Research

Molecular Wire Fabrication and Characterization: hybrid quantum dot/organic chemical structures

With Lindsay Subers (Haverford Physics '06) & Adolphe Alexander (Haverford Physics '05)

Under Construction

Computational Studies of Symbiotic Evolution

Physics majors Jim Duncan ’03 (now with Americorps Northern Arizona Conservation Corps)and Liz Janus '04 (now in a Biophysics PhD program at U. Michigan) have been the primary contributors to our research project aimed at modeling how endosymbiosis could have influenced the evolution of eukayrotic lifeforms early in the Earth's history. In this astrobiology/artificial life project, we were trying to build upon existing methodology used in computational studies of evolution and population biology to examine how endosymbiotic evolution (the evolution of two previously-freeliving individual cells into a single organism because of environmental constraints that produce mutual fitness benefits) could take place in a plausible environmental setting consistent with what we know about the evolution of early life. We came up with a detailed model of how this might have occurred in the vicinity of hot springs and volcanic hot vents in the ocean and we’ve compiled properties of likely model ancestral organisms. We also have computer programs which allow the physical modeling of the relevant environmental variables (temperature, light levels for photosynthesis, chemical nutrient distribution near the hot springs, etc.) and the coevolution of the different populations. We have completed our study of how endosymbiotic lifeforms could emerge from parent photosynthetic cyanobacteria and a chemotrophic single-celled organism, along the lines hypothesized for the evolution of chloroplasts. We will extend this model this spring to include the effects of catastrophes and how the different parent and symbiotic organisms respond.

The following two projects are related to our Packard Interdisciplinary Program in Protein-Based Nanomaterials for Biotechnology

Mechanical Measurements of Biopolymers

Research in my lab this year involves the use of atomic force microscopy (AFM) and laser tweezers to perform mechanical measurements on the polymer properties of synthetic proteins and DNA. In AFM force spectroscopy, a molecule is attached at either end to a solid surface (the substrate) and to the AFM tip (or cantilever). The cantilever is then retracted, stretching the molecule between the tip and surface. The measured deflection of the cantilever can be converted into the force required to stretch the polymer to a measured extension. In laser tweezing, a microscope objective lens is used to both image a sample and to focus an intense laser beam onto a micron-sized spot on the sample. Small dielectric particles, such as glass beads, organelles, vesicles or even entire cells, can be trapped by the intense electric field gradient at the locus of the focused beam, and these particles may then be manipulated by scanning the beam through the sample, all while both viewing the manipulation through a microscope and measuring the forces applied. Biological polymers can then be stretched and their mechanical response measured if they are attached to polystyrene spheres and/or surfaces for laser tweezer manipulation. In the past, research students Benjamin North (now a Biophysics grad student at University of Pennsylvania), Nicholas Wilder (founder of WebShots.com, one of the web's most successful .coms!) and Todd Kerner (Dartmouth M.D./Ph.D. program) successfully completed the construction of such a device in my laboratory, and demonstrated that it is capable of measuring and maintaining forces with magnitudes of biological interest, as well as performing the relevant manipulations. Adam Ingram-Goble (Physics '02; now working in the computer industry) completed installing a micromanipulator/microinjection system which can be used to manipulate the polystyrene spheres used as handles for manipulating the proteins or DNA. He also worked on the biochemistry involved in fluorescently labeling and attaching the DNA to the microspheres.

These methods provide important and complementary tools both for manipulating single molecules or aggregates, and for exploring the mechanical properties of biopolymers, such as proteins and DNA, as a means of deriving otherwise inaccessible structural information. For example, folding-unfolding transitions of secondary and tertiary structures can be studied in this fashion, and information about the timescales required for their refolding derived. We are presently using them to study long coiled-coil synthetic polypeptides with flexibly defined sequences to study the energetics and dynamics of protein folding. Prototypes of these systems have been synthesized in the research group of collaborator Robert Fairman (Biology Department, Haverford College). For example, the forces exerted between opposing coiled coils and their binding energy will be established by mechanically separating the polypeptide strands, akin to published results for separating the strands of DNA and unfolding the domains of the protein titin. These results will be interpreted with a combination of stereochemistry and mathematical modeling in collaboration with collaborators at Haverford College.

So far, working with physics major Theresa Horne '03 and Rob Manning (Haverford Mathematics) we have shown that AFM images of coiled coil myosin II rods taken by Fairman's group can be analyzed to derive a plot of radius of gyration vs. contour length. This plot has been analyzed using standard worm-like-chain models of polymer chain statistics to derive preliminary estimates of persistence length. Our preliminary data shows good agreement between this simple model and the actual experimental results. We will be extending this work in the fall to analyzing images taken of myosin II rod domains under a variety of deposition conditions and using a variety of measures of conformational statistics. Our goal is to correlate information derived from image analysis of AFM images with our studies of the force spectroscopy of these molecules.

