In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD accumulation observed in the mutants, that this phenotype occurs in above ground tissues and is present throughout the early growth stages of the plant. Further examination of lrt1 mutant plants has allowed a preliminary understanding of the role LRT1 may play in LD regulation. Taken together, the results of this study point towards some promising strategies to increase LD content in plant tissues.

The objective of this thesis is to develop an easy-to-use and computationally economical numerical tool to investigate the flow field in the Pulsed Pressure Chemical Vapour Deposition (PP-CVD) reactor. The PP-CVD process is a novel thin film deposition technique with some advantages over traditional CVD methods. The numerical modelling of the PP-CVD flow field is carried out using the Quiet Direct Simulation (QDS) method, which is a flux-based kinetic-theory approach. Two approaches are considered for the flux reconstruction, which are the true directional manner and the directional splitting method. Both the true directional and the directional decoupled QDS codes are validated against various numerical methods which include EFM, direct simulation, Riemann solver and the Godunov method. Both two dimensional and axisymmetric test problems are considered. Simulations are conducted to investigate the PP-CVD reactor flow field at 1 Pa and 1 kPa reactor base pressures. A droplet flash evaporation model is presented to model the evaporation and transport of the liquid droplets injected. The solution of the droplet flash evaporation model is used as the inlet conditions for the QDS gas phase solver. The droplet model is found to be able to provide pressure rise in the reactor at the predicted rate. A series of parametric studies are conducted for the PP-CVD process. The numerical study confirms the hypothesis that the flow field uniformity is insensitive to the reactor geometry. However, a sufficient distance from the injection inlet is required to allow the injected precursor solution to diffuse uniformly before reaching the substrate. It is also recommended that placement of the substrate at the reactor’s centre axis should be avoided.

Present work is focused on the numerical study of evaporation of sessile liquid droplets on top of smooth solid substrates. The process of evaporation of a sessile liquid droplet has lots of different applications both in industry and research area. This process has been under study for many years, and still it is an actual problem, solution of which can give answers on some fundamental and practical questions. Instantaneous distribution of mass and heat fluxes inside and outside of an evaporating sessile droplet is studied in this research using computer simulations. The deduced dependences of instantaneous fluxes are applied for self-consistent calculations of time evolution of evaporating sessile droplets. The proposed theory of evaporating sessile droplets of liquid has been validated against available experimental data, and has shown a good agreement. Evaporation of surfactant solution droplets is studied experimentally. The theory, proposed for two stages of evaporation, fits experimental data well. An additional evaporation stage, specific for surfactant solutions, is observed and described. Mathematical modelling of this stage requires further research on surfactant adsorption and its influence on the value of receding contact angle. Numerical study of the evaporation of microdroplets is conducted in order to evaluate the significance of different evaporation mechanisms (diffusive and kinetic models of evaporation) and different physical phenomena (Kelvin s equation, latent heat of vaporization, thermal Marangoni convection, Stefan flow).

Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.

The present study was undertaken to investigate the effects of the T-type calcium channel blocker, Mibefradil, on white adipocytes. Unexpected for a T-type channel blocker, Mibefradil was found to increase intracellular calcium levels, cause lipid droplet fusion, and result in cell death. Calcium imaging of white adipocytes showed an increase of calcium concentration by Mibefradil at concentrations ranging from10-50 µM. The elevation in calcium by Mibefradil was significantly reduced by pretreatment of cells with Thapsigargin, an endoplasmic reticulum (ER) specific Ca ATPase inhibitor. Additionally, lipid droplet fusion and cell death were also attenuated by Thapsigargin pretreatment in white adipocytes. We conclude that Mibefradil elevated intracellular calcium levels, induced lipid droplet fusion and cell death in white adipocytes via mobilizing intracellular calcium stores from the ER. These results describe novel effects of Mibefradil on white adipocytes and may provide new insight into how this drug might be repurposed in obesity research.

Effective design of liquid fuel injection systems is a function of good understanding of liquid breakup mechanisms. A transient liquid breakup model is developed on the classical interfacial breakup theory by modifying the classical linear perturbation process to include time-dependent base and perturbed flow parameters. The non-isothermal condition on liquid jet instability and breakup is theoretically modelled; with the particular consideration of a spatially variation of surface tension along the liquid-gas interface. The model combines the classical interface hydrodynamic instability and breakup theory and heat-transfer through semi-infinite medium. Analytical liquid breakup model, which combines transient and non-isothermal effects on liquid jet breakup, is suggested. The suggested model could be simplified to the transient breakup model and the non-isothermal breakup model equivalents. A novel mechanistic model, which is based on a simple momentum balance between the injected jet and the aerodynamic drag force, is suggested for breakup length. A new model, which combines energy criterion and dual-timescale for turbulent shear in droplet dispersion, is suggested for droplet breakup criteria on the basis of critical Webber number. All developed models showed good predictions of available experimental data, and established empirical correlation, within the operational conditions of contemporary ICEs, specifically diesel engines. Continued research in these areas could benefit the development of the next generation of liquid fuel injectors and combustors – by accounting for transient effects and non-isothermal conditions in liquid jet breakup, and turbulent shear in droplet breakup.

