Shp-1 (Src homology 2 domain-containing phosphatase 1) est une tyrosine phosphatase retrouvée principalement chez les cellules hématopoïétiques, mais également chez les cellules épithéliales. Bien que Shp-1 soit reconnue comme étant un régulateur négatif de plusieurs voies de signalisation intracellulaire chez les cellules hématopoïétiques, son rôle dans les cellules épithéliales a été jusqu’ici très peu étudié. Afin de mieux comprendre son rôle dans les cellules épithéliales intestinales, nous avons généré un modèle murin de délétion conditionnelle de Shp-1 spécifiquement dans l’épithélium intestinal (Shp-1CEI-KO). De manière intéressante, dès l’âge de 6 semaines, les souris expérimentales présentent une intestinalomégalie associée à une légère augmentation de la prolifération cryptale. La taille des cellules épithéliales est également augmentée, suggérant de l’hypertrophie cellulaire chez les souris invalidées pour Shp-1. Parallèlement, la voie de signalisation PI3K/Akt/mTor est activée dans l’épithélium des souris mutantes. Nous avons également noté une production accrue de cellules caliciformes et de leurs précurseures, les cellules intermédiaires, en absence de Shp-1. Par contre, la maturation des cellules de Paneth semble grandement compromise vu la baisse importante d’expression du lysozyme et des RegIIIβ et RegIIIγ, de même que la faible densité de leurs granules de sécrétion. La comparaison du phénotype intestinal des souris Shp-1CEI-KO avec celui des souris PtenCEI-KO suggère que l’hyperactivation de la voie PI3K/Akt/mTor est responsable en partie des altérations phénotypiques observées chez la souris invalidée pour Shp-1. En conclusion, nos résultats montrent que la tyrosine phosphatase Shp-1 est un régulateur important de l’homéostasie de l’épithélium intestinal en contrôlant notamment la croissance cellulaire et la différenciation des cellules de la lignée sécrétrice.

O transiente hidráulico na adução de Pequenas Centrais Hidrelétricas (PCH) é um importante tema de estudo para a correta escolha e dimensionamento das estruturas de amortecimento, que protegem o circuito adutor da variação abrupta da pressão interna causada pela variação na velocidade média do escoamento. Este trabalho propõe a utilização da hidrodinâmica computacional (CFD) para avaliar a oscilação de massa ou nível d\'água no interior da estrutura de proteção denominada Chaminé de Equilíbrio e comparar esses resultados com a solução teórica analítica, com os critérios de dimensionamento definidos em norma técnica e com os dados de medições experimentais. O problema foi modelado no software Ansys CFX que utiliza o método dos volumes finitos (MVF) para resolver os campos de velocidade e pressão do escoamento, tanto em regime permanente, quanto em regime transiente. Aplicando-se o modelo CFD proposto ao circuito hidráulico didático, os resultados foram validados com a formulação teórica e com as medições experimentais do nível d\'água na chaminé. O modelo foi aplicado num estudo de caso que consiste num circuito hidráulico de PCH. Os resultados da oscilação de massa no interior da chaminé para este estudo de caso confirmam o emprego da fluidodinâmica computacional para avaliar tais escoamentos em regime transitório. / The hydraulic transient in the circuit of Small Hydroelectric Power Plants (SHP) is an important study subject for the correct choice and design of damping structure which protects the conduit from an abrupt variation of the internal pressure caused by the changes in the average flow velocity. This work proposes the use of computational fluid dynamics (CFD) to evaluate the mass oscillation or water level inside the protection structure called Surge Tank and compares these results with theory, design criteria defined by the technical standard, and experimental data. The problem was modeling in Ansys CFX software that uses the finite volume method (FVM) to solve velocity and pressure flow fields, in both steady state and transient flow. The proposed model used in a hydraulic circuit led to results validation with experimental measurements of water level surge tank. For the proposed case study, the response of the numerical CFD simulation confirms the use of computational fluid dynamics to evaluate such transient flows.

