Abstract In mice, gene targeting has become a useful tool for resolving the functions of proteins and for creating new animal models. Cre/loxP technology has been used broadly for generation of genetically modified mice. The Cre recombinase recognizes a specific DNA sequence, called loxP, and removes any DNA fragment between two loxP-sites. The activity of the Cre recombinase can be controlled spatially and temporally with cell- or tissue-specific promoters and synthetic inducing agents, such as tamoxifen or tetracycline. In this thesis, we employed tamoxifen-induced Cre recombination in vitro. Cre-ERTM mice were crossed to ROSA26LacZ reporters and Cre-recombination induced by 4OH-TM was monitored by LacZ staining. 0.5 μM 4OH-TM treatment was competent for tamoxifen-induced Cre-mediated activation of LacZ both in kidney cultures and in experimentally induced kidney mesenchymes. Wnt4 is a secreted signaling molecule, which is necessary for the development of several organs including kidney, ovary, adrenal gland, mammary and pituitary glands. Wnt4 is crucial for kidney development and conventional Wnt4-/- mice die soon after birth, likely due to renal failure. In this thesis, two different Wnt4 alleles, Wnt4EGFPCre and floxed Wnt4, were generated and analyzed to learn more about the Wnt4 functions and to apply these mouse lines to renal functional genomics. In the Wnt4EGFPCre, the EGFPCre fusion cDNA was targeted into exon I of the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates from E12.5 embryonic kidney onwards. Further characterization by crossing with the floxed ROSA26LacZ and yellow fluorescent protein (YFP) reporter lines demonstrated that in addition to the kidney, reporter expression was observed in the gonad, spinal cord, lung and the adrenal gland. The expression pattern of the Cre activity recapitulates well the known pattern of the Wnt4 gene. Time-lapse analysis in organ culture settings showed that the Wnt4 expressing cells contributed to the nephrons, some cells near the stalk of the developing ureter, as well as a number of positive supposed medullary stromal cells. In the conditional Wnt4 knock-out, loxP sites were placed to flank exons 3 to 5. The Wnt4 gene was specifically inactivated with CAGCre and Wnt4EGFPCre lines. In both of these crosses deletion of Wnt4 gene function led to impaired kidney development. In conclusion, we identified the culture conditions that can be used for the tamoxifen-induced conditional mutagenesis in tissue cultures. In addition, the created Wnt4 mouse lines serve as new useful tools for addressing the roles of Wnt4 function in diverse tissues and in different stages of development. / Tiivistelmä Hiirillä geenikohdennuksesta on muodostunut hyödyllinen väline proteiinien tehtävien selvittämisessä ja uusien eläinmallien luomisessa. Cre/loxP -tekniikkaa on käytetty laajasti muuntogeenisten hiirien tuottamisessa. Cre-rekombinaasi tunnistaa spesifisen DNA-jakson, niin kutsutun loxP:n, ja poistaa kaikki DNA-jaksot kahden loxP-sekvenssin väliltä. Cre-rekombinaasin aktiivisuutta voidaan säädellä paikallisesti ja ajallisesti solu- tai kudosspesifisillä promoottoreilla ja synteettisillä indusoivilla kemikaaleilla, kuten tamoksifeenillä tai tetrasykliinillä. Tässä väitöskirjassa hyödynsimme tamoksifeenin aiheuttamaa Cre-rekombinaatiota in vitro -kudosviljelmissä. Cre-ERTM-hiirilinja risteytettiin ROSA26LacZ-reportterilinjan kanssa, ja 4-hydroksitamoksifeenin indusoima Cre-rekombinaasin aktiivisuutta monitoroitiin LacZ–värjäyksellä. 