Cirp function and characterisation in RNA and microRNAs

In order to search for novel genes involved in cell proliferation, the hypothesis was that by infecting primary cells with a cDNA library of immortal cells would render immortalizing genes. Consequently it has been discovered CIRP (Cold inducible RNA-binding protein). Mammalian cells exposed to mild hypothermia show a general inhibition of protein synthesis and a concomitant increase in the expression of a small number of cold-shock mRNAs and proteins. Rbm3, another RNA binding protein belonging to the same family, has been postulated to facilitate protein synthesis at mild cold shock. To investigate if the same occurs for CIRP, CIRP was overexpressed in primary cells and protein sintesis was measured. Interestingly, CIRP increased protein synthesis, however, such increase did not involve an increase in the polysome fraction or affected the ribosome profile. In addition, the effect caused by CIRP inhibition or knockdown was also analyzed. Different siRNAs against CIRP were tested. Once checked their efficiency by decreasing CIRP at mRNA and protein levels, proliferation was tested by BrdU, cell number (DAPI) and proliferation curves were performed. Interestingly, CIRP provoke a decreased proliferation in primary cells: MEFs, HMEC; and cancer cells: TERA2 and HeLa. In conclusion, we describe for the first time that CIRP bypasses replicative senescence when over-expressed at physiological temperature (37ºC) by increasing a general protein synthesis. This effect is achieved through ERK1/2 activation in MEFs.The decrease in growth rate found in mammalian cells treated with mild cold stress is not entirely attributable to arrested metabolism. This decrease may also involve an active process in which CIRP and other stress-responsive proteins play a fundamental role in stimulating proliferation. Although most cell proteins are down-regulated or inhibited with cold stress, CIRP is activated to maintain cells in an active proliferative status and its overexpression at 37°C might be potentially oncogenic.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.

3-M syndrome associated with growth hormone deficiency: 18 year follow-up of a patient.

3-M syndrome is a rare autosomal recessive disorder that causes short stature, unusual facial features and skeletal abnormalities. Mutations in the CUL7, OBSL1 and CCDC8 genes could be responsible for 3-M syndrome.Here we describe the growth and evolution of dismorphic features of an Italian boy with 3-M syndrome and growth hormone deficiency (GHD) from birth until adulthood. He was born full term with a very low birth weight (2400 g=-3.36 standard deviation score, SDS) and length (40.0 cm =-6.53 SDS). At birth he presented with a broad, fleshy nose with anteverted nostrils, thick and patulous lips, a square chin, curvilinear shaped eyebrows without synophrys, short thorax and long slender bones. Then, during childhood tall vertebral bodies, hip dislocation, transverse chest groove, winged scapulae and hyperextensible joints became more evident and the diagnosis of 3-M syndrome was made; this was also confirmed by the finding of a homozygous deletion in exon 18 of the CUL7 gene, which has not been previously described.The patient also exhibited severe GHD (GH <5 ng/ml) and from the age of 18 months was treated with rhGH. Notwithstanding the early start of therapy and good compliance, his growth rate was always very low, except for the first two years of treatment and he achieved a final height of 132 cm (-6.42 SDS).

Brain damage in methylmalonic aciduria: 2-methylcitrate induces cerebral ammonium accumulation and apoptosis in 3D organotypic brain cell cultures.

BACKGROUND: Methylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage. METHODS: To explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose-response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3. RESULTS: Among the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose-response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA. CONCLUSION: Surprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Resting heart rate and incident heart failure and cardiovascular mortality in older adults: role of inflammation and endothelial dysfunction: the PROSPER study.

