Assessment of particulate exposure and surface characteristics in association with urinary levels of 8-hydroxy-2'-deoxyguanosine, considered as marker of oxidative stress

Epidemiological studies have demonstrated that exposure to fine particles is associated to adverse health effects, including cancer, respiratory and cardiovascular diseases. However, mechanisms by which particles induce health effects remain unclear. According to one of the most investigated hypotheses, particles cause adverse effects through the production of reactive oxygen species (ROS), which are very hazardous compounds able to attack directly biological structures, including the DNA strand or the lipid bilayer of the cells. If the defense mechanisms, constituted of antioxidants, are not able to counter ROS, then these compounds will cause in the body a range of oxidation reactions called "oxidative stress". The aim of the present research project was to better understand mechanisms by which exposure to fine particles induces oxidative stress. The first point of this project was to check whether exposure to high levels of fine particles is directly linked to oxidative stress, and whether this oxidative stress is accompanied by the activation of the defense mechanisms (antioxidants). The second point was to study the role played by the particle surface characteristics in the oxidative stress process. For that purpose, a study was conducted in bus depots with the participation of 40 mechanics. First, occupational exposure to particles (PM4) and to other pollutants (NOx, O3) was measured over a two-day period. Then, urine samples of mechanics were collected in order to measure levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and antioxidants. 8OHdG is a molecule formed by the oxidation of DNA and allowing to assess the oxidative stress status of the mechanics. Finally, particles were collected on filters, and functional groups located on the particle surface were analyzed in the laboratory using a Knudsen flow reactor. This technique allows not only to quantify functional groups on the particle surface, but also to measure the reaction kinetics. Results obtained during the field campaign in bus depots showed that mechanics were exposed to rather low levels of PM4 (20-85 μg/m3) and of pollutants (NOx: 100-1000 ppb; O3: <15 ppb). However, despite this low exposure, urinary levels of the oxidative stress biomarker (8OHdG) increased significantly for non-smoking workers over a two-day period of shift. This oxidative stress was accompanied by an increase of antioxidants, indicating the activation of defense mechanisms. On the other hand, the analysis of functional groups on the particle surface showed important differences, depending on the workplace, the date and the activities of workers. The particle surface contained simultaneously antagonistic functional groups which did not undergo internal reactions (such as acids and bases), and was usually characterized by a high density of carbonyl functions and a low density of acidic sites. Reaction kinetics measured using the Knudsen flow reactor pointed out fast reactions of oxidizable groups and slow reactions of acidic sites. Several exposure parameters were significantly correlated with the increase of the oxidative stress status: the presence of acidic sites, carbonyl functions and oxidizable groups on the particle surface; reaction kinetics of functional groups on the particle surface; particulate iron and copper concentrations; and NOx concentration.

Role of Particle Shape on the Stress Propagation in Granular Packings

We present an experimental and numerical study on the influence that particle aspect ratio has on the mechanical and structural properties of granular packings. For grains with maximal symmetry (squares), the stress propagation in the packing localizes forming chainlike forces analogous to the ones observed for spherical grains. This scenario can be understood in terms of stochastic models of aggregation and random multiplicative processes. As the grains elongate, the stress propagation is strongly affected. The interparticle normal force distribution tends toward a Gaussian, and, correspondingly, the force chains spread leading to a more uniform stress distribution reminiscent of the hydrostatic profiles known for standard liquids

Validation of the antenatal perceived stress inventory

This article presents the Antenatal Perceived Stress Inventory. The originality of this scale is to assess the impact of events experienced during pregnancy on the stress perceived by mothers. Scale validation was performed using data from 150 French-speaking nulliparous mothers and collected between 36 and 39 weeks of gestation (T1), and between 2 days (T2) and 6 weeks postpartum (T3). Factor analysis revealed a hierarchical three-factor structure that closely fit the data, including medical and obstetric risks/fetal health (F1), psychosocial changes (F2), and the prospect of childbirth (F3). The Antenatal Perceived Stress Inventory is a valid French prenatal stress scale with good psychometric properties.

