Comparative effects of teriparatide and ibandronate on spine bone mineral density (BMD) and microarchitecture (TBS) in postmenopausal women with osteoporosis: a 2-year open-label study.

Treatment effects over 2 years of teriparatide vs. ibandronate in postmenopausal women with osteoporosis were compared using lumbar spine bone mineral density (BMD) and trabecular bone score (TBS). Teriparatide induced larger increases in BMD and TBS compared to ibandronate, suggesting a more pronounced effect on bone microarchitecture of the bone anabolic drug. INTRODUCTION: The trabecular bone score (TBS) is an index of bone microarchitecture, independent of bone mineral density (BMD), calculated from anteroposterior spine dual X-ray absorptiometry (DXA) scans. The potential role of TBS for monitoring treatment response with bone-active substances is not established. The aim of this study was to compare the effects of recombinant human 1-34 parathyroid hormone (teriparatide) and the bisphosphonate ibandronate (IBN), on lumbar spine (LS) BMD and TBS in postmenopausal women with osteoporosis. METHODS: Two patient groups with matched age, body mass index (BMI), and baseline LS BMD, treated with either daily subcutaneous teriparatide (N = 65) or quarterly intravenous IBN (N = 122) during 2 years and with available LS BMD measurements at baseline and 2 years after treatment initiation were compared. RESULTS: Baseline characteristics (overall mean ± SD) were similar between groups in terms of age 67.9 ± 7.4 years, body mass index 23.8 ± 3.8 kg/m(2), BMD L1-L4 0.741 ± 0.100 g/cm(2), and TBS 1.208 ± 0.100. Over 24 months, teriparatide induced a significantly larger increase in LS BMD and TBS than IBN (+7.6 % ± 6.3 vs. +2.9 % ± 3.3 and +4.3 % ± 6.6 vs. +0.3 % ± 4.1, respectively; P < 0.0001 for both). LS BMD and TBS were only weakly correlated at baseline (r (2) = 0.04) with no correlation between the changes in BMD and TBS over 24 months. CONCLUSIONS: In postmenopausal women with osteoporosis, a 2-year treatment with teriparatide led to a significantly larger increase in LS BMD and TBS than IBN, suggesting that teriparatide had more pronounced effects on bone microarchitecture than IBN.

Epithelial to mesenchymal transition in the pathogenesis of uterine malignant mixed Müllerian tumours: the role of ubiquitin proteasome system and therapeutic opportunities.

Malignant mixed Müllerian tumours (malignant mixed mesodermal tumours, MMMT) of the uterus are metaplastic carcinomas with a sarcomatous component and thus they are also called carcinosarcomas. It has now been accepted that the sarcomatous component is derived from epithelial elements that have undergone metaplasia. The process that produces this metaplasia is epithelial to mesenchymal transition (EMT), which has recently been described as a neoplasia-associated programme shared with embryonic development and enabling neoplastic cells to move and metastasise. The ubiquitin proteasome system (UPS) regulates the turnover and functions of hundreds of cellular proteins. It plays important roles in EMT by being involved in the regulation of several pathways participating in the execution of this metastasis-associated programme. In this review the specifi c role of UPS in EMT of MMMT is discussed and therapeutic opportunities from UPS manipulations are proposed.

Concomitant chemoradiotherapy versus acceleration of radiotherapy with or without concomitant chemotherapy in locally advanced head and neck carcinoma (GORTEC 99-02): an open-label phase 3 randomised trial.

