientation corresponds to the direction of migration of the mesodermal cells. Interestingly, the radial pattern of fibronectin is found in the regions where the ectodermal cells are contracted and develop radially oriented forces. This observation suggests that the supracellular assembly of the extracellular materials could be influenced by the mechanical activity of adjacent cells. Possible modulations of the supracellular organization of extracellular matrix by other factors, e.g. diffusible metabolites, is also discussed. The presence of characteristically organized extracellular matrix components, of spatially differentiated cell activities and of reciprocal interactions between them makes the young chick blastoderm an excellent system for physiological studies of the coordinated cellular activities that lead to changes in form, complexity and function.

Role of topological exclusion in formation and organization of chromosomal territories

Chromosomes of eukaryotic organisms are composed of chromatin loops. Using Monte Carlo simulations we investigate how the topological exclusion between loops belonging to different chromosomes affects chromosome behaviour. We show that in a confined space the topological exclusion limiting catenation between loops belonging to different chromosomes entropically drives the formation of chromosomal territories. The same topological exclusion in a connection with interchromosomal binding via transcription factories explains why actively transcribed genes are found preferentially at the peripheries of their chromosomal territories. This paper is based in part on the results presented in J. Dorier and A. Stasiak, Nucl. Acids Res. 37 (2009), 6316 and 38 (2010), 7410.

Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.

In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.

The Complete Chromosomal Organization of the Reference Strain of the Leishmania Genome Project, L. major ;Friedlin'.

In recent years, analysis of the genomes of many organisms has received increasing international attention. The bulk of the effort to date has centred on the Human Genome Project and analysis of model organisms such as yeast, Drosophila and Caenorhabditis elegans. More recently, the revolution in genome sequencing and gene identification has begun to impact on infectious disease organisms. Initially, much of the effort was concentrated on prokaryotes, but small eukaryotic genomes, including the protozoan parasites Plasmodium, Toxoplasma and trypanosomatids (Leishmania, Trypanosoma brucei and T. cruzi), as well as some multicellular organisms, such as Brugia and Schistosoma, are benefiting from the technological advances of the genome era. These advances promise a radical new approach to the development of novel diagnostic tools, chemotherapeutic targets and vaccines for infectious disease organisms, as well as to the more detailed analysis of cell biology and function.Several networks or consortia linking laboratories around the world have been established to support these parasite genome projects[1] (for more information, see http://www.ebi.ac.uk/ parasites/paratable.html). Five of these networks were supported by an initiative launched in 1994 by the Specific Programme for Research and Tropical Diseases (TDR) of the WHO[2, 3, 4, 5, 6]. The Leishmania Genome Network (LGN) is one of these[3]. Its activities are reported at http://www.ebi.ac.uk/parasites/leish.html, and its current aim is to map and sequence the genome of Leishmania by the year 2002. All the mapping, hybridization and sequence data are also publicly available from LeishDB, an AceDB-based genome database (http://www.ebi.ac.uk/parasites/LGN/leissssoft.html).

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.