Discordances between phylogenetic and morphological patterns in alpine leaf beetles attest to an intricate biogeographic history of lineages in postglacial Europe.

Pleistocene glacial and interglacial periods have moulded the evolutionary history of European cold-adapted organisms. The role of the different mountain massifs has, however, not been accurately investigated in the case of high-altitude insect species. Here, we focus on three closely related species of non-flying leaf beetles of the genus Oreina (Coleoptera, Chrysomelidae), which are often found in sympatry within the mountain ranges of Europe. After showing that the species concept as currently applied does not match barcoding results, we show, based on more than 700 sequences from one nuclear and three mitochondrial genes, the role of biogeography in shaping the phylogenetic hypothesis. Dating the phylogeny using an insect molecular clock, we show that the earliest lineages diverged more than 1 Mya and that the main shift in diversification rate occurred between 0.36 and 0.18 Mya. By using a probabilistic approach on the parsimony-based dispersal/vicariance framework (MP-DIVA) as well as a direct likelihood method of state change optimization, we show that the Alps acted as a cross-roads with multiple events of dispersal to and reinvasion from neighbouring mountains. However, the relative importance of vicariance vs. dispersal events on the process of rapid diversification remains difficult to evaluate because of a bias towards overestimation of vicariance in the DIVA algorithm. Parallels are drawn with recent studies of cold-adapted species, although our study reveals novel patterns in diversity and genetic links between European mountains, and highlights the importance of neglected regions, such as the Jura and the Balkanic range.

Genetic survey of "spinturnix sp" (Acari, Mesostigmata) and their bat hosts in Western Palearctic

SUMMARY : The coevolution between two intimately associated organisms, like host and parasite, is a widely investigated theme in evolutionary biology. Recently, the use of genetic data in the study of host-parasite systems evidences that the genetic information from some parasites can complement genetic data from their hosts and thus may help to better understand their host's evolutionary history. Phylogenetic and population genetic aspects of bat parasites have been poorly investigated. Spinturnicid mites are highly specialized ectoparasites, exclusively associated with bats and therefore represent an ideal model to extant our knowledge on bat and parasite biology and on their coevolutionary history. In this thesis, I developed several molecular markers (mitochondrial DNA) to compare the genetic patterns of Spinturnix mites with their bat hosts at different levels. The molecular co-phylogeny between Spinturnix sp. and their bat hosts suggests a partial cospeciation and the occurrence of failure to speciate events and multiple host switches. Thus, Spinturnix mites do not exactly mirror the phylogenetic pattern of their hosts, despite their intimate association. Similar roosting habits of the hosts seem to promote host switches between different species, as far as ecological conditions are favourable. The phylogeographic study of the Maghrebian bat M. punicus in the Mediterranean area confirms the presence of M. punicus in North Africa, Corsica and Sardinia and highlights that islands and mainland are genetically highly divergent. The comparison between the parasitic mite S. myoti and the Maghrebian bat suggests that the phylogeographic pattern of the mite is moulded by its host, with open water as main barrier for host and parasite dispersal. Moreover, the unique presence of a European S. myoti lineage on M. punicus from Corsica strongly suggests the former presence of mouse-eared bats (M. myotis and/or M. blythii) in Corsica. By highlighting the probable presence of a nowadays locally extinct host species, S. myoti may represent a good proxy for inferring complex evolutionary history of bat hosts. Finally, population genetic surveys of S. myoti and S. bechsteinii suggest that these mites benefit from close contacts between individuals during the mating season and/or hibernation to disperse among remote colonies. The contrasted genetic patterns of these two distinct bat-mite systems evidence that bat social structure is a determinant factor of the genetic structure of mite populations. Altogether, this PhD thesis demonstrates the usefulness of parasites to gather information about their bat hosts. In addition, my results illustrate how different ecological and biological characteristics of bat species allow the emergence of a surprising diversity in the genetic patterns of the parasites, which may contribute to the diversification and speciation of parasites. RESUME : La co-évolution entre deux organismes intimement liés, comme un parasite et son hôte, fait partie des questions largement étudiées en biologie évolutive. Récemment, l'utilisation de données génétique dans l'étude des interactions hôte-parasite a montré que l'information génétique de certains parasites peut compléter les données génétiques de l'hôte et ainsi peut éclairer l'histoire évolutive de leur hôte. Très peu études ont étudié les interactions entre les chauves-souris et leurs parasites d'un point de vue moléculaire. Les acariens du genre Spinturnix sont des ectoparasites très spécialisés exclusivement associés aux chauves-souris. Ils représentent donc un model idéal pour élargir nos connaissances tant sur l'écologie des parasites de chauves-souris que sur leur coévolution. Durant cette thèse, plusieurs marqueurs moléculaires (ADN mitochondrial) ont été développés pour ainsi comparer la distribution de la variation génétique des parasites du genre Spinturnix avec celle de leurs hôtes, et ceci à différents niveaux. Tout d'abord, la co-phylogénie moléculaire entre les espèces de Spinturnix et les leurs hôtes révèle une co-spéciation partielle ainsi que la présence d'événement de non spéciation et de transferts horizontaux. Ces parasites ne reflètent donc pas entièrement l'histoire évolutive de leurs hôtes, malgré leurs intimes associations. La cohabitation de plusieurs espèces de chauves-souris dans un même gîte permet aux parasites un transfert entre différentes espèces, atténuant ainsi leur degré de co-spéciation. Deuxièmement, l'étude phylogéographique du marin du Maghreb dans le bassin Méditerranéen confirme sa présence en Afrique du Nord, en Corse et en Sardaigne. La comparaison avec un de ses parasites S. myoti suggère que la répartition génétique de S. myoti est façonnée par celle de leurs hôtes, avec les étendues d'eau comme barrière principale tant à la dispersion de l'hôte que de son parasite. De plus, la présence unique d'une lignée européenne de ces parasites sur des marins du Maghreb de Corse suggère fortement la présence du grand ou petit marin en Corse dans le passé. En reflétant la présence potentielle à un endroit donné d'une espèce de chauve-souris actuellement disparue, S. myoti peut représenter une bonne alternative pour comprendre l'histoire évolutive complexe des chauves-souris. Finalement, l'étude des structures génétiques des populations des parasites S. myoti et S. bechsteinii suggère que les contacts corporels entre chauves-souris durant la saison de reproduction ou l'hibernation peuvent permettre la dispersion des parasites entre des colonies éloignées géographiquement. La différence de structure génétique entre ces deux associations particulières montre que la structure génétique des populations de parasites dépend fortement des traits d'histoire de vie de son hôte. Dans l'ensemble, cette thèse démontre l'importance des parasites pour amener des informations sur leurs hôtes, les chauves-souris. Elle illustre aussi comment les différences écologique et biologique des différentes espèces de chauves-souris peuvent amener une étonnante diversité de structure génétique au sein de populations de parasites, ce qui peut peut-être contribuer à la diversification et à la spéciation des parasites.

