A Versatile ΦC31 Based Reporter System for Measuring AP-1 and Nrf2 Signaling in Drosophila and in Tissue Culture

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase

CAPILLARY FORCES ON SEDIMENT PARTICLES EXPERIMENTAL MEASUREMENTS AND THEORETICAL ESTIMATES

At the Hanford Site, radioactive wastes have been disposed into the subsurface. These radionuclides move with infiltrating water both in solution and via adsorption on mobile colloidal soil particles. Extensive research has been directed to understand the mechanism of colloid transport: at field-scale in lysimeters, at laboratory scale in columns and by indirect field measurements. This dissertation investigates experimental and theoretical methods to determine capillary forces on millimeter-scale particles in the subsurface. The specific objectives of this dissertation were:1. Measure capillary forces on irregularly shaped sediment particles and compare with theoretical estimates.Capillary forces due to a moving air-water interface were measured experimentally on three PTFE particles of standard shape (sphere, circular disc, square tent) and seven natural sediment particles (basalt, granite, hematite, magnetite, mica, milky quartz, quartz) using a tensiometer. Theoretical calculations were done to estimate maximum capillary forces, assuming the particles to be spherical, cylindrical, and ellipsoidal. The ellipsoidal model was found to give the best approximation of the capillary forces.2. Measure capillary forces on model particles of standard shapes and compare specific features of the force-position curves with theoretically reconstructed curves.Capillary forces due to a moving air-water interface were measured experimentally on nine model polyacrylate particles. Particles were divided into three categories (rounded, fixed, and tapered) based on cross-section. The theoretical reconstruction of the force-position curves indicated that the pinning, snap-in and snap-off of the air-water interface on sharp edges and variation of cross-section along z-axis dictated the shape of the force-position curve.3.Develop a numerical model to predict capillary forces on subsurface particles.Surface roughness features of basalt and quartz sediment particles were reconstructed at triangle resolutions of 3%, 5% and 10% of maximum (root mean squared) dimensions using scanning electron microscopy. Force-position curves were measured experimentally and calculated theoretically for comparison to determine an optimum mesh resolution. The results provided a lower size-limit of roughness features which can affect capillary forces on mm-scale particles interacting with a moving air-water interface

Identification and characterization of proteins with novel functions in Nrf2 signaling

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2015.Oxidative stress causes widespread damage to biomolecules, leads to different pathological conditions and contributes to aging. The Nrf2 transcription factor, a major mediator of oxidative stress responses, controls gene expression programs that protect multiple organs from oxidative damage, delay the onset of some age-associated diseases and promote longevity at least in some organisms. In an unstressed condition, Nrf2 interacts with its cytoplasmic inhibitor Keap1, which targets it for proteasomal degradation. Oxidative stress prevents Keap1-mediated degradation of Nrf2, resulting in its accumulation and nuclear translocation. In the nucleus, Nrf2 dimerizes with a small Maf protein, binds to ‘Antioxidant Response Elements’ and induces multiple antioxidant and detoxification genes. Complete understanding of the molecular mechanisms of Nrf2 regulation is important to assess its role in normal physiology and disease. The key components of Nrf2 signaling are conserved in Drosophila where CncC is the homolog of mammalian Nrf2. In order to study mechanisms of Nrf2 function in Drosophila, cell-based and in vivo transcriptional reporters for Nrf2 were developed. A cell-based dsRNA library screen was carried out to find novel regulators of Nrf2 signaling. Among others we identified Cdk12 and Fs(1)h. Cdk12, a RNA PolII-CTD kinase, was found to be required for CncC target gene expression in a cell-autonomous manner and to be important for oxidative stress resistance. In contrast, Fs(1)h, the sole member of the BET protein family in Drosophila, was identified as an inhibitor of CncC. Bromodomain-containing BET proteins have complex functions in chromosome organization and the control of gene expression. Fs(1)h was found to physically interact with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and induces protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from that of Keap1. Consistent with this, combinations of drugs that can specifically target Keap1 and Fs(1)h cause a synergistic and specific activation of CncC dependent gene expression. This synergism might be exploitable for the design of combinatorial therapeutic approaches, targeting Nrf2 in various diseases

Transgenic ARE reporters and TRE reporters are specifically responsive to Nrf2 and JNK signaling respectively.

<p>Loss of dKeap1 function specifically activates Nrf2 activity <i>in vivo </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034063#pone.0034063-Sykiotis3" target="_blank">[24]</a>. Consequently, expressing a Keap1-specific RNAi under the control of the ubiquitously active arm-Gal4 driver activated the ARE-green reporter, but not the TRE-red reporter. Conversely, arm-Gal4 driven expression of RNAi targeting Puc, the negative regulator of JNK signaling, stimulated TRE-red reporter activity but did not affect the ARE-green reporter. The upper panels show red and green fluorescence in adult 5-day-old female flies. These fluorescence images were superimposed onto the bright-field images of the same flies, as shown in the lower panels. 15 to 20 flies were analyzed for each genotype and all showed similar results. The images shown show representative, randomly selected specimens.</p

Stimulation of cell-based reporters by sulforaphane, oltipraz and stressors.

