Role of Scyba in Neural Development
Eric Armour, '07, and Nancy Wall, Biology
Chemokines are responsible for directing movement of immune system cells. One such chemokine, known as BRAK or CXCL14 in humans, has an ortholog, Scyba, in zebrafish. This gene has been studied extensively in adult humans because its expression is down regulated in many types of cancer, most specifically in squamous cell carcinoma. Scyba serves as a chemattractant for both dendritic cells and monocytes, and is up regulated in inflammatory cells. However, many chemokines are expressed early in embryonic development, long before the immune system is active. Because of the attention to its role in cancer, very little research has looked for the embryonic role of scyba, even though a specific gene expression pattern has been identified. Scyba is expressed 12 hours post fertilization in the midbrain-hindbrain boundary, a very important signaling center in embryonic development. In addition scyba is expressed in the otic placodes, precursors to the adult ear, and a portion of the diencephalon. Unpublished data has suggested that down regulation of scyba has fatal consequences to the developing embryo, causing severe neural abnormalities. Using morpholinos, an antisense oligonuceotide that down regulates gene functions, we hope to see what changes occur in the developing nervous system. With in situ hybridization, a method used to locate specific gene expression, we can monitor the morphological changes within the embryo due to down regulation of scyba. Future studies should determine the effects of up regulation of the gene. Using these data, the role of scyba in early embryonic development can be elucidated.
Site Mutagenesis of mGDF1 for Biochemical and Functional Analysis in
Zebrafish
Elizabeth K. McLeod, '06, and Nancy Wall, Biology
Transforming Growth Factor (TGF) genes play a role in establishing body axes and germ layers in the vertebrate embryo. Vg1 is a member of the TGF superfamily and has a role in left-right patterning of the vertebrate organs. This gene has been closely analyzed in the Xenopus, zebrafish and chick model systems, but a mammalian Vg1 ortholog has not been identified. In a study by Wall et al. (2000), a mouse expression library was screened with a Vg1-specific monoclonal antibody, and mouse GDF1 (mGDF1) was identified as the single cross reactive clone. Sequence analysis showed that while mGDF1 shares a higher homology with Vg1 of zebrafish, chick, and Xenopus than other TGF proteins, the level of homology is lower than expected for an ortholog. However, mGDF1 functions like Vg1 in Xenopus embryos. Further sequence analysis showed that the region for the putative receptor binding loops of GDF1 and the Vg1 orthologs are highly conserved. Of the five residues that make up the binding domain, GDF1 was identical to 4 out of 5 residues in most cases and was identical to chick Vg1 at all five positions. Former students have performed PCR mutagenesis of the mGDF1 sequence, specifically in this region, to make the protein more like a different member of TGF family, BMP4. They have made PCR products that must be ligated into vectors and sequenced to confirm whether or not the site mutations were introduced into mGDF1. After control reactions were performed to be sure all reagents were reliable, I successfully ligated the PCR fragments into vectors. I have also prepared mRNA using in vitro transcription encoding for mGDF1 (without PCR mutagenesis) which will be used to repeat the mGDF1 functional analyses that were done in Xenopus using zebrafish as a model system.
Motion of Non-Uniform Strings
Claire Weiss, '07, Erik Garbacik, '08, and David Cook, Physics
A well-known problem studied in physics is the motion of a string of uniform density, specifically the study of its normal modes of oscillation and its relative frequencies. What happens if the string does not have a uniform density? For example, a cello string that has varying density (either due to poor manufacturing, rosin build-up, or the elastic properties of the string) may vibrate differently than a uniform cello string, and the instrument could therefore sound quite different due to a shift in the positions of the higher harmonics. In this study, we explore solutions to the wave equation describing the motion of a string tied down at both ends for a uniform and a non-uniform string. We employ two different numerical approximation methods. Both methods begin by dividing up the length of the string into small elements. Using the finite difference method and the computer program IDL (Interactive Data Language), we find the normal modes of a string whose density varies linearly from one end to the other end. Using the finite element method and the computer program MARC/Mentat, we find the normal modes of a string whose density changes abruptly at certain locations along the string and whose elasticity influences the tension. When we set the density to be uniform along the string, our approximation methods yield results that agree with those known for a uniform string. When we set the density so that it varies along the string, we find that various properties of the solution change. For example, we find that as the string becomes denser, the speed of propagation of the wave slows down. We also find that the relative frequencies change (the frequencies are no longer multiples of the fundamental frequency), the wavelengths shorten, and the curvature increases.