AIMS: Resting heart rate is a promising modifiable cardiovascular risk marker in older adults, but the mechanisms linking heart rate to cardiovascular disease are not fully understood. We aimed to assess the association between resting heart rate and incident heart failure (HF) and cardiovascular mortality, and to examine whether these associations might be attributable to systemic inflammation and endothelial dysfunction. METHODS AND RESULTS: We studied 4084 older adults aged 70-82 years with known cardiovascular risk factors or previous cardiovascular disease, without pre-existing HF or beta-blockers in the PROSPER study. Over a 3.2-year follow-up period, we examined incident HF hospitalization and cardiovascular mortality according to resting heart rate, along with C-reactive protein (CRP), interleukin-6 (IL-6), tissue plasminogen activator (tPA), and von Willebrand factor (vWf). Mean heart rate was 67 b.p.m. for men and 70 b.p.m. for women. CRP, IL-6, tPA, and vWf levels were all positively correlated with heart rate. After multivariate adjustment, heart rate was associated with HF hospitalization [hazard ratio (HR) 1.78 for highest vs. lowest distribution third, 95% confidence interval (CI) 1.21-2.63, P= 0.003] and cardiovascular mortality (HR 1.74, 95% CI 1.23-2.47, P= 0.002). Further adjustment for both IL-6 and vWf levels decreased HR to 1.60 (95% CI 1.08-2.38, P= 0.020) for HF and to 1.50 (95% CI 1.04-2.15, P= 0.028) for cardiovascular mortality. CONCLUSION: Increased heart rate is associated with increased systemic inflammation and endothelial dysfunction. These factors are likely to contribute to, but do not fully explain, the risk of HF and cardiovascular death associated with increased heart rate in older age.

Identification et caractérisation de gènes suppresseurs de tumeurs dans le glioblastome

Introduction: Glioblastoma (WHO Grade IV glioma) is the most frequent and most¦malignant primary tumor of the brain. With a mean survival of 15 months despite¦multidisciplinary management combining surgery, chemo- and radiotherapy, the prognosis¦is poor. Different studies measured a down-regulation of Wnt Inhibitory Factor 1 (WIF1)¦expression in a majority of gliobastoma due to genetic and epigenetic regulation. Recently,¦a focus on chromosome 12 identified WIF1 as a potential tumor suppressor gene. In¦previous results, transfected glioblastoma cells with ectopic expression of WIF1 had a¦decreased growth rate and adopted a senescence-like phenotype. In this report, we first¦investigated the effect of WIF1 re-expression in glioblastoma cell lines to see if Wnt¦inhibition by WIF1 can lead to senescence. To look further, we assessed p21 and c-Myc¦expression. p21 has a key role in senescence onset and is directly inhibited by c-Myc,¦itself a target of Wnt-pathway. We thus looked if a variation of expression of these genes is¦triggered by WIF1 activity. Finally, as autophagy is thought to play a role in senescence¦onset, we analyzed the expression of different autophagy genes. We therefore looked for¦an association between autophagy activity and senescent phenotype in WIF1-¦overexpressing cell lines.¦Methods: WIF1-overexpressing clones were selected after transfection of stable¦glioblastoma cell lines. Analysis were made through quantitative Polymerase Chain¦Reaction (qPCR), Fluorescence-activated Cell Sorting (FACS) and histochemistry.¦IGFBP7 and ALDH1A3 have been selected to reflect senescence. ATG5, ATG7 and ULK3¦have been selected to reflect autophagy activity.¦Results: Using FACS analysis, we found a higher percentage of large cells with increased¦granularity amongst WIF1-overexpressing cell lines, which are characteristics of¦senescence. In addition, histochemistry showed a higher percentage of multi-nucleated,¦beta-galactosidase positive cells in the same cell lines. An increased expression of genes¦associated with senescence was found as well. All characteristics were correlated with¦levels of WIF1 expression. We did not find any association between p21 and WIF1¦expression. No correlation between WIF1 and c-Myc expression was noticed either. In one¦of the two cell lines analyzed, the expression of autophagy genes showed some¦correlation with expression of WIF1 and expression of genes associated with senescence.¦Discussion: After investigations and characterizations on multiple levels, we have¦evidence for a senescence phenotype upon WIF1-overexpressing cell lines. This gives a¦role to Wnt pathway in the tumorigenicity of glioblastoma. Further experiments are¦required to investigate how Wnt inhibition leads to senescence. The role of autophagy in¦our senescent cells is here still unclear. Some correlations can be found, letting us think¦that there is indeed some involvement of autophagy. However, it is yet to soon to explain¦this relationship. Further experiments are required again to confirm the preliminary results¦and analyze the variations of autophagy activity within time.

Cyclic AMP induces differentiation in vitro of human melanoma cells.

