Molecular self-assembly of thiols onto gold is a powerful method for surface modification in biomaterials research. For instance, thiolated sugars can form glycan-functionalized surfaces for carbohydrate microarray and biosensor fabrication. To illuminate glycosylated surface composition and structure, we utilized a model system consisting of mixed-thiol self-assembled monolayers (SAMs) of a synthetic tetrasaccharide and oligo(ethylene glycol) on gold(111). Here, we show that atomic force microscopy (AFM) analysis of these mixed SAMs reveals two distinct domains that appear to coalesce and phase separate over time. X-ray photoelectron spectroscopy demonstrates a relationship between the compositions of sugar on the surface to its molar ratio in solution. These results suggest that clustering of sugar is thermodynamically favorable, occurring within mixed sugar/oligo(ethylene glycol) SAMs.

The purpose of this study is to create new research tools for characterizing extracellular compounds that are produced by plant endophytes that are difficult to culture. There has been recent evidence that suggest some endophytes produce compounds that are similar to hydrocarbons, which could be useful for making bio-fuels. To detect traces of hydrocarbons, we are using Polydimethylsiloxane (PDMS), which is a polymer material that strongly absorbs organic materials from water, thus concentrating the organics for easier detection. Our procedure involves forming a nanometer thin layer of PDMS on the surface of a Quartz Crystal Microbalance (QCM) crystal and measuring the mass of absorbed organic material as a function of solution concentration. The proportionality constant between absorbed organic material and its solution concentration is the partition coefficients (K). So far, we have been working on how to make uniform nanometer thin PDMS layer on the surface of a QCM crystal. We have found that PDMS and catalyst at 0.5% (weight/volume) in a hexane solvent can create thin layers of PDMS on the QCM crystal when we drop-cast it on a hydrophobic functionalized surface. We are performing further tests of the % weight per volume of the PDMS and catalyst with hexane to ensure we obtain good uniform nanometer thin PDMS layers, which would allow more accurate measurements of K.

Animals control their movement by integrating sensory information from a range of modalities, including visual and mechanical information. In flies, modified hindwings, called halteres, that flap out of phase with the forewings provide gyroscopic sensory information that complements visual information. When in flight, Coriolis forces act on the halteres to produce deflections. Sensory organs at the base of the halteres interpret the deflections and rapidly integrate information from the Coriolis forces into the flight pattern. Halteres respond to stimuli much faster than the visual system, allowing flies to alter their wingstrokes within milliseconds. While recent research establishes a neural basis for the encoding of Coriolis forces in halteres, the biomechanical stimuli that occur naturally on halteres remain unknown. We seek to quantify the strain patterns on halteres and ultimately explore the link between biomechanical inputs and the neural circuits. To do so, we calculate the flexural stiffness of collected halteres by applying point forces to the halteres and measuring their resulting deflections using digital photography. The flexural stiffness is critical to determining the strain that is placed on the sense organs at the base of the halteres, which stimulate neurons. We digitize high-speed videos of oscillating halteres to determine their resonant frequencies. Data from these experiments, as well as high-speed video of halteres in free-flight, is used in a three-dimensional finite element model. The finite element model enables us to find the strain patterns on the halteres, as these patterns cannot be determined experimentally. Through construction of this model, we expect to quantify the direct stimulus to the nervous system. When coupled to known neural circuits of flies, these data will indicate what stimuli are required for specific neural outputs.

The timing and strength of muscle activation underlies the control of animal movement. This is particularly important in maneuverability of animals in complex environments. Insect flight is an ideal model for examining motor control because we have considerable insight into the mechanical basis for flight and muscle activity is easily recorded and analyzed. Wing motions are produced primarily by two pairs of power muscles driven at a constant phase and modulated by a suite of highly variable steering muscles. However, phase of activity in power muscles could have important control consequences, since the timing of muscle activity relative to wing strokes is an essential component of muscle function and can significantly alter work output. We hypothesized that the control of turning maneuvers is a product of steering muscles, while power muscles express their presumed constant timing of activation. We examined left-right timing differences of bilateral power muscles, specifically the dorso-ventral muscles (DVMs) and dorso-longitudinal muscles (DLMs), in induced turns to determine if visual induced neural feedback could significantly alter power muscle function. We visually stimulated tethered hawkmoths with a projected vertical-banding pattern and recorded action-potentials from DVMs and DLMs using implanted tungsten electrodes. We found that both pairs of power muscles varied significantly in their phase of activation during visually induced maneuvers (p< 0.001). The time separation of the muscles was significantly correlated with the visual stimulus (p< 0.001) with a delay (~100ms) consistent with visual processing. These results reject the hypothesis that power muscles maintain an invariant power-generating function. We found that Manduca alter the timing of power muscles to significantly modulate muscle work output. By exploring the means of controlling maneuverability, we begin to unravel the complexities of locomotor control for bio-inspired applications.