Investigating Inflammation and Infection in Primary Macrophages
Megan Wilson, '06, Laurel Benson, '08, and David Hall, Chemistry
Given that virus-induced asthma has been resistant to therapy, knowledge concerning the cellular and physiological processes that are critical in the development of virus-induced asthma is likely to lead to the generation of new forms of intervention. In this regard, previous studies have implicated the macrophage as playing an important role in the elaboration of the inflammatory environment observed in rhinovirus-induced exacerbation of asthma. However, little is known about the cell signaling events that are initiated upon macrophage infection with rhinovirus. Major and minor group rhinovirus are genetically similar although they bind to two different cellular receptors. To determine if the attachment of major (HRV16) and minor group (HRV1a) rhinovirus to the human monocyte-derived macrophages elicits different kinetics of activation for ERK5 and p38, macrophages cells were exposed to HRV16 or HRV1a at an MOI of 10 for 15,30,60, 90 and 120 min. Cell lysates were generated, separated by SDS-PAGE and immunoblotted with anti-phospho-ERK5 or p38. For MAP kinase p38, major and minor group rhinovirus stimulated phosphorylation occurred after 15 min and sustained phosphorylation up to 60 min. Interestingly at the 90 and 120 min time points, there was no p38 phosphorylation for the major group rhinovirus and sustained phosphorylation stimulated by the minor group rhinovirus. A similar kinetics profile holds true for the MAP kinase ERK5. Furthermore, activation of these two MAP kinases and the small molecular weight G-protein RAC are important in the elaboration of the inflammatory mediators MCP-1 and RANTES.
A Summer of Silica: Investigations in Mesoporous Silica
Aaron Jubb, '06, and David Thompson, Chemistry
This summer we have been studying the various physical properties of mesoporous silica, especially bulk material morphology, through a series of experiments. These experiments have been primarily focused upon the calcination step in the synthesis of mesoporous silica. Mesopores are pores that have diameters falling in the 2-50 nanometers range. We began the summer investigating the contents of the calcination effluent produced during the synthesis of mesoporous silica. We hoped to observe how the contents of this calcination effluent changed as we altered the mesoporous silica. During an attempt to synthesize mesoporous silica spheres, we observed previously unreported bulk morphology due to accidental procedure variations. This mesoporous silica was characterized by scanning electron microscopy (SEM) to have a sphere template like morphology. Further investigations are being done to determine why such morphology resulted and how to control the transformation between sphere morphology and this sphere template type morphology. We are doing this by synthesizing multiple samples in parallel and varying the synthesis parameters such as temperature.
Mesoporous Materials: Condensation Kinetics
Tahmida Nazreen, '06, and David Thompson, Chemistry
We observed the response of GC Mass Spectrometer to tertraethyloxysilane (TEOS) in order to study the condensation kinetics for the synthesis of SBA-15 (Santa Barbara Material No. 15) using acidic condition. The results obtained from the GCMS were studied and the limitations of the GC Mass Spectrometer were noticed. One of the ways to overcome the problem of saturation of the GCMS was to change the split ratio. This change has been seen to give more promising results. As future works, we plan to measure the loss of the reactant, tertraethyloxysilane (TEOS), in the synthesis of SBA 15 as a function of initial time, temperature and concentration. We also plan to observe the effects on morphology of SBA-15 if reactants are added as the reaction proceeds to maintain a constant concentration of reactants throughout the reaction.
The Effects of Zebra Mussles (Dreissena Polymorpha) on the Aquatic Ecosystems
of Green Bay, Lake Michigan and Lake Winnebago, WI, USA
Will Daniels, '07, Travis Haas, '07, Michael Schrimpf, '06, and Bart De Stasio, Biology
To determine the effects of zebra mussels (Dreissena polymorpha) on the aquatic ecosystems of Green Bay, Lake Michigan and Lake Winnebago, WI, USA, we measured various biological, chemical, and physical variables in both bodies of water. Water samples were collected and taken to the laboratory to measure zooplankton and phytoplankton species diversity, size structure, and standing stock, as well as chlorophyll a, PO4-3, NO3-, and NH4+ concentrations. Temperature, conductivity, pH, oxidative reductive potential, turbidity, light penetration, and dissolved O2 concentration were also measured in situ with submersible probes. These data will be compared with similar data obtained in the 1980's, prior to the arrival of zebra mussels in the early 1990's. Incorporating our data with data on the benthic environment and the fish populations, which were gathered by researchers at University of Wisconsin-Green Bay, and the Wisconsin Department of Natural Resources, ecosystem modeling software will then be used to produce a food web model of the two bodies of water in pre- and post-zebra mussel conditions. Although data are still being collected and analyzed, zooplankton species composition can already be seen to have changed since the invasion and may be closely associated with algal blooms. If zebra mussels are therefore shown to have a significant impact on the level or intensity of algal blooms, it is possible the mussels will have a significant impact on higher trophic levels in the food web.