0.5 µM:n 4OH-TM konsentraatiolla LacZ-reportterigeeni saatiin aktivoitua tehokkaasti Cre-rekombinaasin avulla sekä munuaisviljelmissä että munuaismesenkyymiviljelmissä. Wnt4 on erittyvä signalointimolekyyli, jolla on keskeinen rooli useiden elinten, kuten munuaisen, munasarjan, lisämunuaisen, rintarauhasen ja aivolisäkkeen kehittymisessä. Wnt4-geenillä on ratkaisevan tärkeä rooli munuaisen kehityksessä, ja poistogeeninen Wnt4-/-hiiri kuolee pian syntymän jälkeen, todennäköisesti munuaisen vajaatoimintaan. Tässä väitöskirjatyössä tuotettiin kaksi eri Wnt4 alleelia, Wnt4EGFPCre ja konditionaalinen Wnt4. Nämä hiirilinjat analysoitiin, jotta saisimme lisää tietoa Wnt4-geenin toiminnasta ja pystyisimme soveltamaan kyseisiä hiirikantoja munuaisten toiminnan selvittämisessä. Wnt4EGFPCre-alleelissa EGFPCre-fuusio -cDNA kohdennettiin osaksi endogeenisen Wnt4-geenin ykköseksonia. Vihreän fluoresoivan proteiinin (EGFP) aktiivisuus havaittiin varhaisen munuaisen kehityksen aikana. Wnt4EGFPCre-alleelin lisäkarakterisointi reportterilinjoilla (Rosa26LacZ ja Rosa26YFP) osoitti, että Wnt4-geenin ilmentyminen havaittiin munuaisen lisäksi sukurauhasissa, selkäytimessä, keuhkoissa sekä lisämunuaisessa. Wnt4EGFPCre-alleeli ilmentyi niissä kudoksissa, joissa endogeenisen Wnt4-geenin tiedetään olevan aktiivinen. Time-lapse -analyysin avulla osoitettiin, että Wnt4-geeniä ilmentävät solut muodostavat tiettyjä rakenteita munuaisen kehityksen aikana. Wnt4-geeni ilmentyi nefroneissa, kehittyvän virtsajohtimen soluissa sekä useissa medullaarisissa stroomasoluissa. Konditionaalisessa (ehdollisessa) Wnt4 knock-out-hiirilinjassa loxP-sekvenssit sijoitettiin eksonien kolme sekä viisi ympärille. Wnt4-geenin toiminta inaktivoitiin CAGCre- ja Wnt4EGFPCre-hiirilinjojen avulla. Näissä molemmissa tapauksissa Wnt4-geenin toiminnan poistaminen johti munuaisen kehityshäiriöön. Yhteenvetona voimme todeta, että olemme tunnistaneet ne kasvatusolosuhteet, joita voidaan hyödyntää, kun halutaan aktivoida reportterigeenejä tai kehityksen kannalta tärkeitä geenejä tamoksifeenin aiheuttamaa Cre/loxP -rekombinaatiota hyväksikäyttäen kudosviljelmissä. Samoja olosuhteita ja menetelmää käyttäen voidaan myös poistaa jonkun kehityksen kannalta tärkeän geenin toiminta ja tutkia sitä kudosviljelmässä. Tuotetut Wnt4-hiirikannat ovat lisäksi uusia hyödyllisiä työkaluja, kun halutaan tutkia Wnt4-geenin toimintaa erilaisissa kudoksissa ja eri kehitysvaiheiden aikana.

Electrical methods of geophysical survey are known to produce results that are hard to predict at different times of the year, and under differing weather conditions. This is a problem which can lead to misinterpretation of archaeological features under investigation. The dynamic relationship between a ‘natural’ soil matrix and an archaeological feature is a complex one, which greatly affects the success of the feature’s detection when using active electrical methods of geophysical survey. This study has monitored the gradual variation of measured resistivity over a selection of study areas. By targeting difficult to find, and often ‘missing’ electrical anomalies of known archaeological features, this study has increased the understanding of both the detection and interpretation capabilities of such geophysical surveys. A 16 month time-lapse study over 4 archaeological features has taken place to investigate the aforementioned detection problem across different soils and environments. In addition to the commonly used Twin-Probe earth resistance survey, electrical resistivity imaging (ERI) and quadrature electro-magnetic induction (EMI) were also utilised to explore the problem. Statistical analyses have provided a novel interpretation, which has yielded new insights into how the detection of archaeological features is influenced by the relationship between the target feature and the surrounding ‘natural’ soils. The study has highlighted both the complexity and previous misconceptions around the predictability of the electrical methods. The analysis has confirmed that each site provides an individual and nuanced situation, the variation clearly relating to the composition of the soils (particularly pore size) and the local weather history. The wide range of reasons behind survey success at each specific study site has been revealed. The outcomes have shown that a simplistic model of seasonality is not universally applicable to the electrical detection of archaeological features. This has led to the development of a method for quantifying survey success, enabling a deeper understanding of the unique way in which each site is affected by the interaction of local environmental and geological conditions.