Peripheral T-cell lymphoma with t(6;14)(p25;q11.2) translocation presenting with massive splenomegaly.

Recurrent chromosomal translocations associated to peripheral T-cell lymphomas (PTCL) are rare. Here, we report a case of PTCL, not otherwise specified (NOS) with the karyotype 46,Y,add(X)(p22),t(6;14)(p25;q11) and FISH-proved breakpoints in the IRF4 and TCRAD loci, leading to juxtaposition of both genes. A 64-year-old male patient presented with mild cytopenias and massive splenomegaly. Splenectomy showed diffuse red pulp involvement by a pleomorphic medium- to large-cell T-cell lymphoma with a CD2+ CD3+ CD5- CD7- CD4+ CD8+/- CD30- TCRbeta-F1+ immunophenotype, an activated cytotoxic profile, and strong MUM1 expression. The clinical course was marked by disease progression in the bone marrow under treatment and death at 4 months. In contrast with two t(6;14)(p25;q11.2)-positive lymphomas previously reported to be cytotoxic PTCL, NOS with bone marrow and skin involvement, this case was manifested by massive splenomegaly, expanding the clinical spectrum of PTCLs harboring t(6;14)(p25;q11.2) and supporting consideration of this translocation as a marker of biological aggressiveness.

Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits.

Peripheral nerve injuries with loss of nervous tissue are a significant clinical problem and are currently treated using autologous nerve transplants. To avoid the need for donor nerve, which results in additional morbidity such as loss of sensation and scarring, alternative bridging methods have been sought. Recently we showed that an artificial nerve conduit moulded from fibrin glue is biocompatible to nerve regeneration. In this present study, we have used the fibrin conduit or a nerve graft to bridge either a 10 mm or 20 mm sciatic nerve gap and analyzed the muscle recovery in adult rats after 16 weeks. The gastrocnemius muscle weights of the operated side were similar for both gap sizes when treated with nerve graft. In contrast, muscle weight was 48.32 ± 4.96% of the contra-lateral side for the 10 mm gap repaired with fibrin conduit but only 25.20 ± 2.50% for the 20 mm gap repaired with fibrin conduit. The morphology of the muscles in the nerve graft groups showed an intact, ordered structure, with the muscle fibers grouped in fascicles whereas the 20 mm nerve gap fibrin group had a more chaotic appearance. The mean area and diameter of fast type fibers in the 20 mm gap repaired with fibrin conduits were significantly (P<0.01) worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. Furthermore, the 10 mm gaps repaired with either nerve graft or fibrin conduit showed similar muscle fiber size. These results indicate that the fibrin conduit can effectively treat short nerve gaps but further modification such as the inclusion of regenerative cells may be required to attain the outcomes of nerve graft for long gaps.

Division of labour influences the rate of ageing in weaver ant workers.