The Effects of Mecamylamine, A Nicotinic Acetylcholine Receptor Antagonist,
on Flash-Evoked Potentials (FEPs) Recorded From Both the Visual Cortex (VC)
and Superior Colliculus (SC) of Male Hooded Rats
Elizabeth Hermanson, '06, and Bruce Hetzler, Psychology
In order to explore the role of acetylcholine in the functions of the visual system, this experiment investigated the effects of mecamylamine, a nicotinic acetylcholine receptor antagonist, on flash-evoked potentials (FEPs) recorded from both the visual cortex (VC) and superior colliculus (SC) of male hooded rats with chronically implanted electrodes. FEPs were recorded 20 and 35 minutes after intraperitoneal injections of saline, 0.3, 3.0, and 10.0 mg/kg mecamylamine, with different dosages administered on different days. Preliminary examination of the FEP data suggests that the amplitude and latency of several FEP components are affected by mecamylamine administration. Gross body movement inside the testing chamber was recorded during presentation of the stimulus, and was altered by dose and time after mecamylamine administration. Body temperature was recorded 40 minutes after injection of the drug, and it appears that mecamylamine produces a dose-dependent hypothermia. Following body temperature recording, behavior in an open-field maze was observed for 4 minutes, and it appears that behavioral changes are dose-dependent, with a dose of 0.3 mg/kg producing hyperactivity, while the higher doses (3.0 and 10.0 mg/kg) produce lethargy in comparison to the control condition.
Effect of Kairmone on Protein Expression in Daphnia Pulex
Calistie Meyer, '06, and Elizabeth DeStasio, Biology
Daphnia pulex is a common species of water flea found in eutrophic and freshwater environments such as lakes, ponds, and marshes. This species can produce both sexually and asexually in a process called parthenogenesis, and development consists of five embryonic stages and four instars upon release from the mother. During the second and third instars of development, the D. pulex is susceptible to its predator Chaoborus. The larvae of Chaoborus release a kairmone that induces a morphological change in the developing Daphnia. Exposure to the kairmone causes the development of spines called “neckteeth” along the dorsal region of the neck. This, in effect, protects the Daphnia from being preyed upon by the Chaoborus, until it has reached maturity and is no longer susceptible to its predator. Although there has been much study on ecological effects of this chemomorphosis, little is known on a molecular level. Through two-dimensional gel electrophoresis, changes in protein expression will be determined and identified between developing daphnia in controlled and kairmone-induced environments. Identification of these proteins will help indicate what is occurring at the genetic level during the morphological changes. Thus far, the practice of two-dimensional gel electrophoresis and staining has been successful. However, the D. pulex protein is degraded on the gels. Modifications are being made to the concentrations of protein being used per gel, along with changes in the procedures of protein extraction. Once active, defined protein is seen on the gels, we will be able to start experimentation with the kairmone exposure.
Extraction and Characterization of Chaoborus Kairomone
Steve Van Der Naalt, '06, and Jerold Lokensgard, Chemistry
Numerous species of the genus Daphnia, specifically Daphnia pulex, prove to produce morphological changes in development in the presence of larval Chaoborus americanus. Daphnia develop protrusions, termed neck teeth, on both their helmets and ventral spines. A chemical signal, or kairomone, is credited with the observed changes induced by Chaoborus. Information gained by others through chemical modifications and filtrations demonstrates that the chemical signal is polar in nature and of a molecular weight lower than 500 mass units. Using large quantities of Chaoborus larvae the chemical signal was extracted through boiling and subsequently filtered and isolated through use of a solid-phase extraction (SPE). Previous studies determined that using SPE with increasing concentrations of methanol yield active fractions at 60-80% methanol. The 'active' SPE fractions from the sample of Chaoborus were then concentrated and evaluated using a tandem MS instrument to gain information regarding the chemical structure of the active molecule. Although no significant structural information was discovered with the samples tested, information for subsequent research opportunities was gained.