Control of movement in animals requires the coordination of input from multiple sensory systems with output of multiple actuators. This coordination is particularly crucial in the control of flight in insects. While wing motions are thought to dominate flight forces, relatively little attention has been given to the role of other control surfaces, including abdominal motions in flight control– either as an aerodynamic rudder or as a way of shifting the center of mass relative to the center of lift. To understand the potential role of abdominal flexion in flight control, prior studies have focused on either tethered preparations or on observations of natural abdominal motions during flight maneuvers. However, recent advances in radio technology, along with the development of neural and muscle stimulation methods provides new methods for understanding movement control. We use radio telemetric methods to deliver stimuli to the ventral nerve cord in the hawkmoth Manduca sexta. Stimulation of the ventral nerve cord elicits motor neuron activation of muscles controlling the position of the abdomen. This remote control of abdominal motions provides direct tests of the potential role of the abdomen in determining flight trajectories. We used a PIC combined with an ultra-miniature radio receiver to deliver pulse-width modulated, bipolar stimulation (100 Hz, duty cycle 50%, ±2.5 – 3V) to freely flying moths. We stimulated the moth by placing tungsten electrodes and Flexible Multisite Electrodes (FMEs) in contact with the ventral nerve cord. Animals showed a change in pitch (nose up or nose down) or yaw (lateral roations) in response to that stimulation and resulting abdominal flexion. These results show that we can elicit abdominal movement remotely in freely-flying animals (both open air and wind tunnel), and further indicate that such abdominal flexion is indeed involved in the control of flight paths.

Due to the plentiful and diverse solutes found in cells, solute-solute interactions are an important part of cellular chemistry. In 2003, Zheng and Pollack observed that charged microspheres suspended in water avoided a region up to 100 µm away from a gel-water boundary. This suggests that solute-solute interactions extend for a much larger distance than previously believed. Although the mechanism of this “exclusion zone” phenomenon is unclear, one possible cause is the presence of a pH gradient around the gel-water boundary, which could have exerted a force on the charged microspheres directed away from the boundary. The Chiu lab seeks to test the viability of the pH gradient theory as an explanation of the exclusion zone phenomenon via microchips. The experiment consists of two stages: first, a pH gradient is established inside a microfluidic channel via the laminar flow of phosphate buffers with differing pH. Second, the behavior of charged microspheres is observed with respect to the pH boundary via microscopy. The preference of the charged microspheres for one side of the pH gradient would confirm pH gradients as the cause of exclusion zones, thus significantly contributing to current scientific knowledge of solute-solute interactions and cellular chemistry. Specifically, the forces caused by a pH gradient could be used to understand cellular chemotaxis: a cellular transport mechanism where the cells migrate towards areas of lower pH to escape their own metabolic waste products. Ultimately, this research could be used to create a novel microfluidic purification method which utilizes pH gradients as the basis to separate the components of extremely small chemical mixtures by charge.

Fascioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant myopathy characterized by a truncation of a repeat region on chromosome 4, called D4Z4. The D4Z4 repeat is also located on chromosome 10; however, truncation of this repeat does not confer the disease. The open reading frame located in the D4Z4 repeat region is called Dux4 and is toxic to muscle cells. In patients without FSHD the D4Z4 locus is heterochromatinized and Dux4 is not expressed, despite evidence for active transcription in the region. In FSHD patients, the D4Z4 repeat is not heterochromatinized and Dux4 can be detected. This project aims to test the hypothesis that a bidirectional promoter in each D4Z4 drives the expression of Dux4 within its own repeat and an antisense transcript from the previous repeat. The balance of sense and antisense transcripts may regulate Dux4 expression. To test the bidirectional transcriptional activity of the Dux4 promoter, we created a plasmid that had the Dux 4 promoter transcribe renilla luciferase in the forward direction and firefly luciferase in the reverse direction. This allowed us to measure the Dux 4 promoter activity by measuring the relative luminescence of renilla and firefly luciferase. Assays of the Dux4 promoter have shown forward transcription in differentiated cells. In undifferentiated cells; however, the Dux4 promoter activity is stronger in the reverse direction therefore showing bidirectional activity. By testing the bidirectional activity of the Dux4 promoter we are looking for differences in bidirectional activity between normal cell lines and diseased cell lines. Differences in the bidirectional activity between normal cell lines and diseased cell lines will show us that a truncation in the D4Z4 region changes the balance between sense and antisense transcripts, therefore, regulating the expression of Dux4.