O transiente hidráulico na adução de Pequenas Centrais Hidrelétricas (PCH) é um importante tema de estudo para a correta escolha e dimensionamento das estruturas de amortecimento, que protegem o circuito adutor da variação abrupta da pressão interna causada pela variação na velocidade média do escoamento. Este trabalho propõe a utilização da hidrodinâmica computacional (CFD) para avaliar a oscilação de massa ou nível d\'água no interior da estrutura de proteção denominada Chaminé de Equilíbrio e comparar esses resultados com a solução teórica analítica, com os critérios de dimensionamento definidos em norma técnica e com os dados de medições experimentais. O problema foi modelado no software Ansys CFX que utiliza o método dos volumes finitos (MVF) para resolver os campos de velocidade e pressão do escoamento, tanto em regime permanente, quanto em regime transiente. Aplicando-se o modelo CFD proposto ao circuito hidráulico didático, os resultados foram validados com a formulação teórica e com as medições experimentais do nível d\'água na chaminé. O modelo foi aplicado num estudo de caso que consiste num circuito hidráulico de PCH. Os resultados da oscilação de massa no interior da chaminé para este estudo de caso confirmam o emprego da fluidodinâmica computacional para avaliar tais escoamentos em regime transitório. / The hydraulic transient in the circuit of Small Hydroelectric Power Plants (SHP) is an important study subject for the correct choice and design of damping structure which protects the conduit from an abrupt variation of the internal pressure caused by the changes in the average flow velocity. This work proposes the use of computational fluid dynamics (CFD) to evaluate the mass oscillation or water level inside the protection structure called Surge Tank and compares these results with theory, design criteria defined by the technical standard, and experimental data. The problem was modeling in Ansys CFX software that uses the finite volume method (FVM) to solve velocity and pressure flow fields, in both steady state and transient flow. The proposed model used in a hydraulic circuit led to results validation with experimental measurements of water level surge tank. For the proposed case study, the response of the numerical CFD simulation confirms the use of computational fluid dynamics to evaluate such transient flows.

Abstract Multiphase Droplet Interactions with a Single Fiber By: Noor M. Farhan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2019 Director: Hooman V. Tafreshi, Professor, Department of Mechanical and Nuclear Engineering Formulating the physics of droplet adhesion to a fiber is interesting intellectually and important industrially. A typical example of a droplet–fiber system in nature is the dew droplets on spider webs, where droplets first precipitate and grow on the fibers, but they eventually fall when they become too heavy. Obviously, quantifying the force of adhesion between a droplet and a fiber is crucial in designing fog harvesting devices or manufacturing filtration media for liquid–gas or liquid–liquid separation, among many other industrial applications. This study is aimed at developing a mathematical framework for the mechanical forces between a droplet and a fiber in terms of their physical and wetting properties. To this end, a series of experiments were conducted to detach ferrofluid droplets of varying volumes from fibers with different diameters and Young–Laplace contact angles (YLCAs) in a controlled magnetic field. The force of detachment was measured using a sensitive scale and used along with the results of numerical simulations to develop a semi-analytical expression for the force required to detach a droplet from a fiber. This universally-applicable expression allows one to predict the force detachment without the need to run an experiment or a computer simulation. This work also reports on the use of magnetic force to measure the force of detachment for nonmagnetic droplets for the first time. This is accomplished by adding a small amount of a ferrofluid to the original nonmagnetic droplet to create a compound droplet with the ferrofluid nesting inside or cloaking the nonmagnetic droplet. The ferrofluid is then used to induce a body force to the resulting compound droplet and thereby detach it from the fiber. The recorded detachment force is used directly (the case of nesting ferrofluid) or after scaling (the case of cloaking ferrofluid) to obtain the force of detachment for the original nonmagnetic droplet. The accuracy of these measurements was examined through comparison with numerical simulations as well as available experimental data in the literature. In addition, a simple method is developed to directly measure the intrinsic contact angle of a fiber (i.e., Young–Laplace Contact angle of the fiber material) with any arbitrary liquid. It is shown that the intrinsic contact angle of a fiber can be obtained by simply measuring the angle between the tangent to the fiber surface and the tangent to the droplet at the contact line, if the droplet possesses a clamshell conformation and is viewed from the longitudinal direction. The novelty of the proposed method is that its predictions are not affected by the volume of the droplet used for the experiment, the wettability of the fiber, the surface tension of the liquid, or the magnitude of the body force acting on the droplet during the experiment. Also, a liquid droplet interaction with granular coatings is simulated and the droplet apparent contact angle (ACA) and the transition from Cassie (fully dry) to Wenzel (fully wet) state as a function to the roughness wavelength have been studied. For a fixed droplet volume, two different granular coatings have been used, spherical and hemispherical bumps. It is demonstrated that the chemistry (YLCA) and geometrical parameters for the granular microtexture play an important effect on the droplet ACA and its transition from Cassie to Wenzel state.