Scanning Tunneling Spectroscopy of Single Walled Carbon Nanotubes
Henry McNeil, '06, and Jeffrey Collett, Physics
Carbon nanotubes were deposited on a substrate and were imaged with a scanning tunneling microscope until atomic resolution was attained. Scanning tunneling spectroscopy was conducted on single walled carbon nanotubes. Using numerical differentiation procedures, spectroscopy data were examined for the presence of van Hove singluarities.
Fox River Water Quality Monitoring: PCB Analysis and Nutrient Testing
Cherisse Hall, '07, Benjamin Glover,'08, Richard Amankwah, '06, and Karen Nordell, Chemistry
Organic pollutants and metals have been identified in many local aquatic environments including the Fox River. Many of these species, including PCBs and heavy metals such as mercury are toxic to organisms due to their bioaccumulation. Due to their toxicity, many of these pollutants are monitored by regulatory agencies such as the EPA and the remediation of these toxic species is an active area of research. This summer, using the soxhlet extractor and a gas chromatograph, we have successfully extracted PCB's from spiked soil sediments collected from Apple Creek, north of Appleton. We are continuing extraction experiments on core sediment samples as well as zebra mussels from Lake Winnebago. In addition to the extraction and identification of PCBs, we are exploring various colorimetric methods to test soluble reactive phosphate levels in water samples from the Fox River and several area creeks. Most of the samples contained concentrations of phosphate below 0.30 mg/L.
Coumadin and Anticoagulation Therapy
A.J. Ow, '06, and Nicholas Maravolo, Biology
Coumadin (Warfarin) has been used as an anticoagulant since 1954. Originally used as rat poison, Coumadin was found to be an affective and fairly safe anticoagulant for humans. Patients suffering from atrial fibrillation, deep vein thrombosis, and heart valve replacement surgery are some of the patients that require anticoagulant therapy. Patients on coumadin have significant lifestyle changes since coumadin is a vitamin K antagonist and has many concomitant drug interactions. Because of these interactions, a patient's clotting is difficult to maintain and regulate. Clinics use prothrombin time and the international normalized ratio to manage a patient's clotting capabilities. Blood tests are given monthly in order to manage their anticoagulant therapy. When a patient has a non-therapeutic INR, it is difficult to say whether the patient is non-compliant, or does not have the proper understanding of their medication. Studies have shown that patients in coumadin clinics have less bleeding episodes and are within the desired INR range more often, when compared to a privately managed coumadin patient. These clinics have specific programs for patients on coumadin, including an education program. An education program does not exist for patients at Appleton Medical Center. The goal of this study is to determine if the lack of education for a patient relates to the patient's INR test results. A quiz was given to illustrate a patient's understanding of coumadin at Appleton Medical center. The quiz results will be used to correlate varying INR's with quiz scores.
Characterization of a Male Mating Defect in C. Elegans
Patrick McEachern, '06, and Elizabeth De Stasio, Biology
Certain gain-of-function alleles of the Caenorhabtitis elegans gene unc-93 confer a distinct type of paralysis known as the “rubberband” phenotype. We use one such allele, unc-93(e1500), as a background to study a suppressor of this phenotype; loss-of-function alleles of the sup-9 gene. The sup-9(lr164) allele is a "male mating defect" alleles that has the peculiar effect of conferring a mating disability in male worms, in tandem with the rescue of the rubberband paralysis. This phenomenon suggests a special role for sup-9 in males, and our research aims to characterize sup-9 and its protein product by discovering the mechanism of this male mating defect. Sup-9 is known to encode a potassium channel that is expressed in neurons and body wall and sex muscles of hermaphrodite worms. We use tissue-specific promoters and GFP tagging to investigate the effects of sup-9(wt) and sup-9(lr164) expression in males. So far, analysis of muscle-specific expression driven by the strong myo-3 promoter has been complicated by morphological defects that seem to be caused by the resulting over-expression of the transgenic sup-9 alleles. These morphological defects are accompanied by varying degrees of paralysis and thus prevent effective assays of male mating ability, but the presence of a morphological defect may itself provide a clue about the nature of the mating defect.