Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene.

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.

Biological control of soil-borne pathogens by fluorescent pseudomonads.

Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.

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.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans.

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Genetic differentiation in Silene dioica metapopulations: estimation of spatiotemporal effects in a successional plant species

Silene dioica is a diploid, dioecious, perennial, insect-pollinated herb and part of the deciduous phase of primary succession in Skeppsvik Archipelago, Gulf of Bothnia, Sweden. These islands are composed of material deposited and left underwater by melting ice at the end of the last ice age. A rapid and relatively constant rate of land uplift of 0.9 cm per year continually creates new islands available for colonization by plants. Because the higher deposits appear first, islands differ in age. Because it is possible to estimate the ages of islands and populations of plant species belonging to early stages of succession, the genetic dynamics occurring within an age-structured metapopulation can be investigated in this archipelago. Fifty-two island populations of S. dioica of known ages, sizes, and distances from each other were studied through electrophoretic data. A number of factors increase the degree of genetic differentiation among these island populations relative to an island model at equilibrium. Newly founded populations were more differentiated than those of intermediate age, which suggests that colonization dynamics increase genetic variance among populations. The very old populations, which decrease in size as they approach extinction, were more differentiated than intermediate-aged populations. Isolation by distance occurs in this system. Colonizers are likely to come from more than one source, and the migrant pool model best explains colonization events in the archipelago. Degree of environmental exposure also affects population differentiation.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Mitochondrial DNA variation along an altitudinal gradient in the greater white-toothed shrew, Crocidura russula.

The distribution of mitochondrial control region-sequence polymorphism was investigated in 15 populations of Crocidura russula along an altitudinal gradient in western Switzerland. High-altitude populations are smaller, sparser and appear to undergo frequent bottlenecks. Accordingly, they showed a loss of rare haplotypes, but unexpectedly, were less differentiated than lowland populations. Furthermore, the major haplotypes segregated significantly with altitude. The results were inconsistent with a simple model of drift and dispersal. They suggested instead a role for historical patterns of colonization, or, alternatively, present-day selective forces acting on one of the mitochondrial genes involved in metabolic pathways.

Effects of arbuscular mycorrhizal fungi and phosphorus fertilization on post vitro growth of micropropagated Zingiber officinale roscoe

The rhizomes of Zingiber officinale Roscoe (ginger) are widely used for their medicinal and flavoring properties, whereas the influence of root symbionts on their growth is poorly understood. In this study, the effects of phosphate fertilization and inoculation with a mixture of arbuscular mycorrhizal fungi (AMF) (isolates Glomus clarum RGS101A, Entrophospora colombiana SCT115A and Acaulospora koskei SPL102A) on survival, growth and development of micropropagated ginger were investigated. After transplanting to post vitro conditions, the ginger microplants were subjected to the following treatments: a) AMF mixture, b) P addition (25 mg kg-1), c) AMF + P, and d) non-mycorrhizal control without P addition. After eight months of growth, survival ranged from 86 to 100 % in the AMF and AMF+P treatments versus 71 % survival in control and P treatments. In the AMF, P and AMF+P treatments, the shoot, root and rhizome biomass production were significantly larger than in the control plants. In the non-mycorrhizal control plants the leaf number, leaf area, number of shoots/plants, and shoot length were significantly lower than in the AMF, P and AMF+P treatments. Root colonization ranged from 81 to 93 % and was not affected by P application. The data confirmed the response of several growth variables of micropropagated ginger to mycorrhizal colonization and P addition.

Genetic exchange in an arbuscular mycorrhizal fungus results in increased rice growth and altered mycorrhiza-specific gene transcription.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts with most terrestrial plants. They improve plant nutrition, particularly phosphate acquisition, and thus are able to improve plant growth. In exchange, the fungi obtain photosynthetically fixed carbon. AMF are coenocytic, meaning that many nuclei coexist in a common cytoplasm. Genetic exchange recently has been demonstrated in the AMF Glomus intraradices, allowing nuclei of different Glomus intraradices strains to mix. Such genetic exchange was shown previously to have negative effects on plant growth and to alter fungal colonization. However, no attempt was made to detect whether genetic exchange in AMF can alter plant gene expression and if this effect was time dependent. Here, we show that genetic exchange in AMF also can be beneficial for rice growth, and that symbiosis-specific gene transcription is altered by genetic exchange. Moreover, our results show that genetic exchange can change the dynamics of the colonization of the fungus in the plant. Our results demonstrate that the simple manipulation of the genetics of AMF can have important consequences for their symbiotic effects on plants such as rice, which is considered the most important crop in the world. Exploiting natural AMF genetic variation by generating novel AMF genotypes through genetic exchange is a potentially useful tool in the development of AMF inocula that are more beneficial for crop growth.