Treating human melanoma lines with dibutyryl adenosine 3':5'-cyclic monophosphate (dbc AMP) resulted in morphologic changes associated with the altered expression of cell surface antigens. After treatment, cells developed long cellular projections characteristic of mature melanocytes and showed the presence of an increased number of Stage II premelanosomes. In addition, induction of melanin synthesis, detected as brown perinuclear pigmentation, was observed. The AMP further drastically reduced the growth rate of the five melanoma cell lines that were tested. The influence of dbc AMP was completely reversible 3 days after the agent was removed from the culture medium. The antigenic phenotype of the melanoma lines was compared before and after dbc AMP treatment. This was done with four monoclonal antibodies directed against major histocompatibility complex (MHC) Class I and II antigens and 11 monoclonal antibodies defining eight different melanoma-associated antigenic systems. Treatment with dbc AMP reduced the expression of human leukocyte antigen (HLA)-ABC antigens and beta-2-microglobulin in five of five melanoma lines. In the two HLA-DR-positive cell lines dbc AMP reduced the expression of this antigen in one line and enhanced it in the other. No induction of HLA-DR or HLA-DC antigens was observed in the Class II negative cell lines. Furthermore, dbc-AMP modulated the expression of the majority of the melanoma antigenic systems tested. The expression of a 90-kilodalton (KD) antigen, which has been found to be upregulated by interferon-gamma, was markedly decreased in all the five cell lines. A similar decrease in the expression of the high molecular weight proteoglycan-associated antigen (220-240 KD) was observed. The reduced expression of Class I and II MHC antigens as well as the altered expression of the melanoma-associated antigens studied were shown to be reversible after dbc AMP was removed. Our results collectively show that the monoclonal antibody-defined melanoma-associated molecules are linked to differentiation. They could provide useful tools for monitoring the maturation of melanomas in vivo induced by chemical agents or natural components favoring differentiation.

Age over 40 years increases the failure rate of non-operative management of blunt splenic injuries

Objective: Non-operative management (NOM) of blunt splenic injuries (BSI) is nowadays considered the standard treatment. The study aimed to determine the criteria applied for NOM and to identify risk factors for its failure. Methods: Review of all adult patients with BSI treated at the University Hospital Bern, Switzerland, between 2000 and 2008. Results: There were 206 patients (146 men, 70·9%) with a mean age of 38·2 ± 19·1 years and an Injury Severity Score of 30·9 ± 11·6. The American Association for the Surgery of Trauma classification of the splenic injury was: grade I, n=43 (20·9%); grade II, n=52 (25·2%); grade III, n=60 (29·1%); grade IV, n=42 (20·4%) and grade V, n=9 (4·4%). 47 patients (22·8%) required immediate surgery. Five or more units of red cell transfusions (P<0·001), Glasgow Coma Scale<11 (P=0·009) and age ≥55 years (P=0·038) were associated with primary operative management (OM). 159 patients (77·2%) qualified for NOM, which was successful in 89·9% (143/159). The overall splenic salvage rate was 69·4% (143/206). Multivariate analysis found age ≥40 years to be the only factor independently related to the failure of NOM (P=0·001). Conclusion: Advanced age is associated with an increased failure rate ofNOM in patients with BSI.

Social capital, government expenditures, and growth

Countries with greater social capital have higher economic growth. We show that socialcapital is also highly positively correlated across countries with government expenditureon education. We develop an infinite-horizon model of public spending and endogenousstochastic growth that explains both facts through frictions in political agency whenvoters have imperfect information. In our model, the government provides servicesthat yield immediate utility, and investment that raises future productivity. Voters aremore likely to observe public services, so politicians have electoral incentives to underprovidepublic investment. Social capital increases voters' awareness of all governmentactivity. As a consequence, both politicians' incentives and their selection improve.In the dynamic equilibrium, both the amount and the efficiency of public investmentincrease, permanently raising the growth rate.

An eceriferum locus, cer-zv, is associated with a defect in cutin responsible for water retention in barley (Hordeum vulgare) leaves.