BACKGROUND: Concomitant chemoradiotherapy and accelerated radiotherapy independently improve outcomes for patients with locally advanced head and neck squamous-cell carcinoma (HNSCC). We aimed to assess the efficacy and safety of a combination of these approaches. METHODS: In our open-label phase 3 randomised trial, we enrolled patients with locally advanced, stage III and IV (non-metastatic) HNSCC and an Eastern Cooperative Oncology Group performance status of 0-2. We randomly allocated patients centrally with a computer program (with centre, T stage, N stage, and localisation as minimisation factors) in a 1:1:1 ratio to receive conventional chemoradiotherapy (70 Gy in 7 weeks plus three cycles of 4 days' concomitant carboplatin-fluorouracil), accelerated radiotherapy-chemotherapy (70 Gy in 6 weeks plus two cycles of 5 days' concomitant carboplatin-fluorouracil), or very accelerated radiotherapy alone (64·8 Gy [1·8 Gy twice daily] in 3·5 weeks). The primary endpoint, progression-free survival (PFS), was assessed in all enrolled patients. This trial is completed. The trial is registered with ClinicalTrials.gov, number NCT00828386. FINDINGS: Between Feb 29, 2000, and May 9, 2007, we randomly allocated 279 patients to receive conventional chemoradiotherapy, 280 to accelerated radiotherapy-chemotherapy, and 281 to very accelerated radiotherapy. Median follow-up was 5·2 years (IQR 4·9-6·2); rates of chemotherapy and radiotherapy compliance were good in all groups. Accelerated radiotherapy-chemotherapy offered no PFS benefit compared with conventional chemoradiotherapy (HR 1·02, 95% CI 0·84-1·23; p=0·88) or very accelerated radiotherapy (0·83, 0·69-1·01; p=0·060); conventional chemoradiotherapy improved PFS compared with very accelerated radiotherapy (0·82, 0·67-0·99; p=0·041). 3-year PFS was 37·6% (95% CI 32·1-43·4) after conventional chemoradiotherapy, 34·1% (28·7-39·8) after accelerated radiotherapy-chemotherapy, and 32·2% (27·0-37·9) after very accelerated radiotherapy. More patients in the very accelerated radiotherapy group had RTOG grade 3-4 acute mucosal toxicity (226 [84%] of 268 patients) compared with accelerated radiotherapy-chemotherapy (205 [76%] of 271 patients) or conventional chemoradiotherapy (180 [69%] of 262; p=0·0001). 158 (60%) of 265 patients in the conventional chemoradiotherapy group, 176 (64%) of 276 patients in the accelerated radiotherapy-chemotherapy group, and 190 (70%) of 272 patients in the very accelerated radiotherapy group were intubated with feeding tubes during treatment (p=0·045). INTERPRETATION: Chemotherapy has a substantial treatment effect given concomitantly with radiotherapy and acceleration of radiotherapy cannot compensate for the absence of chemotherapy. We noted the most favourable outcomes for conventional chemoradiotherapy, suggesting that acceleration of radiotherapy is probably not beneficial in concomitant chemoradiotherapy schedules. FUNDING: French Ministry of Health.

The response of a Swiss university hospital's palliative care consult team to assisted suicide within the institution.

In January 2006 the Centre Hospitalier Universitaire Vaudois (CHUV), a large university hospital in Lausanne, Switzerland, became the first hospital in Switzerland to allow assisted suicide (AS) in exceptional cases within its walls. However, euthanasia is illegal. This decision has posed several ethical and practical dilemmas for the hospital's palliative care consult service. To address these, the team embarked on a formal process of open dialogue amongst its members with the goal of identifying a collective response and position. This process involved meetings every 4 to 6 weeks over the course of 10 months. An iterative process unfolded. One of the principal dilemmas relates to finding a balance between the team's position against AS and the patient's autonomy and the institution's directive. Although all team members expressed opposition to AS, there were mixed opinions as to whether or not the team members should be present during the act if requested so by patients. Some thought this could be misinterpreted as complicity in the act and could send out mixed messages to the public and other health professionals about palliative care. Others felt that the team's commitment to nonabandonment obliged them to be present even if they did not provide the drug or give any advice or assistance. The implications of nonabandonment are explored, as are several other questions such as whether or not the teams are obliged to provide detailed information on AS when requested by patients.

Maintenance of neuronal expression of calbindin by a muscular extract in cultures of chick dorsal root ganglion cells.