Understanding the interactions between a body of liquid and a curvy surface is important for many applications such as underwater drag force reduction, droplet filtration, self-cleaning, and fog harvesting, among many others. This study investigates ways to predict the performance of granular and fibrous surfaces for some of the above applications. More specifically, our study is focused on 1) modeling the mechanical stability of the air-water interface over submerged superhydrophobic (SHP) surfaces and their expected drag reduction benefits, and 2) predicting the mechanical stability of a droplet on a fiber in the presence of an external body force. For the first application, we modeled the air–water interface over submerged superhydrophobic coatings comprised of particles/fibers of different diameters or Young–Laplace contact angles. We developed mathematical expressions and modeling methodologies to determine the maximum depth to which such coatings can be used for underwater drag reduction as well as the magnitude of the depth-dependent drag reduction effect of the surface. For the second application, we studied the force required to detach a droplet from a single fiber or from two crossing fibers. The results of our numerical simulations were compared to those obtained from experiment with ferrofluid droplets under a magnetic field, and excellent agreement was observed. Such information is of crucial importance in design and manufacture of droplet–air and droplet–fluid separation media, fog harvesting media, protective clothing, fiber-reinforced composite materials, and countless other applications.

Nous avons effectué dans ce mémoire des études de magnéto-luminescence sur des boîtes quantiques individuelles de GaAs insérées dans une matrice de GaAlAs, boîtes qui se distinguent de la plupart des systèmes étudiés par l’absence des contraintes. Nous avons pu mesurer l’impact de l’orientation cristalline du substrat sur lequel s’effectue la croissance de ces boîtes sur les propriétés magnéto-optiques.Nous avons pu présenter les premières expériences pratiquées sur des structures à boîtes quantique GaAs « gouttelettes » à charge ajustable, élaborées sur substrat orienté (001). La structure fine des excitons a été étudiée en détail et analysée au moyen d’un modèle analytique y compris en champ transverse permettant d’établir les états et énergies propres. Nous avons observé la polarisation dynamique des noyaux à champ nul dans les boîtes quantiques sans contraintes élaborées sur substrat (111)A. Les décalages Overhauser mesurés s’élèvent à 15 – 16 μeV, correspondant à un champ nucléaire de 0.25 T environ. En champ magnétique transverse, nous avons observé des déviations significatives par rapport àl’effet Hanle normal dans le domaine des faibles champs / In this thesis the magneto-optical properties of single GaAs semiconductor quantum dots in AlGaAs barriers are presented. The strain free dots are grown by original Volmer-Weber ("droplet") epitaxy techniques in a molecular beam epitaxy system at the National Institute for Material Science NIMS, Tsukuba, Japan. We showed the first optical investigation of symmetric GaAs quantum dots grown on (111)A substrates. The inherently small neutral exciton fine structure splitting makes this a promising systemfor the generation of polarisation entangled photons via the exciton-biexciton radiative cascade. In photoluminescence spectra in longitudinal magnetic fields applied along the growth axis, we observe in addition to the expected bright states also nominally dark transitions for both charged and neutral excitons. We uncover a strongly non monotonic, sign-changing field dependence of the bright neutral exciton splitting resulting from the interplay between exchange and Zeeman effects. We present a microscopic theory developed in close collaboration with the A. F. Ioffe Institute (St. Petersburg,Russia) of the magnetic field induced mixing of heavy-hole states with angular momentum projections 3/2 in GaAs droplet dots grown on (111)A substrates.Chapter 4 of this thesis is focused on the charge tuneable structures grown on n+-(100) GaAs substrate. In non-intentionally doped samples, due to charge fluctuations, the neutral X0 exciton and the positively (negatively) charged exciton X+(X-) are observed simultaneously in time integrated spectra. We present here deterministic charging of droplet dots with single electrons. Detailed studies in transverse magnetic fields allowed unambiguously identifying the charge states and determining the exciton fine structure. The neutral exciton fine structure was tuned to zero in finite transverse fields, a crucial property for achieving efficient polarization entangled two photon sources. In chapter 5, we focus on the nuclear spin effects in [111] grown quantum dots under optical pumping conditions. An optically oriented electron spin can transfer its polarization to a nucleus (Overhauser effect). In the well-studied strained InGaAs dots in GaAs, dynamic nuclear polarization (DNP) at zero applied magnetic field is possible due to screening of the nuclear dipole-dipole interaction by strong nuclear quadrupole effects. Here we present the first observation of DNP in strain free dots, i.e. in theabsence of nuclear quadrupole effects. We investigated in detail the role of the strong effective magnetic field acting on the nuclei due to the presence of a well-oriented electron spin (Knight field). This Knight field in the order of 15 mT for most dots is an important ingredient for the observed DNP at zero field. The intricate interplay between the Knight field and the Overhauser field is studied in a transverse magnetic field. These Hanle measurements performed on single dots allow us to determine the sign of the confined electron g-factor and spin relaxation time

