2001 National Science Foundation (NSF)-supported Participants
|Ashlee Baker, Muskingum College
(Mentor: Dr. Alan Waggoner)
Optical biosensors: Investigations of the solvatochromic properties of fluorescent transducer dyes
1. A structure that confers both recognition and selective binding of the target molecule at its physiological concentration
The first dye that was used was fluorescein which is a pH sensitive dye. The structure is:
The IADEANS did not label the protein very well. The protein was labeled with the CPM, however the fluorescence measurements did not detect a large enough signal to show any emission changes.
The polarization of fluorescein was tested as well. Polarization is related to the molecular tumbling rate, which will be slowed when the receptor binds and restricts the motion of the dye. Polarization measurements did not show this behavior. Since fluorescein is highly hydrophobic it may have been pulled into the binding site, restricting its movement before the target was introduced.
The carboxy-functional dye was isolated and purified and its absorption measured. The dye was then activated and reacted with the maleimide-bearing linker. The resulting maleimide-functionalized dye was purified and its structure confirmed by 1H-NMR spectroscopy. Reacting it with papein, a protein which contains a free thiol group, tested the dye’s labeling ability. With the successful labeling of papein, protein 213C can now be labeled and the fluorescence measured.
|Jennifer Baranowski, Grove City College
(Mentor: Dr. Peter Berget)
cDNA Analysis of CD-tagged NIH 3T3 Cell Lines
|Tasha Breaux, Northwestern State University
(Mentor: Dr. Amy Csink)
A Comparison of Satellite Sequences Found in Drosophila melanogaster and its Sibling Species and an Analysis of these Species' Ability to Interspecifically Mate
The goals of this project have been two-fold. First D. melanogaster was crossed with two sibling species (D. simulans and D. mauritiana) and the ability of this cross to produce progeny was examined. While crosses of this type have been previously performed, they have not been done with the brownDominant gene present. These hybrid flies were then backcrossed with the sibling species to create a fly of a sibling species to D. melanogaster that contains the brownDominant gene but not the satellite sequence AAGAG, since this particular sequence is not seen in the pericentric heterochromatin of the second chromosome in the sibling species. To verify this, the second aspect of this project used fluorescent in situ hybridization to determine the presence or absence of various satellite sequences in the specific fly lines that were involved in the interspecific mating.
Despite the presence of the brownDominant gene, F1 progeny were obtained from the interspecific mating of D. melanogaster with each of the sibling species. At this time, the backcross of these interspecific flies with the sibling lines is underway. Also, a great deal of variation among the satellite sequences was detected between the different species of flies examined as well as between different lines of flies within the same species.
|Tara Brownlee, Lebanon Valley College
(Advisor: Dr. Frederick Lanni)
Imaging and Mathematical Analysis of Collagen Gel Deformations
In Dr. Lanni’s lab the interest is in the mechanics of cells within a collagen gel. This involves how cells move and reshape the ECM. Collagen is used as a model ECM and, in order to understand how cells shape the ECM, there first needs to be an understanding of collagen in terms of its mechanical properties.
This summer project involved making collagen gels of known concentration, imaging the gel network under a microscope, and then producing deformations in the gel. A glass microneedle in a motorized micromanipulator was used to apply a highly localized load (force) tangent to the surface of the gel. After imaging the deformations with a video camera attached to the microscope and imaging software called STC-View, the next step was to input the image files into the Deformation Quantification Algorithm (DQA), a computer program developed by Steven Vanni in Dr. Lanni’s lab. The DQA takes the sequential image pairs and produces a field of vectors that show how the gel moved when the load was applied.
The main goal this summer was to produce deformations that would (1) give the best output from the DQA and (2) correspond to known idealized solutions of the elasticity equations. Different types of movements with the microneedle were used to get deformations that the DQA was capable of tracking well. The reason for getting good DQA output was to enable my collaborator on the project, Jennifer Airone, to match my data to her simulated materials with greater accuracy.
From the different types of movements made with the needle and through much trial and error, it was discovered that to produce the best deformations, and subsequently the best DQA output, the needle needed to be (1) moved to the edge of the field of view and (2) moved perpendicular to the edge of the field of view as opposed to parallel. Also, the best output was achieved when a mask was used to block out the site of the needle for the DQA. Lastly, the most important thing discovered was that small, controlled movements, such as those made by using the step feature on the micromanipulator, produced the best output from the DQA.
After discovering how to produce the DQA output, the next step will be to match the actual data to computed mathematical simulations of the movement of the collagen gel. Once the mechanical properties of collagen are known, they will be used to predict the pattern of movement for cells in the collagen gel. This will help researchers to better understand the movement of fibroblasts and other non-muscle locomoting cells not only in collagen gel but also in real tissues and organs.
|Pauline Chugh, Millikin University
(Mentor: Dr. Jonathan Minden)
Investigation of developmental cell death in wildtype and mutant Drosophila embryos
In this project, I studied the process of apoptosis during Drosophila melanogaster embryogenesis. Apoptosis is a highly regulated process that is required for proper development. During development, apoptosis ensures that tissues develop with the correct number of cells. The regulation of apoptosis may be linked to cell proliferation. For example, if there is ectopic proliferation, cell death may increase to compensate whereas a reduction in proliferation would result in a corresponding reduction in cell death. Dead cells are removed by phagocytic cells (macrophage) by a process called phagocytosis, or engulfing of the cells.