Calbindin D-28K is a calcium-binding protein which is expressed by subpopulations of dorsal root ganglion cells cultured from 10-day-old (E10) chick embryos. After 7 or 10 days of culture, more than 20% of the ganglion cells are immunostained by an anticalbindin-antiserum; however, after 14 days of culture, the proportion drops to 10%. This fall can be prevented by addition of muscle extract to cultures at 10 days. Thus the transitory expression of calbindin-immunoreactivity by responsive sensory neurons would be not only induced but also maintained by a differentiation factor of muscular origin.

Cognitive efficacy of quetiapine in early-onset first-episode psychosis: a 12-week open label trial.

Twenty-three adolescents with psychotic disorders, aged from 13 to 18 years, participated in a 12-week open label trial (17 adolescents completed the study) in order to examine the impact of quetiapine on clinical status and cognitive functions (encompassing processing speed, attention, short-term memory, long-term memory and executive function). An improvement in Clinical Global Impression and Positive and Negative Symptom Scale (P's ≤ 0.001) was observed. In addition, after controlling for amelioration of symptoms, a significant improvement was observed on one executive function (P = 0.044; Trail Making Part B). The remaining cognitive abilities showed stability. In addition, we observed an interaction between quetiapine doses (>300 mg/day or <300 mg/day) and time, where lower doses showed more improvement in verbal short-term memory (P = 0.048), inhibition abilities (P = 0.038) and positive symptoms (P = 0.020). The neuropsychological functioning of adolescents with psychotic disorders remained mainly stable after 12 weeks of treatment with quetiapine. However, lower doses seemed to have a better impact on two components of cognition (inhibition abilities and verbal short-term memory) and on positive symptoms.

Changes of beta-amyloid precursor protein splice patterns in brain cell aggregate cultures.

The splice pattern of beta-amyloid precursor protein (beta-APP) has been studied in a variety of neuronal and glial cells and in brain cell aggregate cultures by the polymerase chain reaction (PCR). The brain-typical pattern, in which beta-APP695 is the dominant form, has been found only in aggregate cultures but not in any of the other cell types including neuronal cell lines. Selective elimination of glial cells from aggregates resulted in increased quantities of beta-APP695, whereas removal of neurons led to a reduction of beta-APP695 and to an elevation of beta-APP751 and beta-APP770. This shift of splice pattern was not observed in cocultures of the neuronal cell line PC 12 with primary astrocytes combined in a variety of cellular ratios. Blood serum, which is an essential component of these cultures, tested on aggregates, did not reduce the amount of beta-APP695 or have any marked effects on splice patterns generally. From these results it is concluded that investigations on brain-typical splicing of beta-APP require primary neurons. Neuronal cell lines may be no suitable model systems. Splicing events favoring production of beta-APP695 may mark an important, very early step of amyloid formation in the brain.

Overuse of upper gastrointestinal endoscopy in a country with open-access endoscopy: a prospective study in primary care.

BACKGROUND: This prospective observational study was aimed at evaluating the appropriateness of use of upper gastrointestinal endoscopy (UGE) in primary care in a country with open access to and high availability of the procedure. METHODS: Outpatients were consecutively included in two clinical settings: Setting A (20 primary care physicians during 4 weeks) and B (university-based outpatient clinic during 3 weeks). In patients undergoing UGE, appropriateness of referral was judged by explicit Swiss criteria developed by the RAND/UCLA panel method. RESULTS: Patient visits (8135) were assessed. Six hundred eleven patients complained of upper gastrointestinal symptoms. Physicians decided to perform UGE in 63 of these patients. Twenty-five (40%) of the endoscopies were rated appropriate, 7 (11%) equivocal, and 31 (49%) inappropriate. Overuse of UGE occurred in 5.1% (setting A: 4.7%; setting B:6.5%; p = 0.39) of the patients who presented with upper gastrointestinal symptoms. The decision to perform UGE in previously untreated dyspeptic patients was the most common clinical situation resulting in overuse. CONCLUSIONS: Inappropriate use of UGE is high in Switzerland. However, to better reflect primary care decision making, overuse should be related not only to patients referred for a medical test, but also to the number of patients who complain of the symptoms that would be investigated by the procedure.