Drought limits plant growth and threatens crop productivity. A barley (Hordeum vulgare) ethylene imine-induced monogenic recessive mutant cer-zv, which is sensitive to drought, was characterized and genetically mapped in the present study. Detached leaves of cer-zv lost 34.2 % of their initial weight after 1 h of dehydration. The transpiration was much higher in cer-zv leaves than in wild-type leaves under both light and dark conditions. The stomata of cer-zv leaves functioned normally, but the cuticle of cer-zv leaves showed increased permeability to ethanol and toluidine blue dye. There was a 50-90 % reduction in four major cutin monomers, but no reduction in wax loads was found in the cer-zv mutant as compared with the wild type. Two F(2) mapping populations were established by the crosses of 23-19 × cer-zv and cer-zv × OUH602. More polymorphisms were found in EST sequences between cer-zv and OUH602 than between cer-zv and 23-19. cer-zv was located in a pericentromeric region on chromosome 4H in a 10.8 cM interval in the 23-19 × cer-zv map based on 186 gametes tested and a 1.7 cM interval in the cer-zv × OUH602 map based on 176 gametes tested. It co-segregated with EST marker AK251484 in both maps. The results indicated that the cer-zv mutant is defective in cutin, which might be responsible for the increased transpiration rate and drought sensitivity, and that the F(2) of cer-zv × OUH602 might better facilitate high resolution mapping of cer-zv.

Externalities and interdependent growth: Theory and evidence

I formulate and estimate a model of externalities within countriesand technological interdependence across countries. I find that externalreturns to scale to physical capital within countries are 8 percent; thata 10 percent increase of total factor productivity of a country's neighborsraises its total factor productivity by 6 percent; and that a 2 percentannual growth rate of labor productivity can be explained as an endogenousresponse to an exogenous 0.2 percent annual growth rate of total factorproductivity in the steady--state.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

ABSTRACT: BACKGROUND: Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. RESULTS: Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000. CONCLUSIONS: A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Habitat-quality effects on metapopulation dynamics in greater white-toothed shrews, Crocidura russula.

The effects of patch size and isolation on metapopulation dynamics have received wide empirical support and theoretical formalization. By contrast, the effects of patch quality seem largely underinvestigated, partly due to technical difficulties in properly assessing quality. Here we combine habitat-quality modeling with four years of demographic monitoring in a metapopulation of greater white-toothed shrews (Crocidura russula) to investigate the role of patch quality on metapopulation processes. Together, local patch quality and connectivity significantly enhanced local population sizes and occupancy rates (R2 = 14% and 19%, respectively). Accounting for the quality of patches connected to the focal one and acting as potential sources improved slightly the model explanatory power for local population sizes, pointing to significant source-sink dynamics. Local habitat quality, in interaction with connectivity, also increased colonization rate (R2 = 28%), suggesting the ability of immigrants to target high-quality patches. Overall, patterns were best explained when assuming a mean dispersal distance of 800 m, a realistic value for the species under study. Our results thus provide evidence that patch quality, in interaction with connectivity, may affect major demographic processes.