Downhole production measurements are periodically acquired in hydrocarbon reservoirs to monitor and diagnose fluid movement in the borehole and the near-borehole region. However, because of the complexity involved with physical modeling and numerical implementation of borehole and formation multiphase flow behavior, inference of near-borehole petrophysical properties from production measurements is limited to simplified single-phase reservoir models. This dissertation develops a new transient coupled borehole-formation fluid flow algorithm to numerically simulate two-phase production logs (PL) acquired across heterogeneous rock formations penetrated by vertical and deviated boreholes. Subsequently, the coupled flow algorithm is used to estimate relevant dynamic petrophysical properties from borehole production measurements. The developed reservoir-borehole fluid flow model is based on an isothermal, one-dimensional (borehole axis) version of two-fluid formulation that simulates simultaneous flow of two fluid phases in oil-water, oil-gas, and gas-water flowing systems. Linkage of borehole and formation fluid flow models is carried out by introducing additional source terms into borehole mass conservation equations. Transient simulation of two-phase production measurements indicates the presence of borehole cross-flow when performing a shut-in test across differentially-depleted multilayer reservoirs. In a two-layer synthetic reservoir model penetrated by a vertical borehole, only two hours of through-the-borehole cross-communication of differentially-depleted layers gives rise to more than 14% increase in volume-averaged oil-phase relative permeability of the low-pressure layer. Simulated borehole fluid properties in the presence of cross-flow are used to estimate formation average pressure from two-phase selective-inflow-performance analysis. A new inversion-based interpretation algorithm is developed to estimate near-borehole absolute permeability and fluid-phase saturation from two-phase production logs. The inversion algorithm integrates production logs acquired in time-lapse mode to construct a near-borehole reservoir model that describes depth variations of skin factor over the elapsed time. Feasibility studies using synthetic reservoir models show that the estimated petrophysical properties are adversely influenced by the large volume of investigation associated with PL measurements. Moreover, undetectable fluid production across low-permeability layers decreases the sensitivity of production logs to layer incremental flow rate, thus increasing estimation uncertainty. Despite these limitations, the estimated fluid saturation and permeability across high-permeability layers are within 25% and 20% of the corresponding actual values, respectively. Oil-water and oil-gas flowing systems are additionally studied to quantify the added value of remedial workover operations (e.g., water and gas shut-off). Simulation of a gas shut-off performed in a gas-oil field example recommends a minimum bottom-hole pressure to prevent high gas production caused by (i) gas coning effects, and (ii) released gas from oil solution. Maintaining bottom-hole pressure above that limit gives rise to more than 60% reduction of downhole gas production. / text

Master of Science / Department of Geology / Abdelmoneam Raef / Advancements, applications, and success of time-lapse (4D) seismic monitoring of carbonate reservoirs is limited by these systems’ inherent heterogeneity and low compressibility relative to siliciclastic systems. To contribute to the advancement of 4D seismic monitoring in carbonates, an investigation of amplitude envelope across frequency sub-bands was conducted on a high-resolution 4D seismic data set acquired in fine temporal intervals between a baseline and eight monitor surveys to track CO₂-EOR from 2003-2005 in the Hall-Gurney Field, Kansas. The shallow (approximately 900 m) Plattsburg ‘C Zone’ target reservoir is an oomoldic limestone within the Lansing-Kansas City (LKC) supergroup – deposited as a sequence of high-frequency, stacked cyclothems. The LKC reservoir fluctuates around thin-bed thickness within the well pattern region and is susceptible to amplitude tuning effects, in which CO₂ replacement of initial reservoir fluid generates a complex tuning phenomena with reduction and brightening of amplitude at reservoir thickness above and below thin-bed thickness, respectively. A thorough analysis of horizon snapping criteria and parameters was conducted to understand the sensitivity of these autonomous operations and produce a robust horizon tracking workflow to extend the Baseline Survey horizon data to subsequent Monitor Surveys. This 4D seismic horizon tracking workflow expedited the horizon tracking process across monitor surveys, while following a quantitative, repeatable approach in tracking the LKC and maintaining geologic integrity despite low signal-to-noise ratio (SNR) data and misties between surveys. Analysis of amplitude envelope data across frequency sub-bands (30-80 Hz) following spectral decomposition identified geometric features of multiple LKC shoal bodies at the reservoir interval. In corroboration with prior geologic interpretation, shoal boundaries, zones of overlap between stacked shoals, thickness variation, and lateral changes in lithofacies were delineated in the Baseline Survey, which enhanced detail of these features’ extent beyond capacity offered from well log data. Lineaments dominated by low-frequency anomalies within regions of adjacent shoals’ boundaries suggest thicker zones of potential shoal overlap. Analysis of frequency band-to-band analysis reveals relative thickness variation. Spectral decomposition of the amplitude envelope was analyzed between the Baseline and Monitor Surveys to identify spectral and tuning changes to monitor CO₂ migration. Ambiguity of CO₂ effects on tuning phenomena was observed in zones of known CO₂ fluid replacement. A series of lineaments highlighted by amplitude brightening from the Baseline to Monitor Surveys is observed, which compete with a more spatially extensive effect of subtle amplitude dimming. These lineaments are suggestive of features below tuning thickness, such as stratigraphic structures of shoals, fractures, and/or thin shoal edges, which are highlighted by an increased apparent thickness and onset of tuning from CO₂. Detailed analysis of these 4D seismic data across frequency sub-bands provide enhanced interpretation of shoal geometry, position, and overlap; identification of lateral changes in lithofacies suggestive of barriers and conduits; insight into relative thickness variation; and the ability of CO₂ tuning ambiguity to highlight zones below tuning thickness and improve reservoir characterization. These results suggest improved efficiency of CO₂ -EOR reservoir surveillance in carbonates, with implications to ensure optimal field planning and flood performance for analogous targets.