Phylogeographic patterning of mtDNA in the widely distributed harvest mouse (Micromys minutus) suggests dramatic cycles of range contraction and expansion during the mid- to late Pleistocene

We examined sequence variation in the mitochondrial cytochrome b gene (1140 bp, n = 73) and control region (842-851 bp, n = 74) in the Eurasian harvest mouse (Micromys minutus (Pallas, 1771)), with samples drawn from across its range, from Western Europe to Japan. Phylogeographic analyses revealed region-specific haplotype groupings combined with overall low levels of inter-regional genetic divergence. Despite the enormous intervening distance, European and East Asian samples showed a net nucleotide divergence of only 0.36%. Based on an evolutionary rate for the cytochrome b gene of 2.4%(.)(site(.)lineage(.)million years)(-1), the initial divergence time of these populations is estimated at around 80 000 years before present. Our findings are consistent with available fossil evidence that has recorded repeated cycles of extinction and recolonization of Europe by M. minutus through the Quaternary. The molecular data further suggest that recolonization occurred from refugia in the Central to East Asian region. Japanese haplotypes of M. minutus, with the exception of those from Tsushima Is., show limited nucleotide diversity (0.15%) compared with those found on the adjacent Korean Peninsula. This finding suggests recent colonization of the Japanese Archipelago, probably around the last glacial period, followed by rapid population growth.

Characterization of gravitational rock slope deformations at different spatial scales based on field, remote sensing and numerical approaches