Pollution stress on aquatic microbial communities

Thousands of chemical compounds enter the natural environment but many have unknown effects and consequences, in particular at low concentrations. This thesis work contributes to our understanding of pollution effects by using bacteria as test organisms. Bacteria are important for this question because some of them degrade and transform pollutants into less harmful compounds, but secondly because they themselves can be inhibited in their reproduction by exposure to toxic compounds. When inhibitory effects occur this may change the composition of the microbial com¬munity in the long run, leading to altered or diminished ecosystem services by those communities. As a result chemicals of anthropogenic origin may accumulate and per¬sist in the environment, and finally, affect higher organisms as well. In addition to acquiring basic understanding of pollutant effects at low concentrations on bacterial communities an applied goal of this thesis work was to develop bacteria-based tests to screen new organic chemicals for toxicity and biodégradation. In the first part of this work we developed a flow cytometry-based assay on SYT09 plus ethidium-bromide or propidium-iodide stained cells of Pseudomonas ûuorescens exposed or not to a variety of pollutants under oligotrophic growth conditions. Flow cytometry (FC) allows fast and accurate counting of bacterial cells under simul¬taneous assessment of their physiological state, in particular in combination with different fluorescent dyes. Here we employed FC and fluorescent dyes to monitor the effect that pollutants may exert on Pseudomonas ûuorescens SV3. First we designed an oligotrophic growth test, which enabled us to follow population growth at low densities (104 - 10 7 cells per ml) using 0.1 mM sodium acetate as carbon source. Cells in the oligotrophic milieu were then exposed or not to a variety of common pollutants, such as 2-chlorobiphenyl (2CBP), naphthalene (NAH), 4-chlorophenol (4CP), tetradecane (TD), mercury chloride (HgCl2) or benzene, in different dosages. Exposed culture samples were stained with SYT09 (green fluorescent dye binding nucleic acids, generally staining all cells) in combination with propidium iodide (PI) or ethidium bromide (EB), both dyes being membrane integrity indicators. We ob- served that most of the tested compounds decreased population growth in a dosage- dependent manner. SYT09/PI or SYT09/EB staining then revealed that chemical exposure led to arisal of subpopulations of live and injured or dead cells. By modeling population growth on the total cell numbers in population or only the subpopulation of live cells we inferred that even in stressed populations live cells multiply at rates no different to unexposed controls. The net decrease in population growth would thus be a consequence of more and more cells being not able to multiply at all, rather than all cells multiplying at slower rates. In addition, the proportion of injured cells correlated to the compound dosage. We concluded that the oligotrophic test may be useful to asses toxicity of unknown chemicals on a variety of model bacteria. Mul¬tiple tests can be run in parallel and effects are rapidly measured within a period of 8 hours. Interestingly, in the same exposure tests with P. fluorescens SV3 we observed that some chemicals which did not lead to a reduction of net population growth rates did cause measurable effects on live cells. This was mainly observed in cells within the live subpopulation as an increase of the EB fluorescence signal. We showed that SYT09/EB is a more useful combination of dyes than SYT09/PI because PI fluorescence tend to increase only when cells are effectively dead, but not so much in live cells (less then twofold). In contrast, EB geometric mean fluorescence in live cells increased up to eightfold after exposure to toxic compounds. All compounds even at the lowest concentration caused a measurable increase in EB geometric mean fluorescence especially after 2 h incubation time. This effect was found to be transient for cells exposed to 2CBP and 4CP, but chronic for cells incubated with TD and NAH (ultimately leading to cell death). In order to understand the mechanism underlying the observed effects we used known membrane or energy uncouplers. The pattern of EB signal increase in chemical-exposed populations resembled mostly that of EDTA, although EB fluorescence in EDTA-treated or pasteurized cells was even higher than after exposure to the four test chemicals. We conclude that the ability of cells to efflux EB under equilibrium conditions is an appropriate measure for the potential of a chemical to exert toxicity. Since most bacterial species possess efflux systems for EB that all require cellular energy, our test should be more widely relevant to infer toxicity effects of chemical exposure on the physiological status of the bacterial cell. To better understand the effect of toxicant exposure on efflux defense systems, we studied 2-hydroxybiphenyl toxicity to Pseudomonas azeiaica HBP1. We showed that 2-HBP exerts toxicity even to P. azelaica HBP1, but only at concentrations higher than 0.5 