Open to closed system transition traced through the TDIC isotopic signature at the aquifer recharge stage, implications for groundwater 14C dating

14C dating models are limited when considering recent groundwater for which the carbon isotopic signature of the total dissolved inorganic carbon (TDIC) is mainly acquired in the unsaturated zone. Reducing the uncertainties of dating thus implies a better identification of the processes controlling the carbon isotopic composition of the TDIC during groundwater recharge. Geochemical interactions between gas, water and carbonates in the unsaturated zone were investigated for two aquifers (the carbonate-free Fontainebleau sands and carbonate-bearing Astian sands, France) in order to identify the respective roles of CO2 and carbonates on the carbon isotopic signatures of the TDIC; this analysis is usually approached using open or closed system terms. Under fully open system conditions, the seasonality of the 13C values in the soil CO2 can lead to important uncertainties regarding the so-called "initial 14C activity" used in 14C correction models. In a carbonate-bearing unsaturated zone such as in the Astian aquifer, we show that an approach based on fully open or closed system conditions is not appropriate. Although the chemical saturation between water and calcite occurs rapidly within the first metre of the unsaturated zone, the carbon isotopic contents (δ13C) of the CO2 and the TDIC evolve downward, impacted by the dissolution-precipitation of the carbonates. In this study, we propose a numerical approach to describe this evolution. The δ13C and the A 14C (radiocarbon activity) of the TDIC at the base of the carbonate-hearing unsaturated zone depends on (i) the δ13C and the A 14C of the TDIC in the soil determined by the soil CO2, (ii) the water's residence time in the unsaturated zone and (iii) the carbonate precipitation-dissolution fluxes. In this type of situation, the carbonate δ13C-A 14C evolutions indicate the presence of secondary calcite and permit the calculation of its accretion flux, equal to ~ 4.5 ± 0.5 x 10-9 mol grock-1 yr-1. More generally, for other sites under temperate climate and with similar properties to the Astian sands site, this approach allows for a reliable determination of the carbon isotopic composition at the base of the unsaturated zone as the indispensable "input function" data of the carbon cycle into the aquifer.

Prevention of vitamin K deficiency bleeding with three oral mixed micellar phylloquinone doses: results of a 6-year (2005-2011) surveillance in Switzerland.

In 2003, the Swiss guidelines to prevent vitamin K deficiency bleeding (VKDB) were adapted. As two oral doses (2 mg, hour/day 4) of mixed micellar VK preparation had failed to abolish late VKDB, a third dose (week 4) was introduced. This report summarizes the new guidelines acceptance by Swiss pediatricians and the results of a prospective 6-year surveillance to study their influence on the incidence of VKDB. The new guidelines acceptance by Swiss pediatricians was evaluated by a questionnaire sent to all pediatricians of the Swiss Society of Paediatrics. With the help of the Swiss Paediatric Surveillance Unit, the incidence of VKDB was monitored prospectively from July 1, 2005 until June 30, 2011. Over a 6-year period (458,184 live births), there was one case of early and four cases of late VKDB. Overall incidence was 1.09/10(5) (95 % confidence intervals (CI) 0.4-2.6). Late VKDB incidence was 0.87/10(5) (95 % CI 0.24-2.24). All four infants with late VKDB had an undiagnosed cholestasis at the time of bleeding; parents of 3/4 had refused VK prophylaxis, and in 1/4, the third VK dose had been forgotten. Compared with historical control who had received only two oral doses of mixed micellar VK (18 cases for 475,372 live births), the incidence of late VKDB was significantly lower with three oral doses (Chi(2),Yates correction, P = 0.007). CONCLUSION: VKDB prophylaxis with 3 × 2 mg oral doses of mixed micellar VK seems to prevent adequately infants from VKDB. The main risk factors for VKDB in breast-fed infants are parental VK prophylaxis refusal or an unknown cholestasis.