Rock slope instabilities such as rock slides, rock avalanche or deep-seated gravitational slope deformations are widespread in Alpine valleys. These phenomena represent at the same time a main factor that control the mountain belts erosion and also a significant natural hazard that creates important losses to the mountain communities. However, the potential geometrical and dynamic connections linking outcrop and slope-scale instabilities are often unknown. A more detailed definition of the potential links will be essential to improve the comprehension of the destabilization processes and to dispose of a more complete hazard characterization of the rock instabilities at different spatial scales. In order to propose an integrated approach in the study of the rock slope instabilities, three main themes were analysed in this PhD thesis: (1) the inventory and the spatial distribution of rock slope deformations at regional scale and their influence on the landscape evolution, (2) the influence of brittle and ductile tectonic structures on rock slope instabilities development and (3) the characterization of hazard posed by potential rock slope instabilities through the development of conceptual instability models. To prose and integrated approach for the analyses of these topics, several techniques were adopted. In particular, high resolution digital elevation models revealed to be fundamental tools that were employed during the different stages of the rock slope instability assessment. A special attention was spent in the application of digital elevation model for detailed geometrical modelling of past and potential instabilities and for the rock slope monitoring at different spatial scales. Detailed field analyses and numerical models were performed to complete and verify the remote sensing approach. In the first part of this thesis, large slope instabilities in Rhone valley (Switzerland) were mapped in order to dispose of a first overview of tectonic and climatic factors influencing their distribution and their characteristics. Our analyses demonstrate the key influence of neotectonic activity and the glacial conditioning on the spatial distribution of the rock slope deformations. Besides, the volumes of rock instabilities identified along the main Rhone valley, were then used to propose the first estimate of the postglacial denudation and filling of the Rhone valley associated to large gravitational movements. In the second part of the thesis, detailed structural analyses of the Frank slide and the Sierre rock avalanche were performed to characterize the influence of brittle and ductile tectonic structures on the geometry and on the failure mechanism of large instabilities. Our observations indicated that the geometric characteristics and the variation of the rock mass quality associated to ductile tectonic structures, that are often ignored landslide study, represent important factors that can drastically influence the extension and the failure mechanism of rock slope instabilities. In the last part of the thesis, the failure mechanisms and the hazard associated to five potential instabilities were analysed in detail. These case studies clearly highlighted the importance to incorporate different analyses and monitoring techniques to dispose of reliable and hazard scenarios. This information associated to the development of a conceptual instability model represents the primary data for an integrated risk management of rock slope instabilities. - Les mouvements de versant tels que les chutes de blocs, les éboulements ou encore les phénomènes plus lents comme les déformations gravitaires profondes de versant représentent des manifestations courantes en régions montagneuses. Les mouvements de versant sont à la fois un des facteurs principaux contrôlant la destruction progressive des chaines orogéniques mais aussi un danger naturel concret qui peut provoquer des dommages importants. Pourtant, les phénomènes gravitaires sont rarement analysés dans leur globalité et les rapports géométriques et mécaniques qui lient les instabilités à l'échelle du versant aux instabilités locales restent encore mal définis. Une meilleure caractérisation de ces liens pourrait pourtant représenter un apport substantiel dans la compréhension des processus de déstabilisation des versants et améliorer la caractérisation des dangers gravitaires à toutes les échelles spatiales. Dans le but de proposer un approche plus globale à la problématique des mouvements gravitaires, ce travail de thèse propose trois axes de recherche principaux: (1) l'inventaire et l'analyse de la distribution spatiale des grandes instabilités rocheuses à l'échelle régionale, (2) l'analyse des structures tectoniques cassantes et ductiles en relation avec les mécanismes de rupture des grandes instabilités rocheuses et (3) la caractérisation des aléas rocheux par une approche multidisciplinaire visant à développer un modèle conceptuel de l'instabilité et une meilleure appréciation du danger . Pour analyser les différentes problématiques traitées dans cette thèse, différentes techniques ont été utilisées. En particulier, le modèle numérique de terrain s'est révélé être un outil indispensable pour la majorité des analyses effectuées, en partant de l'identification de l'instabilité jusqu'au suivi des mouvements. Les analyses de terrain et des modélisations numériques ont ensuite permis de compléter les informations issues du modèle numérique de terrain. Dans la première partie de cette thèse, les mouvements gravitaires rocheux dans la vallée du Rhône (Suisse) ont été cartographiés pour étudier leur répartition en fonction des variables géologiques et morphologiques régionales. En particulier, les analyses ont mis en évidence l'influence de l'activité néotectonique et des phases glaciaires sur la distribution des zones à forte densité d'instabilités rocheuses. Les volumes des instabilités rocheuses identifiées le long de la vallée principale ont été ensuite utilisés pour estimer le taux de dénudations postglaciaire et le remplissage de la vallée du Rhône lié aux grands mouvements gravitaires. Dans la deuxième partie, l'étude de l'agencement structural des avalanches rocheuses de Sierre (Suisse) et de Frank (Canada) a permis de mieux caractériser l'influence passive des structures tectoniques sur la géométrie des instabilités. En particulier, les structures issues d'une tectonique ductile, souvent ignorées dans l'étude des instabilités gravitaires, ont été identifiées comme des structures très importantes qui contrôlent les mécanismes de rupture des instabilités à différentes échelles. Dans la dernière partie de la thèse, cinq instabilités rocheuses différentes ont été étudiées par une approche multidisciplinaire visant à mieux caractériser l'aléa et à développer un modèle conceptuel trois dimensionnel de ces instabilités. A l'aide de ces analyses on a pu mettre en évidence la nécessité d'incorporer différentes techniques d'analyses et de surveillance pour une gestion plus objective du risque associée aux grandes instabilités rocheuses.