mM. Above this concentration transient loss of membrane polarization and integrity occurred, which we conclude from staining of growing cells with fluorescent dyes. Cells finally recover and resume growth on 2HBP. The high resistance of P. azelaica HBP1 to 2-HBP was found to be the result of an efficient MexABOprM- type efflux pump system counteracting passive influx of this compound into the membrane and cellular interior. Mutants with disrupted mexA, mexB and oprM genes did no longer grow on 2-HBP at concentrations above 100 μΜ, whereas below this concentration we found 2-HBP-concentration dependent decrease of growth rate. The MexAB-OprM system in P. azeiaica HBP1 is indeed an efflux pump for ethidium bromide as well. By introducing gfp reporter fusions responsive to intracellular 2- HBP concentrations into HBP1 wild-type or the mutants we demonstrated that 2HBP enters into the cells in a similar way. In contrast, the reporter system in the wild-type cells does not react to 2-HBP at an outside concentration of 2.4 μΜ, whereas in mutant cells it does. This suggests that wild-type cells pump 2-HBP to the outside very effectively preventing accumulation of 2-HBP. 2HBP metabolism, therefore, is not efficient enough to lower the intracellular concentration and prevent toxicity. We conclude that P. azelaica HBP1 resistance to 2-HBP is mainly due to an efficient efflux system and that 2HBP in high concentrations exerts narcotic effects on the bacterial membrane. In the part of this thesis, we investigated the possibilities of bacteria to degrade pollutants at low concentrations (1 mg per L and below). As test components we used 2-hydroxybiphenyl, antibiotics and a variety of fragrances, many of which are known to be difficult to biodegrade. By using accurate counting of low numbers of bacterial cells we could demonstrate that specific growth on these compounds is possible. We demonstrated the accuracy of FC counting at low cell numbers (down to 103 bacterial cells per ml). Then we tested whether bacterial population growth could be specifically monitored at the expense of low substrate concentrations, us¬ing P. azelaica HBP1. A perfect relationship was found between growth rate, yield and 2-HBP concentrations in the range of 0.1 up to 5 mg per L. Mixing P. azelaica within sludge, however, suggested that growth yields in a mixed community can be much lower than in pure culture, perhaps because of loss of metabolic intermediates. We then isolated new strains from activated sludge using 2-HBP or antibiotics (Nal, AMP, SMX) at low concentrations (0.1-1 mg per L) as sole carbon and energy sub¬strate and PAO microdishes. The purified strains were then examined for growth on their respective substrate, which interestingly, showed that all strains can not with¬stand higher than 1 or 10 mg per L concentrations of target substrate. Thus, bacteria must exist that contribute to compound degradation at low pollutant concentrations but are inhibited at higher concentrations. Finally we tested whether specific biomass growth (in number of cells) at the expense of pollutants can also be detected with communities as starting material. Hereto, we focused on a number of fragrance chemicals and measured community biomass increase by flow cytometry cell counting on two distinct starter communities: (i) diluted Lake Geneva water, and dilute activated sludge from a wastewater treatment plant. We observed that most of the test compounds indeed resulted in significant biomass increase in the starter community compared to a no-carbon added control, but activated sludge and lake Geneva water strongly differed (almost mutually ex¬clusive) in their capacity to degrade the test chemicals. In two cases for activated sludge the same type of microbial community developed upon compound exposure, as concluded from transcription fragment length polymorphism analysis on community purified and PCR amplified 16S rRNA gene fragments. To properly test compound biodegradability it is thus important to use starter communities of different origin. We conclude that FC counting can be a valuable tool to screen chemicals for their biodegradability and toxicity. - Des milliers de produits chimiques sont libérés dans l'environnement mais beaucoup ont des effets inconnus, en particulier à basses concentrations. Ce travail de thèse contribue à notre comprehension des effets de la pollution en utilisant des bacteries comme des organismes-tests. Les bacteries sont importantes pour etudier cette ques¬tion car certaines d'entre elles peuvent degrader ou transformer les polluants, mais également parce qu'elles-mmes peuvent tre inhibees dans leur reproduction après avoit ete exposees à ces composes toxiques. Quand des effets inhibiteurs ont lieu, la composition de la communauté microbienne peut tre changee à long terme, ce qui mène à une reduction du service d'ecosystème offert par ces communautés. En consequence, après leur liberation dans l'environnement, les produits chimiques d'origine anthropogenique peuvent soit s'y accumuler et per¬sister, exerant ainsi des effets encore inconnus sur les organismes vivants. En plus d'acquérir des connaissances de base