Excessive alcohol consumption in young men: is there an association with their earlier family situation? A baseline-analysis of the C-SURF-study (Cohort Study on Substance Use Risk Factors).

AIMS: To determine whether parental factors earlier in life (parenting, single parent family, parental substance use problem) are associated with patterns of alcohol consumption among young men in Switzerland. METHODS: This analysis of a population based sample from the Cohort Study on Substance Use Risk Factors (C-SURF) included 5,990 young men (mean age 19.51 years), all attending a mandatory recruitment process for the army. These conscripts reported on parental monitoring and rule-setting, parental behaviour and family structure. The alcohol use pattern was assessed through abstention, risky single occasion drinking (RSOD), volume drinking and dependence. Furthermore, the impact of age, family socio-economic status, educational level of the parents, language region and civil status was analysed. RESULTS: A parental substance use problem was positively associated with volume drinking and alcohol dependence in young Swiss men. Active parenting corresponded negatively with RSOD, volume drinking and alcohol dependence. Single parent family was not associated with a different alcohol consumption pattern compared to standard family. CONCLUSION: Parental influences earlier in life such as active parenting (monitoring, rule-setting and knowing the whereabouts) and perceived parental substance use problem are associated with alcohol drinking behaviour in young male adults. Therefore, health professionals should stress the importance of active parenting and parental substance use prevention in alcohol prevention strategies.

Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)

Background: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury. Results:The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest. Conclusions: Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest.

Work-related stress according to the demand-control model and minor psychic disorders in nursing workers

This was a cross-sectional study that aimed to assess the association between work-related stress according to the Demand-Control Model, and the occurrence of Minor Psychic Disorder (MPD) in nursing workers. The participants were 335 professionals, out of which 245 were nursing technicians, aged predominantly between 20 and 40 years. Data were collected using the Job Stress Scale and the Self-Reporting Questionnaire-20. The analysis was performed using descriptive and analytical statistics. The prevalence of suspected MPD was 20.6%. Workers classified in the quadrants active job and high strain of the Demand-Control Model presented higher potential for developing MPD compared with those classified in the quadrant low strain. In conclusion, stress affects the mental health of workers and the aspects related to high psychological demands and high control still require further insight in order to understand their influence on the disease processes of nursing workers.

Stress, coping and presenteeism in nurses assisting critical and potentially critical patients

Objective to verify the associations between stress, Coping and Presenteeism in nurses operating on direct assistance to critical and potentially critical patients. Method this is a descriptive, cross-sectional and quantitative study, conducted between March and April 2010 with 129 hospital nurses. The Inventory of stress in nurses, Occupational and Coping Questionnaire Range of Limitations at Work were used. For the analysis, the Kolmogorov-Smirnov test, correlation coefficient of Pearson and Spearman, Chi-square and T-test were applied. Results it was observed that 66.7% of the nurses showed low stress, 87.6% use control strategies for coping stress and 4.84% had decrease in productivity. Direct and meaningful relationships between stress and lost productivity were found. Conclusion stress interferes with the daily life of nurses and impacts on productivity. Although the inability to test associations, the control strategy can minimize the stress, which consequently contributes to better productivity of nurses in the care of critical patients and potentially critical.


Caring for family caregivers: An analysis of a family-centered intervention

Objective To assess the effectiveness of Problem-Solving Therapy (PST) on family caregivers through the use of scales to measure anxiety, depression and emotional distress; and to explore facilitating factors and obstacles for its use based on the narrative of nurses. Method A clinical trial and an exploratory focus group with the use of mixed analysis methodology. The study was conducted in a primary health care center in Tarragona, Spain, and the sample consisted of 122 family caregivers who were included in the home care service, and 10 nurses who participated in the intervention group. Family caregivers with evident symptoms of anxiety, depression and emotional distress received PST in the intervention group. The intervention group also consisted of a discussion with eight nurses, which was transcribed and submitted to content analysis. Conclusion Problem-Solving Therapy proved to be effective in reducing perceived anxiety, depression and emotional distress. We identified its strong points and obstacles as described by nurses.