During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neuronal circuitry, and subsequent matching of glial numbers and position to that circuitry. Multiple molecular mechanisms act in concert to control neural precursor expansion prior to neurogenesis, and to allow for an exponential generation of neurons while ensuring the maintenance of sufficient precursors to produce later-born neurons, glial cells and adult neural stem cells. Throughout cortical development, these processes are regulated in part by the precursors’ environment as well as intrinsic changes in precursors and their modes of division, which regulate the fate of daughter cells and the balance between self-renewal and differentiation. In the first part of this thesis, the protein tyrosine phosphatase SHP-2 was identified as a novel signaling protein that regulates the neurogenic to gliogenic switch by potentiating neurogenic signals and suppressing gliogenic signals until the appropriate developmental time point for astrogenesis, providing one mechanism whereby precursors integrate conflicting environmental cues. A Noonan Syndrome (NS)-associated activated SHP-2 mutation causes perturbations in neural cell genesis, which may contribute to the mild mental retardation and learning disabilities observed in NS patients. In the second part of this thesis, a novel Rho-regulatory pathway which includes the Rho-GEF Lfc and its negative regulator Tctex-1 were also found to regulate neurogenesis, potentially by directing mitotic spindle orientation during precursor divisions, thereby regulating the symmetric and asymmetric nature of radial precursor divisions.

During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neuronal circuitry, and subsequent matching of glial numbers and position to that circuitry. Multiple molecular mechanisms act in concert to control neural precursor expansion prior to neurogenesis, and to allow for an exponential generation of neurons while ensuring the maintenance of sufficient precursors to produce later-born neurons, glial cells and adult neural stem cells. Throughout cortical development, these processes are regulated in part by the precursors’ environment as well as intrinsic changes in precursors and their modes of division, which regulate the fate of daughter cells and the balance between self-renewal and differentiation. In the first part of this thesis, the protein tyrosine phosphatase SHP-2 was identified as a novel signaling protein that regulates the neurogenic to gliogenic switch by potentiating neurogenic signals and suppressing gliogenic signals until the appropriate developmental time point for astrogenesis, providing one mechanism whereby precursors integrate conflicting environmental cues. A Noonan Syndrome (NS)-associated activated SHP-2 mutation causes perturbations in neural cell genesis, which may contribute to the mild mental retardation and learning disabilities observed in NS patients. In the second part of this thesis, a novel Rho-regulatory pathway which includes the Rho-GEF Lfc and its negative regulator Tctex-1 were also found to regulate neurogenesis, potentially by directing mitotic spindle orientation during precursor divisions, thereby regulating the symmetric and asymmetric nature of radial precursor divisions.

The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transduction pathways, thereby treating cancer. We attempted to synthesize compounds based on the hits obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, history of chemotherapeutic drugs and an introduction to signal transduction pathways. The following two chapters briefly introduce the biological targets in the authors study. Chapter Two describes the role of B-cell lymphoma type xL (Bcl-xL), in apoptosis and the development of drugs targeting Bcl-xL. Examples of Bcl-xL drugs relevant to this study have been provided. Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathways and the recent developments in drug development towards the inhibition of this oncogene. Chapter Four gives a general introduction to microwave-assisted organic synthesis and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author's own findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibitors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL. Our strategy focused on identifying an isoquinolinol lead with increased potency. Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency.

Although the etiology surrounding many autoimmune diseases remains unknown, the underlying characteristic of many of these diseases is a disruption in the balance of pro- and anti- inflammatory cytokines It is well established that the dysregulation of the IL-12 family of cytokines, an increase in IL-12/IL-23 and a decrease in IL-27 production has been implicated in these conditions. We used ELISA, RT-PCR, Immunofluorescence and Western immunoblotting in conjunction with pharmalogical inhibitors and siRNA to demonstrate the role of SHP-1/Src in the regulation of IL-12, IL-23, IL-27 and IL-10 in LPS-stimulated human THP-1 cells, monocytes and MDMs. My results show for the first time that Src kinase activity relies on SHP-1 activity, and together this complex functions in TLR4-mediated MyD88 and TRIF pathways. Furthermore Src exhibits a dual role as a positive regulator for anti-inflammatory IL-10/IL-27 and as a negative regulator of pro-inflammatory IL-12/IL-23 downstream of TLR4. Moreover, the involvement of PI3K and JNK MAPK, dependent on SHP-1/Src complex, in the regulation of IL-12 family and IL-10 downstream of TLR4 was shown.