sur les effets des polluants à basses concentra¬tions sur les communautés microbiennes, un but applique de cette thèse était de développer des tests bases sur les bacteries afin d'identifier de nouveau composes pour leur toxicité ou leur biodégradation. Dans la première partie de ce travail, nous avons developpe un test base sur la cytometrie de flux (FC) sur des cellules de Pseudomonas fluorescens colorees par du bromure d'ethidium ou de l'iodure de propidium et exposees ou non à une palette de polluants sous des conditions de croissance oligotrophique. La cytometrie de flux est une technique qui connaît de nombreuses applications dans la microbiologie environ¬nementale. Cela est principalement du au fait qu'elle permet un comptage rapide et precis ainsi que l'évaluation de l'état physiologique, en particulier lorsqu'elle est combinée h des colorations fluorescentes. Ici, nous avons utilise la technique FC et des colorants fluorescents afin de mesurer l'effet que peuvent exercer certains pollu¬ants sur Pseudomonas ûuorescens SV3 . D'abord nous avons conu des tests oligo- trophiques qui nous permettent de suivre la croissance complète de cellules en culture h des densites faibles (104 -10 7 cellules par ml), sur de l'acetate de sodium à 0.1 mM, en presence ou absence de produits chimiques (2-chlorobiphenyl (2CBP), naphthalène (NAH), 4-chlorophenol (4CP), tetradecane (TD), chlorure de mercure(II) (HgCl2)) à différentes concentrations. Afin de montrer le devenir des bacteries tant au niveau de la cellule individuelle que celui de la population globale, après exposition à des series de composes chimiques, nous avons compte les cellules colorees avec du SYT09 (col¬orant fluorescent vert des acides nucléiques pour la discrimination des cellules par rapport au bruit de fond) en combinaison avec l'iodure de propidium (PI) ou le bromure d'ethidium (EB), indicateurs de l'intégrité de la membrane cellulaire avec FC. Nous avons observe que de nombreux composes testes avaient un effet sur la croissance bacterienne, resultant en une baisse du taux de reproduction de la pop¬ulation. En outre, la double coloration que nous avons utilisee dans cette etude SYT09/PI ou SYT09/EB a montre que les produits chimiques testes induisaient une reponse heterogène des cellules dans la population, divisant celle-ci en sous- populations "saine", "endommagee" ou "morte". Les nombres de cellules à partir du comptage et de la proportion de celles "saines" et "endommagees/mortes" ont ensuite ete utilises pour modeliser la croissance de P. ûuorescens SV3 exposee aux produits chimiques. La reduction nette dans la croissance de population est une consequence du fait que de plus en plus de cellules sont incapables de se reproduire, plutt que du fait d'une croissance plus lente de l'ensemble de la population. De plus, la proportion de cellules endommagees est correllee au dosage du compose chimique. Les résultats obtenus nous ont permis de conclure que le test oligotrophique que nous avons developpe peut tre utilise pour l'évaluation de la toxicité de produits chimiques sur différents modèles bacteriens. Des tests multiples peuvent tre lances en parallèle et les effets sont mesures en l'espace de huit heures. Par ailleurs, nous en déduisons que les produits chimiques exercént un effet sur la croissance des cellules de P. ûuorescens SV3, qui est heterogène parmi les cellules dans la population et depend du produit chimique. Il est intéressant de noter que dans les mmes tests d'exposition avec P. ûuorescens SV3, nous avons observe que certains composes qui n'ont pas conduit à une reduction du taux de la croissance nette de la population, ont cause des effets mesurables sur les cellule saines. Ceci a ete essentiellement observe dans la portion "saine" des cellules en tant qu'augmentation du signal de la fluorescence de 1ΈΒ. D'abord nous avons montre que SYT09/EB était une com¬binaison de colorants plus utile que celle de SYT09/PI parce que la fluorescence du PI a tendance à augmenter uniquement lorsque les cellules sont effectivement mortes, et non pas dans les cellules saines (moins de deux fois plus). Par opposi¬tion, la fluorescence moyenne de l'EB dans les cellules saines augmente jusqu'à huit fois plus après exposition aux composes toxiques. Tous les composes, mme aux plus basses concentrations, induisent une augmentation mesurable de la fluorescence moy¬enne de 1ΈΒ, plus particulièrement après deux heures d'incubation. Cet effet s'est revele tre transitoire pour les cellules exposees aux 2CNP et 4CP, mais est chro¬nique pour les cellules incubees avec le TD et le NAH (entranant la mort cellulaire). Afin de comprendre les mécanismes qui sous-tendent les effets observes, nous avons utilise des decoupleurs d'energie ou de membrane. L'augmentation du signal EB dans les populations causee par des produits chimiques ressemblait à celle exerce par le chelateur des ions divalents EDTA. Cependant, les intensités du signal EB des cellules exposees aux produits chimiques testees n'ont jamais atteint les valeurs des cellules traitees avec l'EDTA ou pasteurises. Nous en concluons que le test oli- gotrophique utilisant la coloration (SYT09/)EB des