<p>S2 cells transiently transfected with TRE-fluc, ARE-fluc or mRE-fluc plasmids, as indicated, were exposed to sodium arsenite (NaAsO₂), Diethyl Maleate (DEM) or oltipraz. The firefly luciferase activity was measured 24 hrs after drug treatment and normalized to renilla luciferase activity expressed from a co-transfected Act5C renilla luciferase construct. Note that both TRE and ARE reporters respond to the stressors, Arsenite and DEM, but only ARE-fluc responds to Oltipraz. The mRE reporter responds neither to oltipraz nor to the stressors. The activity of mRE-fluc reporter under control conditions was set to 1. Error bars indicate standard deviation of triplicate measurements.</p

Signal specificity of cell-based reporters.

<p>A. Artificial stimulation of JNK and Nrf2 signaling in S2 cells specifically activates the TRE-fluc or ARE-fluc reporter, respectively. Expression of a constitutively active mutant of <i>Drosophila</i> JNKK (Hep^act) activated a co-transfected TRE-fluc reporter, but not an ARE-fluc reporter. Conversely, overexpression of CncC increased the activity of the ARE and not the TRE reporter. Firefly luciferase activity was normalized using a cotransfected renilla luciferase construct (Act5C rluc) as a reference. The activity under control conditions (reporters co-transfected with pAct-Gal4) was set to 1. B. Signal-dependent activation of TRE and ARE reporters specifically requires JNK or Nrf2 pathway components. S2 cells were transfected with TRE-fluc reporters and JNK activity was stimulated by co-expression of constitutively active <i>Drosophila</i> JNKK (Hep^act) as indicated. Cells transfected with the ARE-fluc reporter were treated with the specific Nrf2 activator Oltipraz. dsRNA mediated knockdown of CncC, or MafS compromised only Oltipraz-induced ARE-fluc activity. Conversely, dsRNA against Hep (JNKK) and Bsk (JNK) only reduced TRE activity. Firefly luciferase activity was normalized to renilla luciferase activity driven by Act5C promoter. The activity of the activated reporters under control conditions was set to 1. Error bars indicate standard deviation of triplicate measurements.</p

Effect of Particle Shape on Capillary Forces Acting on Particles at the Air–Water Interface

The capillary forces exerted by moving air–water interfaces can dislodge particles from stationary surfaces. The magnitude of the capillary forces depends on particle shape, orientation, and surface properties, such as contact angle and roughness. The objective was to quantify, both experimentally and theoretically, capillary force variations as an air–water interface moves over the particles. We measured capillary forces as a function of position, i.e., force–position curves, on particles of different shape by using force tensiometry. The particles (5 mm nominal size) were made of polyacrylate and were fabricated using a 3D printer. Experimental measurements were compared with theoretical calculations. We found that force–position curves could be classified into in three categories according to particle shapes: (1) curves for particles with round cross sections, such as spheroidal particles, (2) curves for particles with fixed cross sections, such cylindrical or cubical particles, and (3) curves for particles with tapering cross sections, such as prismatic or tetrahedral particles. Spheroidal particles showed a continuously varying capillary force. Cylindrical or cubical particles showed pronounced pinning of the air–water interface line at edges. The pinning led to an increased capillary force, which was relaxed when the interface snapped off from the edges. Particles with tapering cross section did not show pinning and showed reduced capillary forces as the air–water interface line perimeter and displacement cross section continuously decrease when the air–water interface moved over the particles

Effect of Particle Shape on Capillary Forces Acting on Particles at the Air–Water Interface

The capillary forces exerted by moving air–water interfaces can dislodge particles from stationary surfaces. The magnitude of the capillary forces depends on particle shape, orientation, and surface properties, such as contact angle and roughness. The objective was to quantify, both experimentally and theoretically, capillary force variations as an air–water interface moves over the particles. We measured capillary forces as a function of position, i.e., force–position curves, on particles of different shape by using force tensiometry. The particles (5 mm nominal size) were made of polyacrylate and were fabricated using a 3D printer. Experimental measurements were compared with theoretical calculations. We found that force–position curves could be classified into in three categories according to particle shapes: (1) curves for particles with round cross sections, such as spheroidal particles, (2) curves for particles with fixed cross sections, such cylindrical or cubical particles, and (3) curves for particles with tapering cross sections, such as prismatic or tetrahedral particles. Spheroidal particles showed a continuously varying capillary force. Cylindrical or cubical particles showed pronounced pinning of the air–water interface line at edges. The pinning led to an increased capillary force, which was relaxed when the interface snapped off from the edges. Particles with tapering cross section did not show pinning and showed reduced capillary forces as the air–water interface line perimeter and displacement cross section continuously decrease when the air–water interface moved over the particles

Simultaneous measurement of Nrf2 and JNK responses <i>in vivo</i>.

<p>Exposure of flies that carry two copies each of both the TRE-red-2R and the ARE-green-2L reporters to the oxidative stressor paraquat increased both ARE and TRE activities, however with different spatial specificity. Oral application of the cancer chemopreventive drug oltipraz, on the other hand, only induced ARE and not TRE activity. The upper and the middle panels show green and red fluorescence separately in the same flies. These green and red fluorescence images are merged in the lower panel. The control food for oltipraz treatment was supplemented with 1% DMSO, the solvent used for oltipraz. The control reporter stock mRE-red-2L did not respond to either paraquat or oltipraz.</p