La protéine tyrosine phosphatase SHP-2 est connue pour jouer un rôle important dans le maintien de l’homéostasie de plusieurs tissus et organes par ses effets régulateurs sur plusieurs voies de signalisation intracellulaire. Notre laboratoire a récemment démontré que la délétion embryonnaire de SHP-2 (SHP-2CEI-KO) à l’épithélium intestinal entraîne le développement rapide d’une inflammation colique sévère. La délétion ayant lieu au stade embryonnaire, une altération dans le développement intestinal pourrait être en cause dans l’initiation de cette inflammation. Afin d’éliminer cette possibilité, un modèle de délétion conditionnelle inductible de SHP-2 dans les cellules épithéliales intestinales (SHP-2CEI-KOER) a été généré, la délétion ayant été induite par injection de tamoxifène trois mois après la naissance. Ce modèle a permis d’éliminer la composante développementale, la délétion ayant lieu chez la souris adulte. De manière intéressante, les souris subissant la délétion de SHP-2 développent aussi une inflammation colique suite à 18 jours de traitement au tamoxifène. Cette inflammation s’accompagne d’une diminution de l’activité de la signalisation MEK/ERK MAPK et d’une activation de la signalisation JAK/STAT. Des altérations dans la différenciation des cellules caliciformes et de Paneth sont observées, les souris expérimentales présentant une diminution du nombre de cellules caliciformes ainsi qu’une présence aberrante de cellules intermédiaires (précurseurs des cellules caliciformes) dans leur côlon. Cette diminution du nombre de cellules caliciformes est présente dès le 10e jour de traitement au tamoxifène, précédant donc l’apparition des signes d’inflammation colique, ceux-ci débutant seulement au 12e jour de traitement au tamoxifène. Nos résultats sur une lignée cellulaire capable de se différencier dans un phénotype caliciforme, les LS174T, montrent une activation de la voie Notch suite à l’inactivation de la voie ERK/MAPK, cette activation étant associée à une augmentation de l’expression des gènes associés aux cellules caliciformes. Ces résultats corrèlent avec l’observation que MEK/ERK est inhibée dans l’épithélium déficient pour l’expression de SHP-2 alors que celle de Notch est activée. Ces résultats nous indiquent donc un rôle important de la tyrosine phosphatase SHP-2 dans le maintien de l’homéostasie intestinale, et ce, même chez l’adulte. L’inactivation de SHP-2 résulte en effet dans la perte de l’intégrité de la muqueuse colique amenant l’inflammation. De manière intéressante, des polymorphismes (SNP) dans le gène encodant SHP-2 ont été récemment associés à une susceptibilité accrue à développer une colite ulcéreuse chez des patients. Pris ensemble, ces résultats suggèrent donc que SHP-2 pourrait constituer une nouvelle cible dans le traitement des maladies inflammatoires intestinales.

Although the etiology surrounding many autoimmune diseases remains unknown, the underlying characteristic of many of these diseases is a disruption in the balance of pro- and anti- inflammatory cytokines It is well established that the dysregulation of the IL-12 family of cytokines, an increase in IL-12/IL-23 and a decrease in IL-27 production has been implicated in these conditions. We used ELISA, RT-PCR, Immunofluorescence and Western immunoblotting in conjunction with pharmalogical inhibitors and siRNA to demonstrate the role of SHP-1/Src in the regulation of IL-12, IL-23, IL-27 and IL-10 in LPS-stimulated human THP-1 cells, monocytes and MDMs. My results show for the first time that Src kinase activity relies on SHP-1 activity, and together this complex functions in TLR4-mediated MyD88 and TRIF pathways. Furthermore Src exhibits a dual role as a positive regulator for anti-inflammatory IL-10/IL-27 and as a negative regulator of pro-inflammatory IL-12/IL-23 downstream of TLR4. Moreover, the involvement of PI3K and JNK MAPK, dependent on SHP-1/Src complex, in the regulation of IL-12 family and IL-10 downstream of TLR4 was shown.