Time-lapse seismic allows oil/gas reservoir monitoring during production, highlighting compaction and water movement. Time-lapse modeling, using a stress-dependent rock physics model, helps determine the need and frequency of expensive repeat seismic acquisition. We simulate a Gulf of Mexico gas reservoir time-lapse response for depletion and water flooding using uncertainty ranges in water saturation, porosity, stress-induced velocity changes, and pore compressibility. An analysis is conducted to see if a water-swept region could have been predicted. Findings show the swept and un-swept monitor cases amplitude differences range from 6% to 15%, which is higher than the actual monitor seismic noise level. Thus, it is unlikely these cases could be differentiated. However, the modeled amplitude changes from base to monitor cases do not match measured amplitude changes. This suggests the rock property model requires pressure-variance improvement and/or the changes in seismic amplitudes are associated with pressure/porosity, thickness, or saturation cases not modeled.

Un des mécanismes principaux de la morphogenèse des organismes est la contraction des réseaux d’actine sous l’effet du moteur moléculaire Myosine II. L’invagination de l’endoderme postérieur de Drosophila est causée par la contraction apicale des cellules par MyoII, mais la cause de sa déformation polarisée est inconnue. Nous avons découvert une vague de Rho1, MyoII et de déformation qui se propage à l’échelle du tissu et sous-tend la déformation de l’endoderme. MyoII est d’abord activée dans le primordium de l’endoderme par un ligand de GPCR, Fog. L’activation apicale de MyoII se propage ensuite à travers l’épithélium dorsal à 2.2 ± 0.2 µm/min. La dynamique de la vague n’est définie ni par les niveaux de Fog ni par leur motif d’expression. A la place, l’activité de MyoII est nécessaire pour l’activation intracellulaire de Rho1 et sa propagation à travers le tissu, indiquant une boucle de rétroaction. Des simulations d’un matériau viscoélastique contractile montrent qu’une boucle de rétroaction basée sur la tension peut générer une vague. Des perturbations de l’environnement mécanique du tissu avec des moyens génétiques ou mécaniques résultent en une augmentation de l’activité de MyoII et une diminution de la vitesse de la vague. Les déformations ou les forces du tissu procurent donc un rétrocontrôle sur l’activation de Rho1/MyoII lors de la vague, contrôlant sa dynamique. A l’échelle cellulaire, la vague de déformation implique la compression basale des cellules et l’étalement et l’adhésion du cortex apical sur la membrane vitelline, suivi d’un détachement. Ainsi la morphogenèse observée émerge de la propagation stimulée mécaniquement d’une vague de déformation 3D. / One of the main mechanisms of organism morphogenesis is the contraction of actin filament networks powered by non-muscle Myosin II motor proteins (MyoII). Drosophila presumptive posterior endoderm invagination is caused by MyoII-dependent apical constriction, but the cause of its polarized deformation is unknown. We unravelled a tissue scale wave of high Rho1 and MyoII activation and deformation which underlies the polarized deformation of the endoderm. MyoII is first activated medio-apically in cells within the endoderm primordium by the GPCR ligand Fog. Subsequently, apical MyoII activation propagates across the dorsal epithelium at a constant speed of 2.2 ± 0.2 µm/min. MyoII wave dynamics are set neither by Fog levels nor expression pattern. Instead, both intracellular Rho1 activation and its propagation across the tissue require sustained MyoII activity, indicating a positive feedback from contractility into Rho1 activity. Through simulations of a contractile viscoelastic material we found that a stress-based feedback loop could generate a wave. Perturbations of the tissue mechanical environment with both genetic and physical means result in an increase in MyoII activity and a strong reduction of the wave speed. Tissue deformation or stress thus provides a feedback onto Rho1/MyoII activity during the wave, controlling its dynamics. At the cell scale, the deformation wave involves cells basal compression and apical cortex spreading and adhering onto the vitelline membrane, followed by de-adhesion, that correlates with MyoII activation and propagation within cells. Thus the observed morphogenesis emerges from a mechanically driven wave of 3D deformation.