The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

Perceived stress in elderly stroke survivors after hospital discharge to home

AbstractOBJECTIVETo evaluate the relationship between perceived stress and comorbidities, neurological deficit, functional independence and depressive symptoms of stroke survivors after hospital discharge.METHODCross-sectional study with 90 elderly stroke survivors. The National Institutes of Health Stroke Scale instrument, the Functional Independence Measure instrument, the Geriatric Depression Scale and the Perceived Stress Scale were used. Bivariate Pearson correlation, independent t test and multiple regression analysis were used to evaluate the relationship between perceived stress and other variables.RESULTSThe final regression model showed that higher perceived stress was related to less functional independence (p= 0.022) and more depressive symptoms (p <0.001).CONCLUSIONAt hospital discharge, interventions should be planned for the treatment of depressive symptoms and to create adaptation strategies to the reduction of functional independence, in order to reduce the stress of the survivors.

Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation.

After primary growth, most dicotyledonous plants undergo secondary growth. Secondary growth involves an increase in the diameter of shoots and roots through formation of secondary vascular tissue. A hallmark of secondary growth initiation in shoots of dicotyledonous plants is the initiation of meristematic activity between primary vascular bundles, i.e. in the interfascicular regions. This results in establishment of a cylindrical meristem, namely the vascular cambium. Surprisingly, despite its major implications for plant growth and the accumulation of biomass, the molecular regulation of secondary growth is only poorly understood. Here, we combine histological, molecular and genetic approaches to characterize interfascicular cambium initiation in the Arabidopsis thaliana inflorescence shoot. Using genome-wide transcriptional profiling, we show that stress-related and touch-inducible genes are up-regulated in stem regions where secondary growth takes place. Furthermore, we show that the products of COI1, MYC2, JAZ7 and the touch-inducible gene JAZ10, which are components of the JA signalling pathway, are cambium regulators. The positive effect of JA application on cambium activity confirmed a stimulatory role of JA in secondary growth, and suggests that JA signalling triggers cell divisions in this particular context.

Quantitative analysis of crystal/grain sizes and their distributions in 2D and 3D

We review methods to estimate the average crystal (grain) size and the crystal (grain) size distribution in solid rocks. Average grain sizes often provide the base for stress estimates or rheological calculations requiring the quantification of grain sizes in a rock's microstructure. The primary data for grain size data are either 1D (i.e. line intercept methods), 2D (area analysis) or 3D (e.g., computed tomography, serial sectioning). These data have been used for different data treatments over the years, whereas several studies assume a certain probability function (e.g., logarithm, square root) to calculate statistical parameters as the mean, median, mode or the skewness of a crystal size distribution. The finally calculated average grain sizes have to be compatible between the different grain size estimation approaches in order to be properly applied, for example, in paleo-piezometers or grain size sensitive flow laws. Such compatibility is tested for different data treatments using one- and two-dimensional measurements. We propose an empirical conversion matrix for different datasets. These conversion factors provide the option to make different datasets compatible with each other, although the primary calculations were obtained in different ways. In order to present an average grain size, we propose to use the area-weighted and volume-weighted mean in the case of unimodal grain size distributions, respectively, for 2D and 3D measurements. The shape of the crystal size distribution is important for studies of nucleation and growth of minerals. The shape of the crystal size distribution of garnet populations is compared between different 2D and 3D measurements, which are serial sectioning and computed tomography. The comparison of different direct measured 3D data; stereological data and direct presented 20 data show the problems of the quality of the smallest grain sizes and the overestimation of small grain sizes in stereological tools, depending on the type of CSD. (C) 2011 Published by Elsevier Ltd.