The evolutionary theory of ageing predicts that the timing of senescence has been primarily shaped by the extrinsic mortality rate, which causes selection intensity to decline over time. One difficulty in testing the evolutionary theory of ageing is that extrinsic mortality risk is often confounded with body size and fecundity, which may also directly affect lifespan. Social insects with a pronounced division of labour between worker castes provide a unique opportunity to study the direct effect of extrinsic mortality on the evolution of ageing rates independently of body size, reproductive effort and genetic configuration. In the weaver ant, Oecophylla smaragdina, the major (large) workers perform the risky tasks outside the nest, while the minor (small) workers stay within the highly protected arboreal nest. Hence, this pronounced division of labour is associated with high differences in extrinsic mortality risks. The evolutionary theory of ageing predicts that the minor workers should have a longer intrinsic lifespan than the major workers. In line with this prediction, we found that in a protected environment the minor workers lived significantly longer than the major workers did. Hence, the ageing rate appears to have been moulded by variation in the extrinsic mortality rate independently of size, reproductive effort and genetic configuration.

Conservation phylogeography: does historical diversity contribute to regional vulnerability in European tree frogs (Hyla arborea)?

Documenting and preserving the genetic diversity of populations, which conditions their long-term survival, have become a major issue in conservation biology. The loss of diversity often documented in declining populations is usually assumed to result from human disturbances; however, historical biogeographic events, otherwise known to strongly impact diversity, are rarely considered in this context. We apply a multilocus phylogeographic study to investigate the late-Quaternary history of a tree frog (Hyla arborea) with declining populations in the northern and western part of its distribution range. Mitochondrial and nuclear polymorphisms reveal high genetic diversity in the Balkan Peninsula, with a spatial structure moulded by the last glaciations. While two of the main refugial lineages remained limited to the Balkans (Adriatic coast, southern Balkans), a third one expanded to recolonize Northern and Western Europe, loosing much of its diversity in the process. Our findings show that mobile and a priori homogeneous taxa may also display substructure within glacial refugia ('refugia within refugia') and emphasize the importance of the Balkans as a major European biodiversity centre. Moreover, the distribution of diversity roughly coincides with regional conservation situations, consistent with the idea that historically impoverished genetic diversity may interact with anthropogenic disturbances, and increase the vulnerability of populations. Phylogeographic models seem important to fully appreciate the risks of local declines and inform conservation strategies.

Calbindin D-28k- and substance P-immunoreactive primary sensory neurons: peripheral projections in chick hindlimbs.

The peripheral projections of two distinct subpopulations of primary sensory neurons, expressing either calbindin D-28k or substance P, were studied in chick hindlimbs by immunodetecting calbindin D-28k with a rabbit antiserum and substance P with a mouse monoclonal antibody. Calbindin D-28k-immunoreactive axons provided an innervation restricted to specific mechanoreceptors such as muscle spindles, Herbst and Merkel corpuscles, or collars of feather follicles but were absent from Golgi tendon organs. In contrast, substance P-positive axons spread out diffusely in muscles and skin, formed loose plexuses, and extended free branches to the endomysium, arteries, superficial dermis, or dermal pulp of feather follicles. The present results show that calbindin D-28k- and substance P-immunoreactive primary sensory neurons provide distinct modes of innervation to selective targets in peripheral tissues. The results suggest a possible correlation between CaBP-expressing nerve endings and rapidly adapting mechanoreceptors.

The chemokines CCL19 and CCL21 and their role in T and dendritic cell biology

Summary : The chemokines CCL19 and CCL21 and their common receptor CCR7 attract antigenpresenting dendritic cells (DCs) and naive T cells into the T zone of secondary lymphoid organs (SLO) and are therefore critically involved in homeostatic T cell recirculation and the initiation of adaptive immune responses. In addition. CCR7 ligands were proposed to mediate T cell exit from neonatal thymus, allowing colonization of T zones in SLOB. The relative contribution of CCL19 and CCL21 to these processes has remained unclear, as they were studied in mouse models lacking either CCR7 or both ligands. The aim of my thesis was to characterize Cc119-' mice and thereby investigate the relative roles of the two CCR7 ligands in development, homeostasis and immune response. The first study addressed the role of CCR7 ligands in DC biology. We found that CCL19 is dispensable for DC migration to lymph nodes and their localization to T zones. Furthermore, a CCL19-deficient environment did not lead to a defect in DC maturation or T cell priming. Therefore, CCL21 is sufficient to mediate CCR7-dependent processes during the initiation of adaptive immune responses. In the second study we investigated how the two CCR7 ligands affect CCR7 expression and function on naive T cells. We found that in SLOB CCR7 is constantly occupied with CCL19 and CCL21, eventually leading to its internalization. The reduced level of free CCR7 on these cells led to diminished ligand sensitivity and consequently impaired chemotactic responses. This effect was reversible by passage through aCCR7 ligand-free environment like the blood circulation. We propose that the different states of ligand sensitivity in SLOB and blood are important to allow for proper T cell recirculation. In the third study the role of CCL19 in neonatal thymus and spleen was analyzed. While neonatal Cc119-!- mice had no defect in thymic egress, we observed reduced T cell accumulation in the spleen but not lymph nodes. We identified reticular stromal cells in the developing white pulp (WP) as the major CCL 19 source. The development of these WP stromal cells as well as their CCL19 expression were dependent on LTalß2+ B cells. In conclusion, we have found that CCL21 can mostly compensate for lack of CCL19 in homeostasis and immunity. In contrast, during development. CCL19 has anon-redundant function for T cell colonization of the spleen.

Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.