Neotropical woodlice (isopoda) colonizing leaf-litter of pioneer plants in a coal residue disposal environment

The irregular disposal of coal combustion residues has adverse impacts on terrestrial ecosystems. Pioneer plants and soil invertebrates play an important role in the recovery of these areas. The goal of this study was to investigate the colonization patterns of terrestrial isopods (Oniscidea) in leaf litter of three spontaneous pioneer plants (grass - Poaceae, shrub - Euphorbiaceae, tree - Anarcadiaceae) at sites used for fly ash or boiler slag disposal. The experiment consisted of eight blocks (four per disposal site) of 12 litter bags each (four per plant species) that were randomly removed after 6, 35, 70 or 140 days of field exposure. Three isopod species were found in the litter bags: Atlantoscia floridana (van Name, 1940) (Philosciidae; n = 116), Benthana taeniata Araujo & Buckup, 1994 (Philosciidae; n = 817) and Balloniscus sellowii (Brandt, 1833) (Balloniscidae; n = 48). The isopods colonized the three leaf-litter species equally during the exposure period. However, the pattern of leaf-litter colonization by these species suggests a conflict of objectives between high quality food and shelter availability. The occurrence of A. floridana and the abundance and fecundity of B. taeniata were influenced by the residue type, indicating that the isopods have different degrees of tolerance to the characteristics of the studied sites. Considering that terrestrial isopods are abundant detritivores and stimulate the humus-forming processes, it is suggested that they could have an indirect influence on the soil restoration of this area.

Landscape structure affects dispersal in the greater white-toothed shrew: inference between genetic and simulated ecological distances

Animal dispersal in a fragmented landscape depends on the complex interaction between landscape structure and animal behavior. To better understand how individuals disperse, it is important to explicitly represent the properties of organisms and the landscape in which they move. A common approach to modelling dispersal includes representing the landscape as a grid of equal sized cells and then simulating individual movement as a correlated random walk. This approach uses a priori scale of resolution, which limits the representation of all landscape features and how different dispersal abilities are modelled. We develop a vector-based landscape model coupled with an object-oriented model for animal dispersal. In this spatially explicit dispersal model, landscape features are defined based on their geographic and thematic properties and dispersal is modelled through consideration of an organism's behavior, movement rules and searching strategies (such as visual cues). We present the model's underlying concepts, its ability to adequately represent landscape features and provide simulation of dispersal according to different dispersal abilities. We demonstrate the potential of the model by simulating two virtual species in a real Swiss landscape. This illustrates the model's ability to simulate complex dispersal processes and provides information about dispersal such as colonization probability and spatial distribution of the organism's path

How patch configuration affects the impact of disturbances on metapopulation persistence.

Disturbances affect metapopulations directly through reductions in population size and indirectly through habitat modification. We consider how metapopulation persistence is affected by different disturbance regimes and the way in which disturbances spread, when metapopulations are compact or elongated, using a stochastic spatially explicit model which includes metapopulation and habitat dynamics. We discover that the risk of population extinction is larger for spatially aggregated disturbances than for spatially random disturbances. By changing the spatial configuration of the patches in the system--leading to different proportions of edge and interior patches--we demonstrate that the probability of metapopulation extinction is smaller when the metapopulation is more compact. Both of these results become more pronounced when colonization connectivity decreases. Our results have important management implication as edge patches, which are invariably considered to be less important, may play an important role as disturbance refugia.

Brain metastasis: opportunity for drug development?

PURPOSE OF REVIEW: Brain metastases are a common clinical problem, and only limited treatment options exist. We review recent advances in medical brain metastasis research with a focus on the most common tumor types associated with secondary brain colonization: melanoma, breast cancer and lung cancer. We speculate on opportunities for drug development in patients with brain metastases, both as a treatment of established disease and as an adjuvant and prophylactic strategy. RECENT FINDINGS: BRAF inhibitors and the immunomodulatory anticytotoxic T-lymphocyte-associated antigen 4 antibody ipilimumab have shown clinically meaningful activity in melanoma patients with brain metastases. In breast cancer, current studies on drug treatment of brain metastases are mainly focusing on human epidermal growth factor receptor 2 targeting agents such as lapatinib. Emerging data seem to implicate a potential role of targeted agents including antiangiogenic compounds, pazopanib, and epithelial growth factor receptor inhibitors for prevention of brain metastasis formation in breast cancer or nonsmall cell lung cancer. SUMMARY: Novel drugs are beginning to enter clinical practice for selected patients with brain metastases. The promising findings from recent studies may fuel more research on brain metastases and their optimal drug treatment.