Why human islets die? analysis of death signaling pathways during isolation and following cytokine treatments

RESUME Le diabète de type 1 se définit comme un désordre métabolique d'origine auto-immune qui aboutit à la destruction progressive et sélective de la cellule ß-pancréatique sécrétrice d'insuline. Cette maladie représente 10 % des cas de diabète enregistrés dans la population mondiale, et touche les jeunes de moins de 20 ans. Le traitement médical par insulinothérapie corrige le manque d'hormone mais ne prévient pas les nombreuses complications telles que les atteintes cardiaques, neurologiques, rénales, rétiniennes, et les amputations que la maladie provoque. Le remplacement de la cellule ß par transplantation d'îlots de Langerhans est une alternative prometteuse au traitement médical du diabète de type 1. Cependant la greffe d'îlots est encore un traitement expérimental et ne permet pas un contrôle efficace de la glycémie au long terme chez les patients transplantés, et les raisons de cet échec restent mal comprises. L'obstacle immédiat qui se pose est la purification d'un nombre suffisant d'îlots viables et la perte massive de ces îlots dans les premières heures suite à la greffe. Cette tendance presque systématique de la perte fonctionnelle du greffon immédiatement après la transplantation est connue sous le terme de « primary graft non-function » (PNF). En effet, la procédure d'isolement des îlots provoque la destruction des composantes cellulaires et non cellulaires du tissu pancréatique qui jouent un rôle déterminant dans le processus de survie de l'îlot. De plus, la transplantation elle-même expose les cellules à différents stress, notamment le stress par les cytokines inflammatoires qui encourage la mort cellulaire par apoptose et provoque par la suite le rejet de la greffe. L'ensemble de ces mécanismes aboutit a une perte de la masse d'îlot estimée a plus de 60%. Dans ce contexte, nous nous sommes intéressés à définir les voies majeures de stress qui régissent cette perte massive d'îlot par apoptose lors du processus d'isolement et suite à l'exposition immédiate aux cytokines. L'ensemble des résultats obtenus indique que plusieurs voies de signalisation intracellulaire sont recrutées qui s'activent de manière maximale très tôt lors des premières phases de l'isolement. La mise en culture des îlots deux jours permet aux voies activées de revenir aux taux de base. De ce fait nous proposons une stratégie dite de protection qui doit être 1) initiée aussitôt que possible lors de l'isolement des îlots pancréatiques, 2) devrait probablement bloquer l'activation de ces différentes voies de stress mis en évidence lors de notre étude et 3) devrait inclure la mise en culture des îlots purifiés deux jours après l'isolement et avant la transplantation. RESUME LARGE PUBLIC Le diabète est une maladie qui entraîne un taux anormalement élevé de sucre (glucose) dans le sang du à une insuffisance du pancréas endocrine à produire de l'insuline, une hormone qui régule la glycémie (taux de glucose dans le sang). On distingue deux types majeurs de diabètes; le diabète de type 1 ou juvénile ou encore appelé diabète maigre qui se manifeste souvent pendant l'enfance et qui se traduit par une déficience absolue en insuline. Le diabète de type 2 ou diabète gras est le plus fréquent, et touche les sujets de plus de 40 ans qui souffrent d'obésité et qui se traduit par une dysfonction de la cellule ß avec une incapacité à réguler la glycémie malgré la production d'insuline. Dans le diabète de type 1, la destruction de la cellule ß est programmée (apoptose) et est majoritairement provoquée par des médiateurs inflammatoires appelés cytokines qui sont produites localement par des cellules inflammatoires du système immunitaire qui envahissent la cellule ß-pancréatiques. Les cytokines activent différentes voies de signalisation parmi lesquelles on distingue celles des Mitogen-Activated Protein Kinase (MAPKs) composées de trois familles de MAPKs: ERK1/2, p38, et JNK, et la voie NF-κB. Le traitement médical par injections quotidiennes d'insuline permet de contrôler la glycémie mais ne prévient pas les nombreuses complications secondaires liées à cette maladie. La greffe d'îlots de Langerhans est une alternative possible au traitement médical, considérée avantageuse comparée a la greffe du pancréas entier. En effet l'embolisation d'îlots dans le foie par injection intraportale constitue une intervention simple sans complications majeures. Néanmoins