<p>Microscale environments provide significant resolution and distortion challenges with respect to measurement techniques; however, with improvements to existing techniques, it is possible to gather relevant data to better understand the thermal and fluidic mechanisms at such small scales in evaporating droplets.</p> <p> </p> <p>Infrared thermography provides several unique challenges at small scales. A primary issue is that the low native resolution of traditional infrared cameras significantly hamper the collection of details of microscale features. Furthermore, surfaces exhibiting vastly different emissivities, results in inaccurate temperature measurements that can only be corrected with irradiance-based emissivity maps of the surface; however, due to the resolution limitations of infrared thermography, these emissivity maps can also display significant errors. These issues are overcome through the use of multi-frame super-resolution. The enhanced resolution allows for better capture of microscale features, therefore, enhancing the emissivity map. A quantitative error analysis of the system is conducted to quantify the feature size resolution improvement as well as the smoothing effect of super-resolution reconstruction. Furthermore, a sensitivity analysis is conducted to quantify the impact of registration uncertainty on the accuracy of the reconstruction. Finally, the improved emissivity map from super-resolution is demonstrated to show the increased accuracy over low-resolution mapping.</p> <p> </p> <p>When applied to water droplets, particularly on nonwetting surfaces, infrared thermography is confounded by the presence of nonuniform reflectivities due to the spherical curvature of the liquid-air interface. Thus, when measuring the temperature along the vertical axis of a water droplet, it is necessary to correct the reflection. Using a controlled background environment, in conjunction with the Fresnel equations, it is possible to correct the reflective effects on the interface and calculate the actual temperature profile. This allows for a better understanding of the governing mechanisms that determine the thermal transport within the droplet. While thermal conduction is the primary transport mechanism along the vertical axis of the droplet, it is determined that the temperature drop is partially dampened by the convective transport from the ambient air to the liquid interface. From this understanding revealed by the measurements, the vapor-diffusion-based model for evaporation was enhanced to better predict evaporation rates.</p> <p> </p> <p>Further exploration into the mechanisms behind droplet evaporation on nonwetting surfaces requires accurate knowledge of the internal flow behavior. In addition, the influence of the working fluid can have a significant impact on the governing mechanisms driving the flow and the magnitude of the flowrate. While water droplet evaporation has been shown to be governed by buoyancy-driven convection on nonwetting substrates, similar studies on organic liquid droplets are lacking. Particle image velocimetry is effective at generating a velocity flow field, but droplets introduce distortion due to the refraction from the spherical interface of the droplet. As such, velocity correction using a ray-tracing approach was conducted to correct the velocity magnitudes and direction. With the velocity measurements, the flow was determined to be surface-tension-driven and showed speeds that are an order of magnitude higher than those seen in buoyancy-driven flow in water droplets. This resulted in the discovery that advection plays a significant role in the transport within the droplet. As such, the vapor-diffusion-governed evaporation model was adjusted to show a dramatic improvement at predicting the temperature gradient along the vertical axis of the droplet.</p> <p> </p> <p>Armed with the knowledge of flow behavior inside droplets, it is expected that droplets with aqueous solutions should exhibit buoyancy-driven convection. The final part of this work, therefore, leverages this phenomenon to enhance mixing during reactions. Colorimetry is a technique that is widely utilized to measure the concentration of a desired sample within some liquid; the sample reacts with a reagent dye the color change is measured, usually through absorbance measurements. In particular, the Bradford assay is used to measure protein concentration by reacting the protein to a Coomassie^TM Brilliant Blue G-250. The absorbance of the dye increases, most significantly at the 590 nm wavelength, allowing for precise quantitation of the amount of protein in the solution. A droplet-based reaction chamber with buoyancy-enhanced mixing has the potential to speed up the measurement process by removing the need for a separate pre-mixing step. Furthermore, the reduced volume makes the process more efficient in terms of reactant usage. Experimental results of premixed solutions of protein sample and reagent dye show that the absorbance measurement through a droplet tracks strongly with the protein concentration. When the protein sample and dye reagent are mixed <i>in situ</i>, the complex interaction between the reactants, the mixing, and the adsorption of protein onto the substrate creates a unique temporal evolution in the measured absorbance of the droplet. The characteristic peaks and valleys of this evolution track strongly with concentration and provide the framework for measurement of concentration in a droplet-based system.</p> <p> </p> <p>This thesis extends knowledge about droplet thermal and fluidic behavior through enhanced measurement techniques. This knowledge is then leveraged in a novel application to create a simple, buoyancy-driven colorimetric reaction setup. Overall, this study contributes to the field of miniaturized, efficient reaction and measurement devices.</p>

Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposé. / This thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed.