A Deep Bed Filtration model has been developed to quantify the effect of solids invasion from drilling fluids on the permeability of rock formations. The calculated particle-trapping profiles are ...compared directly with experimental profiles from scanning electron microscopy and synchrotron
X
-ray diffraction tomography mapping. The computed permeability reduction as a consequence of particle invasion is in broad agreement with experiment. Backflow was modelled by reversing the flow rate, starting off with a situation where all particles either remain trapped or are all released. It appears that the experimentally observed 30% release of particles upon backflow is reproducible within the limits of the two extreme cases. When erosion is included in the model, a peak in the backflow pressure time series can be observed. This peak may be correlated with the experimentally observed flow initiation pressure, which is the backflow pressure needed to initiate flow after initial inflow filtration. Finally, we conclude that internal reservoir damage, within the limits of our 1-D single phase DBF model, may contribute to the experimentally observed flow initiation pressure.
CRISPR/Cas9 genome engineering strategies allow the directed modification of the
genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or ...fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the
gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the
gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a
-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.
Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR ...'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.
Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single ...amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.
During mud circulation and cementing operations, non-Newtonian completion fluids are injected along the wellbore annular space formed by the gap between the outer wall of the casing and the rock ...face. During such processes, these fluids displace each-other and follow a complex path given by pressure gradients, rheology and density contrasts, casing rotation and reciprocation and by the actual shape and orientation of the annulus. Muds and cement slurries also often exhibit a yield stress which may represent additional challenge for optimal fluid removal and cement coverage. This paper presents a novel approach to solving the 3D flow and displacement of completion fluids in the annulus. In particular, this work extends a model published in Tardy and Bittleston (2015) which solves the flow in the 2D axial-azimuthal plane, to now capture fluid distribution and velocity profiles across the gap width in the 3D axial-azimuthal-radial space. The new 3D model is derived using the so-called narrow-gap approximation for the momentum balance equations. This approximation provides a mean to solve the 3D velocity and concentration fields while solving a 2D-only elliptic pressure equation, which is significantly faster to solve than the original 3D pressure equation, and without suffering any significant loss of accuracy.
•Novel approach allowing the resolution of 3D annular flow of completion fluids using a 2D-only pressure equation.•CPU requirements significantly lower than conventional 3D CFD-type modeling.•Model based on the lubrication technique with the narrow-gap assumption.•Extensive model validation against CFD simulations and experimental data.
The in situ rheology of polymeric solutions has been studied experimentally in etched silicon micromodels which are idealizations of porous media. The rectangular channels in these etched networks ...have dimensions typical of pore sizes in sandstone rocks. Pressure drop/flow rate relations have been measured for water and non-Newtonian hydrolyzed-polyacrylamide (HPAM) solutions in both individual straight rectangular capillaries and in networks of such capillaries. Results from these experiments have been analyzed using pore-scale network modeling incorporating the non-Newtonian fluid mechanics of a Carreau fluid. Quantitative agreement is seen between the experiments and the network calculations in the Newtonian and shear-thinning flow regions demonstrating that the ‘shift factor,’
α, can be calculated a priori. Shear-thickening behavior was observed at higher flow rates in the micromodel experiments as a result of elastic effects becoming important and this remains to be incorporated in the network model.
In this paper, a 3D filtration law for power-law fluids flowing in heterogeneous porous media is derived through stochastic homogenisation. The filtration equation for isotropic porous media is first ...considered, at the local Darcy-scale. This equation possesses a single flow parameter, which depends on the space variables. The spatial variation of this parameter is modelled by a stationary random field and therefore arbitrarily heterogeneous and anisotropic in character. The stochastic homogenisation technique is then applied for averaging the interplay between rock and fluid parameters. A simple analytical and tractable formula is derived which expresses the importance of both rheological and porous medium related parameters on the mean flow. In order to validate the formula, comparisons are made with numerical experiments for 2D flows. The new law is found to be in good agreement with numerical experiments.
Les canaux potassiques à deux domaines P (K2P) contrôlent l’excitabilité cellulaire et jouent un rôle central dans l’établissement et le maintien du potentiel de repos membranaire dans la majorité ...des cellules animales. Depuis leur identification dans les années 90, ces canaux ont été impliqués dans de nombreuses fonctions comme la modulation de l’activité neuronale, l’activité du muscle cardiaque ou encore la physiologie rénale. Malgré l’importance de ces canaux, peu de données existent sur les processus cellulaires qui contrôlent leur fonction in vivo. Au cours de ma thèse, j’ai utilisé des approches génétiques, d’imagerie et d’électrophysiologie pour comprendre comment l’expression, la distribution et l’activité du canal K2P UNC-58 sont contrôlés chez le nématode modèle C. elegans.Pour cela, j’ai effectué un crible suppresseur du phénotype locomoteur du mutant gain de fonction unc-58(e665). J’ai ainsi obtenu 133 mutants présentant une large gamme de niveaux de suppression, suggérant l’implication de plusieurs gènes dans les processus de régulation du canal. En utilisant les technologies de reséquençage complet de génome, j’ai pu cloner six nouveaux gènes requis pour la fonction d’unc 58.J’ai ensuite caractérisé en détail le rôle d’unc-44/ankyrine dans le contrôle du trafic d’unc 58. Ce travail a conduit à 4 conclusions majeures : (1) UNC-58, malgré sa structure de canal potassique, possède en réalité une sélectivité ionique altérée favorisant le passage des ions sodium, (2) l’addition à UNC 58 de protéine fluorescente par approche CRISPR/Cas9 nous a permis pour la première fois d’observer directement la distribution du canal UNC-58 in vivo, (3) l’ankyrine est nécessaire à l’adressage du canal UNC-58 à la surface des muscles et dans les axones des neurones mécanosenseurs ALM. Cette fonction fait intervenir une poche d’interaction lipidique localisée au sein du module Zu5N-Zu5C-UPA d’UNC-44, (4) ce mécanisme est hautement sélectif puisqu’il n’est pas requis pour l’adressage de 6 autres canaux potassiques musculaires. Mon crible a également identifié une interaction génétique entre unc-70/ß-spectrine et unc-44/ankyrine. Toutefois, la nature moléculaire de cette interaction reste encore à préciser
Two-pore potassium channels (K2P) control cell excitability and play a central role in the establishment and the maintenance of the resting membrane potential of almost all animal cells. Since their identification in the late 90s, these channels have been implicated in a large number of functions ranging from neuronal and cardiac activity to kidney physiology. Despite the crucial functions of these channels, comparatively little is known about the cellular processes controlling their function in vivo. During my PhD, I used a wide range of strategies including genetics, microscopy and electrophysiology to understand how the expression, the distribution and the activity of the K2P channel UNC-58 are controlled in the model nematode C. elegans. I have first performed a genetic suppressor screen targeting the locomotion phenotype of the gain of function mutant unc-58(e665). The screen yielded 133 mutants, displaying a wide range of suppression level, suggesting that several genes may be implicated in the channel regulation process. By using whole genome sequencing technologies, I’ve been able to clone six new genes required for the function of UNC-58.Then, I’ve characterized in detail the role of unc-44/ankyrin in the trafficking of UNC 58. This project led to 4 main conclusions : (1) UNC-58, despite its potassium channel structure, has an altered ionic selectivity, allowing preferably sodium ions to pass through the channel (2) the addition of a fluorescent protein to UNC-58 by CRISPR/Cas9 approaches allowed us for the first time to directly observe the addressing of the UNC-58 channel to the muscle surface and axons of ALM mechanosensory neurons. This function involves a lipid binding pocket located within the Zu5N-Zu5C-UPA module of UNC-44, (4) this mechanism is highly selective since it is not required for the addressing of 6 other muscular channels.My screen also identified a genetic interaction between unc-70/ß-spectrin and unc-44/ankyrin. However, the exact molecular nature of this interaction remains to be elucidated
This paper points out two numerical problems linked to the resolution of compositional multiphase flow models for porous media with the finite-volume technique. In particular, we consider fluid ...mixtures featuring fast mass transfers between the phases, hence stiff. In this context, we show how the computation of mass exchange kinetics can be expensive and that erroneous saturation front locations arise. A numerical splitting method is developed which is proven to be stable with advection-type time steps, whatever the stiffness of the mass transfer. The erroneous front location problem is illustrated and shown to be intrinsically linked to the numerical diffusion. If we assume that the fluids are in thermodynamical equilibrium, we find that spurious solutions can be avoided by deriving and solving a new uncoupled hyperbolic equation for the saturation.PUBLICATION ABSTRACT
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