Algal blooms are the swift population increase of algae in an area. Some harmful algal blooms, HABs, can result in mass mortality events in marine life, human poisoning, and hypoxia. Algal bloom occurrences are not solely due to reproduction; researchers hypothesize that these algae are transported by currents and aggregate by swimming to favorable conditions. This algal movement depends on the swimming patterns and stability of the organism. Our focus is Heterosigma akashiwo, a species of algae which develops in a variety of shapes and sizes. We aim to determine whether particular morphologies have greater success in navigating and overcoming turbulence to reach the surface of the ocean. The computer program MATLAB functions as a numerical computing environment to run a turbulence model with various dissipation rates is run with a cell of a particular chosen morphology. MATLAB determines cell movement patterns contingent on varying intensities of turbulence. The cell morphology model is determined by many variable factors including differential density, asymmetries in body structure, center of buoyancy, center of gravity, corrective torque, and steering ability. Models of variations on morphology will then be run with differing turbulence dissipation rates for a set amount of time to determine in what direction they are moving and how they are oriented in the process. This model will allow us to determine why H. akashiwo has evolved with such a diversity of morphologies, and if particular shapes and/or sizes are more beneficial in different conditions or allow more rapid aggregation to sites of favorable conditions for algal blooms. By targeting the formation patterns of HABs, our research will contribute to monitoring these algal blooms and developing preventative measures.

Hydrogen fuel cells are one of the cleanest ways to efficiently convert fuel to electricity. In a hydrogen fuel cell, hydrogen and oxygen react to form water, producing electricity in the process. Understanding fuel cells’ aging processes is a critical step in making them cost-effective. As a fuel cell ages, water build-up in the gas diffusion layer (GDL) increase, constricting oxygen flow into the catalyst layer, where the cell’s reactions take place, reducing efficiency. Therefore, in order to make fuel cells more durable, research in GDL materials is necessary. This research focused on characterizing the wetting properties of the GDL as a function of fuel cell age. The aging process was simulated rapidly using electrochemical oxidation. For sample characterization, capillary pressure measurements quantified the wetting of the GDLs, while cyclic voltammetry determined the extent of surface oxidation. The results showed that increased surface oxidation correlated to enhanced wetting properties. Additionally, the characterization of samples from Los Alamos National Lab that had been “naturally” aged in real fuel cells indicated that factors other than surface oxidation, such as platinum contamination, were also important in changing the wetting properties of GDLs. How the graphitic content of the GDL affects its wetting properties and accelerates aging is being tested. In addition to the techniques mentioned above, Raman spectroscopy is used to determine the graphitic content of the samples. Characterizing the wetting properties of the GDL as a function of different variables, such as age and graphitic content, is key to understanding which materials are the most durable and why. This understanding will lead to improved fuel cell design and increased utility.

The radiotracer, [F-18]-fluoro-3'-deoxy, 3'-L-fluorothymidine (FLT), is used in positron emission tomography (PET) to detect in vivo tumor proliferation. The ability to trace tumor proliferation is a useful tool to determine the effects of cancer treatments on tumor cells. However, a better understanding of FLT efflux in cells is needed for a more accurate measure of cellular proliferation. Although it has been shown that thymidine kinase-1 (TK1) activity does not solely reflect FLT uptake in cells, it is necessary to compare the rate at which FLT is metabolized and the rate at which the native thymidine (TdR) is metabolized. The transport of FLT and other nucleotides and the concentration of free nucleotides in the cell also affect FLT efflux. Determining how TK1 activity relates to these other factors enables a better understanding of how FLT uptake is affected by various cancer treatments. FLT is very similar to TdR in structure with an addition of a fluorine isotope that does not allow it to be incorporated into DNA. TK1 is a cytosolic, cycle dependent enzyme that is active during the synthesis phase of DNA. The enzyme is part of the salvage pathway and its main function is to phosphorylate TdR to thymidine mono-phosphate (TMP) once it enters a cell. Once DNA synthesis is completed, TK1 is degraded. Thus, when cells are in a resting state, no TK1 activity should be measured. FLT metabolism also undergoes the same salvage pathway as TdR. To measure the rate at which TdR gets metabolized in vitro, TK assays were preformed. Radiolabeled TdR were added to cell lysates in order to expose the cytosolic TK1 enzyme. Then, at various time points (usually between 0 – 15 min), samples were taken in order to measure how much the radiolabeled TdR was converted into TMP.