la technique de préparation d'îlots altère la fonction endocrine et cause la perte massive d'îlots pancréatiques. De plus, la transplantation elle-même expose la cellule ß à différents stress, notamment le stress par les cytokines inflammatoires qui provoque le rejet de greffon cellulaire. Dans la perspective d'augmenter les rendements des îlots purifiés, nous nous sommes intéressés à définir les voies majeures de stress qui régissent cette perte massive d'îlot lors du processus d'isolement et suite à l'exposition immédiate aux cytokines après transplantation. L'ensemble de ces résultats indique que le stress induit lors de l'isolement des îlots et celui des cytokines recrute différentes voies de signalisation intracellulaire (JNK, p38 et NF-κB) qui s'additionnent entre-elles pour altérer la fonction et la viabilité de l'îlot. De ce fait une stratégie doit être mise en place pour bloquer toute action synergique entre ces différentes voies activées pour améliorer la viabilité et la fonction de la cellule ß lors du greffon cellulaire. SUMMARY Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the progressive and selective destruction of the pancreatic ß-cells that secrete insulin, leading to absolute insulin deficiency. T1DM accounts for about 10% of all diabetes cases, affecting persons younger than 20 years of age. Medical treatment using daily exogenous insulin injection corrects hormone deficiency but does not prevent devastating complications such as heart attack, neuropathy, kidney failure, blindness, and amputation caused by the disease. Pancreatic islet transplantation (PIT) is one strategy that holds promise to cure patients with T1DM, but purified pancreatic islet grafts have failed to maintain long-term glucose homeostasis in human recipients, the reasons for this failure being still poorly understood. There is however a more immediate problem with islet grafting that is dependent upon poor islet recovery from donors and early islet loss following the first hours of grafting. This tendency of islet grafts to fail to function within a short period after transplantation is termed primary graft non-function (PNF). Indeed, the islet isolation procedure itself destroys cellular and non-cellular components of the pancreas that may play a role in supporting islet survival. Further, islet transplantation exposes cells to a variety of stressful stimuli, notably pro-inflammatory cytokines that encourage ß-cell death by apoptosis and lead to early graft failure. Altogether these mechanisms lead to an estimated loss of 60% of the total islet mass. Here, we have mapped the major intracellular stress signaling pathways that may mediate human islet loss by apoptosis during isolation and following cytokine attack. We found that several stress pathways are maximally activated from the earliest stages of the isolation procedure. Culturing islet for two days allow for the activated pathways to return to basal levels. We propose that protective strategies should 1) be initiated as early as possible during isolation of the islets, 2) should probably target the activated stress pathways that we uncovered during our studies and 3) should include culturing islets for two days post-isolation and prior transplantation.

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.

Raman microstructural analysis of silicon-on-insulator formed by high dose oxygen ion implantation: As-implanted structures

A microstructural analysis of silicon-on-insulator samples obtained by high dose oxygen ion implantation was performed by Raman scattering. The samples analyzed were obtained under different conditions thus leading to different concentrations of defects in the top Si layer. The samples were implanted with the surface covered with SiO2 capping layers of different thicknesses. The spectra measured from the as-implanted samples were fitted to a correlation length model taking into account the possible presence of stress effects in the spectra. This allowed quantification of both disorder effects, which are determined by structural defects, and residual stress in the top Si layer before annealing. These data were correlated to the density of dislocations remaining in the layer after annealing. The analysis performed corroborates the existence of two mechanisms that generate defects in the top Si layer that are related to surface conditions during implantation and the proximity of the top Si/buried oxide layer interface to the surface before annealing.