The National Council Licensure Examination for Registered Nurses (NCLEX-RN) first-time pass rate for an associate degree in nursing (ADN) program was below the national mean, presenting a problem for graduates who failed the NCLEX-RN and could not enter the workforce as registered nurses. Knowles's assumptions of adult learning, Ebbinghaus's forgetting curve, and Zull's neuroscience research served as the theoretical frameworks for this study. The purpose of this study was to identify which academic and time-lapse variables predict the graduate's first-time passage for the NCLEX-RN. Archived academic records and NCLEX-RN results for all 786 graduates from one ADN program who took the NCLEX-RN for the first time in 2015 were reviewed. Binary logistic regression analysis of the data identified multiple academic and one time lag factor as significant predictors for first-time NCLEX-RN passage. Key results included positive relationships between academic variables (prerequisite grade point average [GPA], nursing GPA, cumulative GPA, final course grade in 1 medical surgical course) and NCLEX-RN passage. An inverse relationship was shown between NCLEX-RN pass and the number of nursing component failures and time lag between clinical capstone completion and first NCLEX-RN attempt. Results informed a policy recommendation to provide timely intervention and resources for students at risk, with a goal of promoting success on the first attempt of the NCLEX-RN. Increasing the numbers of graduates who successfully complete the NCLEX-RN on the first attempt and promptly join the nursing workforce will demonstrate positive social change by mitigating the nursing shortage, which promotes safe patient care.

Le milieu karstique, et tout particulièrement sa partie superficielle, est caractérisé par une altération importante responsable sur le site de Lascaux de formations calcaires en plaquettes mais aussi de dissolutions importantes pouvant laisser place à des sables, argiles, etc. La présence de grottes ornées dans ce milieu nécessite la caractérisation du milieu environnant afin de protéger les grottes et contribuer à la conservation des œuvres pariétales. Cette thèse porte sur la caractérisation du milieu karstique à plusieurs échelles au moyen de deux méthodes : la géophysique avec la Tomographie de Résistivité Electrique (TRE), et la caractérisation pétrophysique, à l’échelle du laboratoire, avec la microscopie, la porosimétrie au mercure et des mesures électriques sur échantillons. A l’échelle du massif, nous avons caractérisé l’amont hydraulique de la grotte de Lascaux par un suivi temporel par TRE durant 6 ans et par trois modélisations géostatistique 3D, à trois dates. Le suivi temporel a permis de comprendre le fonctionnement hydrogéologique du site. Le couplage du suivi temporel avec le 3D complété par les mesures sur échantillons a en outre permis de comprendre le fonctionnement d’un drain situé dans l’épikarst susceptible d’alimenter l’émergence à l’entrée de la grotte de Lascaux. La modélisation 3D a aussi permis d’identifier un escarpement du promontoire calcaire au nord-est de la grotte de Lascaux qui laisse place à des formations argilo-sableuse. A l’échelle de la paroi et en lien avec des recherches menées sur la grotte Chauvet-Pont d’Arc, un dispositif original de micro-TRE a été mis en place à la paroi dans une carrière souterraine, permettant une mesure de TRE à une échelle submétrique et une résolution centimétrique. A cette résolution apparaît un effet d’électrode que nous corrigeons lors de l’inversion des données de TRE par l’utilisation d’une Electrode Equivalente Ponctuelle (EEP) placée à une profondeur caractéristique. La paroi étudiée a été soumise à des feux. On montre par un suivi TRE sur plusieurs mois et des analyses pétrophysiques complémentaires que les feux impactent de façon tangible les premiers centimètres de la paroi dont la porosité et la connectivité sont modifiées. On montre en outre que des variations saisonnières de saturation sont mises en évidence à cette petite échelle. Ce dispositif pourrait être appliqué au site de Lascaux pour, à terme, mesurer et prédire les arrivées d’eau à la paroi, en lien ou pas avec la circulation modélisée à l’échelle du massif. Une telle démarche pourrait devenir un outil de conservation de parois rocheuses support d’œuvres pariétales. / The karst environment and especially its superficial part is typified by an important alteration. At the Lascaux site, alteration is responsible