Open elbow arthrolysis for posttraumatic elbow stiffness

Résumé Introduction et objectif : l'articulation du coude est vulnérable à l'enraidissement, en particulier après un traumatisme. Le but de cette étude est d'évaluer les résultats du traitement chirurgical des raideurs post-traumatiques du coude. Type d'étude : étude rétrospective de cohorte. Les patients : dix-huit patients ont été évalués cliniquement par un observateur indépendant dans un intervalle moyen de 16 mois (6 à 43) après avoir subi une arthrolyse ouverte du coude, avec comme indication opératoire une raideur post traumatique du coude. Les traumatismes subis étaient : 11 fractures isolées, 1 luxation isolée, 6 fracture-luxations complexes. Les prises en charge initiales étaient : non-chirurgicales pour 3 patients, résection de la tête radiale pour 1 patient, réduction ouverte et ostéosynthèse pour 14 patients. Les patients ont présenté des types de raideurs mixtes pour la plupart d'entre eux. Type d'intervention : arthrolyse ouverte du coude. Evaluation : la fonction du coude et la satisfaction des patients furent les principales données récoltées. Lacs de l'évaluation, les scores de SECEC (Société Européenne de Chirurgie d'Epaule et du Coude) ont été calculés. Une analyse radiologique a aussi été effectuée. Résultats : trois patients ont présenté des complications postopératoires mineures : 1 déhiscence de plaie, 1 infection sous-cutanée, 1 sérome. Aucune de ces complications n'a eu de répercussion sur le résultat final. Le gain d'amplitude articulaire moyen était de 40°, avec un gain moyen en flexion du coude de 14° (0°-45°), et de 26° en extension (5°-67°) Aucun patient n'a présenté d'instabilité du coude. Il n'y a pas eu de signe de progression d'arthrose sur les clichés radiographiques à la revue. Nous n'avons pas mis en évidence de rapport entre le type de raideur et la voie d'abord sur les résultats. Les patients ayant présentés les raideurs les plus importantes ont bénéficiés des meilleurs résultats en terme de mobilité (p<0.001). Les meilleurs résultats ont été obtenus pour les patients opérés dans la première année aptes le traumatisme initial (p=0.008). Le score SECEC moyen était de 88 points pour les coudes opérés (52-100), et de 96 points pour les coudes sains (88-100). Conclusion : l'arthrolyse ouverte confère de bons résultats pour les patients présentant une raideur post traumatique du coude. Les meilleurs résultats sont obtenus pour les patients présentant une raideur sévère, prise en charge dans l'année qui suit le traumatisme initial.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

Impact of bedside open lung biopsies on the management of mechanically ventilated immunocompromised patients with acute respiratory distress syndrome of unknown etiology.

BACKGROUND: Open lung biopsy (OLB) is helpful in the management of patients with acute respiratory distress syndrome (ARDS) of unknown etiology. We determine the impact of surgical lung biopsies performed at the bedside on the management of patients with ARDS. METHODS: We reviewed all consecutive cases of patients with ARDS who underwent a surgical OLB at the bedside in a medical intensive care unit between 1993 and 2005. RESULTS: Biopsies were performed in 19 patients mechanically ventilated for ARDS of unknown etiology despite extensive diagnostic process and empirical therapeutic trials. Among them, 17 (89%) were immunocompromised and 10 patients experienced hematological malignancies. Surgical biopsies were obtained after a median (25%-75%) mechanical ventilation of 5 (2-11) days; mean (+/-SD) Pao(2)/Fio(2) ratio was 119.3 (+/-34.2) mm Hg. Histologic diagnoses were obtained in all cases and were specific in 13 patients (68%), including 9 (47%) not previously suspected. Immediate complications (26%) were local (pneumothorax, minimal bleeding) without general or respiratory consequences. The biopsy resulted in major changes in management in 17 patients (89%). It contributed to a decision to limit care in 12 of 17 patients who died. CONCLUSION: Our data confirm that surgical OLB may have an important impact on the management of patients with ARDS of unknown etiology after extensive diagnostic process. The procedure can be performed at the bedside, is safe, and has a high diagnostic yield leading to major changes in management, including withdrawal of vital support, in the majority of patients.

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.