cellules exposees ou non à un produit chimique est utile afin d'evaluer l'effet toxique exerce par les polluants sur la physiologie bacterienne. Afin de mieux comprendre la reaction d'un système de defense par pompe à efflux après exposition à une toxine, nous avons étudié la toxicité du 2-hydroxybiphenyl (2-HBP) sur Pseudomonas azeiaica HBP1. Nous avons montre que le 2-HBP exerce une toxicité mme sur HBP1, mais uniquement à des concentrations supérieures à 0.5 mM. Au-dessus de cette concentration, des pertes transitoires d'intégrité et de polarization membranaire ont lieu, comme cela nous a ete montre par coloration des cellules en croissance. Les cellules sont finalement capables de se rétablir et de reprendre leur croissance sur 2-HBP. La forte resistance de P. azeiaica HBP1 h 2-HBP physiologie bacterienne s'est revele tre le résultat d'un système de pompe h efflux de type MexABOprM qui contre-balance l'influx passif de ce compose h travers la membrane. Nous avons montre, en construisant des mutants avec des insertions dans les gènes mexA, mexB and oprM et des fusions avec le gène rapporteur gfp, que l'altération de n'importe quelle partie du système d'efflux conduisait à accroître l'accumulation de 2-HBP dans la cellule, en comparaison avec la souche sauvage HBP1, provoquant une diminution de la resistance au 2-HBP ainsi qu'une baisse du taux de reproduction des cellules. Des systèmes d'efflux similaires sont répandus chez de nombreuses espèces bactériennes. Ils seraient responsables de la resistance aux produits chimiques tels que les colorants fluorescents (bromure d'ethidium) et des antibiotiques. Nous concluons que la resistance de P. azelaica HBP1 à 2-HBP est principalement due à un système d'efflux efficace et que 2-HBP, à des concentrations elevees, exerce un effet deletère sur la membrane bacterienne. En se basant sur le comptage des cellules avec la FC, nous avons developpe ensuite une methode pour evaluer la biodegradabilite de polluants tels que le 2-HBP ainsi que les antibiotiques (acide nalidixique (Nal), ampicilline (AMP) ou sulfamethoxazole (SMX)) à de faibles concentrations lmg par L et moins), par le suivi de la croissance spécifique sur le compose de cultures microbiennes pures et mixtes. En utilisant un comptage precis de faibles quantités de cellules nous avons pu demontrer que la croissance spécifique sur ces composes est possible. Nous avons pu illustrer la precision du comptage par cytometrie de flux à faible quantité de cellules (jusqu'à 10 3 cellules par ml). Ensuite, nous avons teste s'il était possible de suivre dynamiquement la croissance de la population de cellules sur faibles concentrations de substrats, en utilisant P. azelaica HBP1. Une relation parfaite a ete trouvee entre le taux de croissance, le rendement et les concentrations de 2-HBP (entre 0.1 et 5 mg par L). En mélangeant HBP1 à de la boue active, nous avons pu montrer que le rendement en communauté mixtes pouvait tre bien inférieur qu'en culture pure. Ceci étant peut tre le résultat d'une perte d'intermédiaires métaboliques. Nous avons ensuite isole de nouvelles souches à partir de la boue active en utilisant le 2-HBP ou des antibiotiques (Nal, AMP, SMX) h basses concentrations (0.1-1 mg par L) comme seules sources de carbone et d'energie. En combinaison avec ceci, nous avons également utilise des microplaques PAO. Les souches purifiees ont ensuite ete examinees pour leurs croissances sur leurs substrats respectifs. De faon intéressante, toutes ces souches ont montre qu'elles ne pouvaient pas survivre à des concentrations de substrats supérieures à 1 ou 10 mg par L. Ainsi, il existe des bacteries qui contribuent à la degradation de composes à basses concentrations de polluant mais sont inhibes lorsque ces concentrations deviennent plus hautes. Finalement, nous avons cherche à savoir s'il est possible de detecter une croissance spécifique à une biomasse au depend d'un polluant, en partant d'une communauté microbienne. Ainsi, nous nous sommes concentre sur certains composes et avons mesure l'augmentation de la biomasse d'une communauté grce à la cytometrie de flux. Nous avons compte deux communautés de depart distinctes: (i) une dilution d'eau du Lac Léman, et une dilution de boue active d'une station d'épuration. Nous avons observe que la plupart des composes testes ont entrane une augmentation de la biomasse de depart par rapport au control sans addition de source de carbone. Néanmoins, les échantillons du lac Léman et de la station d'épuration différaient largement (s'excluant mutuellement l'un l'autre) dans leur capacité à degrader les composes chimiques. Dans deux cas provenant de la station d'épuration, le mme type de communauté microbienne s'est developpe après exposition aux composes, comme l'a démontré l'analyse TRFLP sur les fragments d'ARN 16S purifie de la communauté et amplifie par PCR. Afin de tester correctement la biodegradabilite d'un compose, il est donc important d'utiliser des communautés de depart de différentes origines Nous en concluons que le comptage par cytometrie de flux peut tre un outil de grande utilité pour mettre en valeur la biodegradabillite et la toxicité des composes chimiques.