for plate limestone and also detrital clayey-sands soils due to dissolution of limestones. The presence of painted caves in this altered environment raises the need to characterize the karst environment in order to protect the caves and contribute to mural paintings conservation. The aim of this PhD thesis is to characterize the karst environment at different scales using two main methods: (1) geophysics and in particular Electrical Resistivity Tomography (ERT) and (2), petrophysics at the Lab scale, including microscopy, mercury porosimetry and electrical measurements. At the field scale, we characterized the calcareous surroundings of the Lascaux cave relying on a 6 years-long time-lapse monitoring by ERT complemented by geostatistical 3D reconstructions of the resistivity values. The time-lapse monitoring permitted a better understanding of the hydrogeology of the site. Combining ERT time-lapse monitoring, 3D models of the resistivity distribution and additional measurements on samples allowed to identify a drain within the epikarst, which is most likely responsible for the emergence of water at the Lascaux cave entrance. The 3D modelling of the resistivity values also allowed to identify a scarp of unaltered limestones, northeast of the Lascaux cave, leaving room for clayey-sand detrital formations. In relation to a current research program on the Chauvet-Pont d’Arc cave, an original micro-ERT acquisition layout was fitted on the wall of an underground quarry, allowing measurements at a sub-metric scale with a centimetric resolution. At such a fine resolution, an unwanted electrode effect appeared. We remedied this electrode effect by inverting ERT data with an Equivalent Electrode Point (EEP) buried in the ground at a characteristic depth. The quarry wall was subjected to fires. Relying on a micro-ERT time-lapse monitoring during few months complemented by petrophysical analyses, we showed that the fires impacted the first centimeters of the quarry wall whose porosity and connectivity were modified. We also showed that seasonal water saturation variations could be detected at such a small scale. Thus, this small scale acquisition layout may ultimately be used at Lascaux to measure and predict water intakes directly at the cave walls in relation, or not, with the water flow modelled at the field scale. Such an approach may become a new tool for the conservation of painted cave walls.

Les bactéries forment des communautés spatiales adhérentes à des surfaces, appelées biofilms. Ces organisations bactériennes sont omniprésentes dans les milieux naturel, industriel et médical et peuvent porter atteinte à notre santé lorsqu'elles hébergent des agents pathogènes, parmi lesquels le médiatique Staphylococcus aureus sur lequel a porté l'ensemble de ce travail de thèse. Cette bactérie est l'une des principales causes d'infections chroniques, mais également d'infections nosocomiales, impliquant le plus souvent des biofilms. Il est aujourd'hui reconnu qu'une telle biostructure est un véritable bouclier à l'action des antimicrobiens et à celle du système immunitaire. Outre les résistances génétiques des bactéries pathogènes aux antibiotiques, l'hétérogénéité chimique et biologique de la structure tridimensionnelle des biofilms pourrait être à l'origine de ces phénomènes de tolérance et de chronicité d'infections. C'est à cette problématique que se rattache ce travail de thèse concernant l'action de la vancomycine sur des biofilms de S. aureus. Alors que les connaissances sur la réactivité de cet antibiotique clef avec S. aureus proviennent essentiellement d'études réalisées sur des cellules planctoniques, l'originalité de notre approche a été d'étudier la diffusion-réaction de la vancomycine in situ dans l'épaisseur des biofilms en utilisant en particulier des outils avancés de microscopie de fluorescence (Time-Lapse, FLIM, FRAP, et FCS). Nous avons ainsi évalué sa biodisponibilité dans la matrice d'exopolymères, ainsi que l'impact de la physiologie spécifique des bactéries incluses en biofilms sur l'activité de cet antibiotique, utilisé seul ou en association avec la rifampicine. Cette approche multidisciplinaire a permis une meilleure compréhension des mécanismes impliqués dans la singulière tolérance de ces biostructures à l'action des antibiotiques, et de souligner l'urgence de développer des approches préventives telles que le diagnostic précoce des infections impliquant des biofilms.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.