I used two different Drosophila mutants to investigate regulation of cell death and phagocytosis. The first of these mutants is the string (stg) mutant. The string protein is responsible for regulating cell division after the 14th division. The first 14 divisions are regulated by maternal proteins. However, cell division in embryos after the 14th division is regulated by the zygotic string protein. String mutants lack the zygotic string protein and therefore there are no more cell divisions following division 14. I used Acridine orange, a fluorescent marker that labels dead cells, to sudy cell death in stg embryos to see if cell death compensates for less cell division in these embryos. The fluorescent marker was injected into both wild-type and mutant embros and they were then analyzed using fluorescence 4D-microscopy.
The second mutant, myoblast city (mbc), has cytoskeletal defects that result in a lack of myoblast fusion. The mbc gene encodes a protein that is nearly homologous to the human protein DOCK180, which is also involved in myoblast fusion. When the mbc gene is missing, the fusion of myoblasts into multinucleate muscles is virtually nonexistent. I hypothesized that the cytoskeletal abnormalities would cause a defect in the removal of dead cells by inhibiting the ability of phagocytic cells to wrap around and engulf dead cells. I studied two different mbc mutations, mbcC1 and mbcC2, and examined the rate of engulfment and macrophage function. Vgal, a fluorescent marker that labels phagocytic cells, was injected into both wild-type and mutant embryos to observe the process of phagocytosis. The injected embryos were then analyzed using fluorescence time-lapse microscopy. The time-lapse movies indicate that macrophage in mbc mutant embryos take nearly twice the amount of time to engulf dead cells and show a reduction in movement compared to wild-type embryos. These results suggest that mbc mutants exhibit defects in phagocytosis as well as myoblast fusion.
|Roslyn Crowder, Florida A&M University
(Advisor: Dr. William Brown)
Single Chain Variable Fragments (ScFv) and the use of Fluorescent Dyes
Single chain variable fragments (ScFv) have been engineered in Dr. William Brown’s laboratory to recognize the bonds between TDI and amino acids, particularly lysine. It was then useful to screen the previously prepared ScFv library to find a specific ScFv that recognized the bond between TDI and the amino acid serine. This is important because with a hydroxyl group with pKa 14, serine is predicted to be the amino acid that is predominantly modified by TDI in vivo. The screening procedure termed phage panning utilizes the ability to specifically recognize an antigen. The antigen in this case encompasses a P-Tolylmonoisocyanate (TMI) conjugated to N-acetyl Serine that is covalently linked to Bovine Serum Albumin (BSA).
After finding such an ScFv, we used it as an in vivo biosensor to locate TDI conjugates. This required the use of fluorescent dyes to follow the ScFv. Therefore, another portion of our research was dedicated to linking Thiol-reactive fluorescent dyes at various cysteine locations engineered around the active site of the ScFvs. We also used enzyme-linked immunosorbent assays (ELISAs) to determine which binding sites can be labeled without affecting target binding. We also tested different fluorescent signals that respond to the binding events, which include pH changes, solvent polarity, and molecular tumbling rate.
TMI only contains a single isocyanate group while TDI has two. Isoelectric focusing (IEF) gels and absorbance readings at 240 nm were to make certain that both the serine and lysine conjugates were similar in conjugation. Using ELISAs with both TMI-Serine and TMI-Lysine conjugates, the ScFv #12 was tested on its binding specificity.
|Jermaine Jones, University of Virginia
(Mentor: Dr. Jonathan Jarvik)
Improving the Efficiency of Retroviral Infection of NIH3T3 Cells using Partially Synchronized Cultures
Cultures of NIH3T3 cells were subjected to a synchronization protocol consisting of serum starvation for forty-eight hours to produce cell-cycle arrest, followed by serum restoration (stimulation) to release the cells from arrest. Twenty-one hours later, when the synchronized cells were about to enter mitosis, they were infected with the Stealth virus. The number of EGFP-positive CD-tagged clones was determined by fluorescence microscopy and compared with that of asynchronous control cells infected in parallel. Preliminary data suggest that there was a greater CD-tagging efficiency in the synchronized cells as compared to the unsynchronized controls.
To examine the effectiveness of the synchronization protocol, asynchronously growing cells were fluorescein-labeled using CMFSE and then serum-starved for two days. Flow cytometric analysis revealed that approximately half of the cells arrested in a G0-like state - but that the other half continued to divide — i.e., they were not growth-arrested and therefore not subject to synchronization when serum was restored. These results suggest that it may be possible to further improve CD-tagging efficiency by employing a more effective synchronization protocol.
|Catherine Lewis, University of Virginia
(Mentor: Dr. Peter Berget)
Construction of Modified Stealth Vectors for CD-Tagging
|Maricel Martinez, Universidad Metropolino
(Mentor: Dr. David Hackney)
Introduction of a second microtubule binding site into conventional kinesin
|Elizabeth Ottesen, Grinnell College
(Mentor: Dr. Gordon Rule)
Characterization of Active Site Dynamics in the Human Glutathione Transferase M2-2