The p53 gene codes for a tumor suppressor protein that regulates the cell cycle. P53 plays an anti-cancer role by activating DNA repair, arresting the cell cycle at the G1/S phase transition for DNA damage recognition, and inducing programmed cell death (apoptosis) if DNA damage is irreparable. The gene has a polymorphism on codon 72 that can cause an Arginine amino acid in the protein to be substituted by a Proline amino acid. Patient genotype can then either be homozygous for the Arginine variant, homozygous for the Proline variant, or heterozygous (having both Proline and Arginine variants). Our lab is working on a project to analyze the genotypes of patients who have been exposed to radiation. This involves taking cells from the blood and infecting them with Epstein-Barr virus to immortalize them so they can be stored for long periods of time. After isolating DNA from these cells, PCR is performed with primers specific for the Arginine or Proline variants. PCR mixes are separated on a gel to identify the presence or absence of products, indicating the patient genotype. We will enter this information into a database and compare their frequencies with the normal population using a chi square test. This statistical analysis allows us to determine whether or not our data is similar to the regular healthy population by measuring the variance.

3'-deoxy-3'-fluorothymidine (FLT) is a newly developed PET imaging tracer. As a radioactive analog of the DNA precursor thymidine, FLT was predicted to concentrate in cancerous tissues, given the high demand rapidly-dividing tumor cells have for thymidine. After preliminary tests, cancer cell-lines (A549 lung cells and TK6 B-lymphocytes) have been shown to uptake FLT through more complex mechanisms than previously believed. As part of our lab’s overall goal to understand these mechanisms I am working on a project to develop background data on the effects anti-cancer treatments have on FLT uptake in cancer cells. The first step is to construct a series of survival curves for A549 cells treated with radiation, Taxol, and 5-fluorouracil, three common agents used in cancer treatment. By adding a known number of cells to a series of growth plates, and treating each plate in the series with increasing doses of a single anti-cancer agent, the resulting surviving cells can be counted and compared to the starting population. This data is then graphed, resulting in a curve showing the thresholds for lethal and sub-lethal doses of a given treatment. Analysis of these curves is then used to determine the optimal dosage needed to affect the behavior of the cancer cells without destroying them. Test cancer cells are then treated with this optimal dosage, followed by FLT exposure. By comparing the resulting differences in FLT uptake between treated and non-treated cells, it is possible to develop a clearer understanding of how FLT uptake is regulated by the cells. From a diagnostic standpoint, this information can be used to better analyze PET images of tumors by knowing how much a given treatment will change a tumor’s FLT uptake.

Currently, cancer treatments use positron emission topography (PET) imaging with 3’-deoxy-3-[F-18]fluorothymidine (FLT) as a tracer to measure tumor growth. We know FLT accumulates in tumors because thymidine uptake is higher in proliferating cells due to increased DNA synthesis during S phase of the cell cycle. We want to quantify the factors that affect FLT uptake so that doctors will know how to interpret FLT-PET images to assess the effectiveness of an anti-tumor treatment and switch patients to new drugs sooner if the current treatment is shown to be ineffective. Many factors affect FLT uptake in cells. The focus here is FLT uptake via Equilibrative Nucleoside Transporters (ENTs), which are transmembrane transport proteins that facilitate the uptake of nucleotides like thymidine into the dividing cell. Previous work at the Schwartz lab has shown that drugs, radiation and growth conditions all affect FLT uptake. We are now looking at changes in ENT activity to characterize how FLT uptake changes in response to drug treatments. We will look to see if transporter quantity changes in response to treatment and whether the number of transporter sites on the cell membrane also varies. We also want to look at the localization of the transporters in the cell in response to anti-cancer treatments, such as radiation, 5-fluoruracil, and paclitaxel.

The dorsal raphe nucleus (DRN) lies in the midbrain and provides serotonergic innervation to multiple areas in the forebrain. One of these areas is the caudate-putamen complex or dorsal striatum (DS) which current research suggests is further divided into distinct subregions. The DS has been implicated in the obsessions that characterize obsessive-compulsive disorder and other addictive behaviors. Since some of these disorders respond to medicines that target the serotonin system, we investigated the serotonergic innervation of the rat DS by stereotaxically injecting retrogradely transported red (excitation at 605 Hz) and green (excitation at 515Hz) fluospheres. We used 35 Sprague Dawley rats ranging in weight from 300-360g. The red and green fluospheres were injected at three different coordinates: anterior dorsal medial, posterior dorsal medial and dorsal lateral striatum. After a five day waiting period, to allow the tracers to reach the DRN, rats were transcardially profused and 40µm coronal sections prepared on a vibratome were mounted on slides to be viewed under a fluorescent microscope. Preliminary data show fluosphere injections in the forebrain successfully transport retrogradely to discrete regions within the DRN and we are performing stereological analysis to quantify these results. Additionally, we will be using immunohistochemistry to verify the identity of these neurons as serotonergic. As an anatomical control, fluorescent signal was detected in the substantia nigra (SN) and signal was strongest in the rostral sections of the DRN corresponding to previous literature. The visualization of discrete subregions of DRN projecting to discrete subregions of the DS provide a rationale for further analysis of this circuitry.