This work is being conducted with support from the Packard Foundation and the National Science Foundation.

Quartz Crystal Microbalance Biosensors
With Karin Akerfeldt (Haverford Chemistry) we investigated constructing biosensors which will allow us to assay for the binding of porphyrin-functionalized peptides to gold surfaces coated with porphyrin-based self-assembled monolayers (SAM). In a preliminary series of experiments, Haverford sophomore Emiliano Salatino showed the feasibility of these measurements by constructing a temperautre stabilized quartz crystal microbalance (QCM) biosensor which allows very sensitive measurements of the frequency shifts due to adsorption of chemicals onto the face of a vibrating quartz crystal. This technique can work in fluid and can easily detect monolayer coatings of proteins or other molecules of biological interest. Sebastian Mankowski(physics '05) and Shilpa Narayan (biology and biophysics '05) performed preliminary measurements with this system in physics 326 (advanced laboratory) and shown that monolayer depositions are feasible with our apparatus. Studies going on in spring 2004 with Bryn Mawr physics major Thida Aye '04 are aimed at using Prof. Akerfeldt's samples to characterize SAM formation with her samples using this approach.

Previous Research

In a collaborative project with Biology professor Jenni Punt, we studied the formation of immunological synapses in T-cells as a means of studying development and differentiation in the immune system. In particular, we stimulated T-cells with microspheres coated with anti-TCR and CD28 as a means of activating the T-cells and inducing capping. The capping regions were labeled with fluorescently-labeled cholera toxin, which preferentially labels lipid microdomains. We performed image analysis of the capping regions and the distributions of the lipid microdomains to see how this distribution behaviors as a function of time. We also analyzed the distribution of lipid rafts and actin to see how these distributions compare with the profiles in the synapse region of TCR and MHC/peptide complexes (components of the central synapse region or cSMAC); and cytoskeletal components such as LFA-1 and ICAM (components of the peripheral synapse region or pSMAC). We used computer modeling to describe how lipid raft assembly proceeds during synapse formation. This research has been presented in a talk at the American Physical Society 2002 March Meeting. Joshua Adelman (Biology '01, now in a biophysics PhD program at UC Berkeley) began this collaborative project. Mark Lee (Physics '02, now working as a research assistant in the Immunology Dept, Univ. Penn.) pursued this work for his senior research in biophysics, and Jim Duncan (Physics '03)continued it as a summer '02 research project.

Since our present time resolution is limited by the necessity of sampling cells which have been incubated with microspheres for long periods of time, we are also working on a new line of experiments which will allow us to stimulate individual T-cells with coated microspheres. Josh has shown it is possible to use our laser tweezer to manipulate the T-cells and microspheres so as to bring the two into juxtaposition at a defined time. We can then monitor the dynamics of capping using video microscopy.

Aaron Clauset (Physics and Computer Science '02, now in a CS PhD program at University of New Mexico) worked on a collaborative project with Prof. Kwabena Boahen (Univ. Penn. Bioengineering) and I on applications of information theory to visual processing. Time-varying digitalized images were analyzed for their Shannon information statistics using a program written by Aaron, then he used this approach to compute the statistical redundancy of the visual information in a series of model images meant to model those encountered by the retina in actual environments. Initial results suggested that a retinal filter could achieve significant data compression without loss of visual information by sampling spatially and temporally in the range of 5-10 adjacent pixels.

In an independent project, senior physics major Peter Ingebretson '02 (now working at Maxis Corp.) worked on the study of genetic algorithms for use in evolving programmable gate arrays integrated circuits. While much research in the past has involved demonstrating that PGA's can solve interesting circuit design problems, Peter's focus was on the characteristics of the genetic algorithms themselves: how does the mutation rate, the distribution of individual circuit designs amongst a population, and other parameters of the algorithm affect the course of the circuit's evolution?

Peter presented a poster on this work titled "Genetic Algorithms and Programmable Circuit Design: Realization in Real Gate Arrays", at the Interface Between Biology and Materials Science Symposium held at the University of Pennsylvania November, 2000, as well as the HHMI summer poster session at Haverford College, Fall 2000.

Carl Knutson (Physics '02, now in a physics PhD program at U. Texas, Austin) performed fabrication and AFM imaging in the laboratory of Prof. David Adams (Columbia University) on patterns made from edge transfer lithography, a technique which uses polymer-based microstamps to transfer microspheres or other particles. Here at Haverford, we worked on the theory of edge transfer lithography, using a model in which the transfer process was assumed to proceed by wetting of the stamp and substrate surface.

Samuel D. Floyd (Philosophy Ph.D. program, University of Pittsburgh and Oxford University) and I completed a study of the influence of local anesthetics on membrane structure, as determined via epifluorescence microscopy which has appeared in Physical Review E. Our work probed the in-plane phase behavior of model membrane systems which incorporate two commonly used local anesthetics, dibucaine and tetracaine. Click here to see an abstract on this research, based on our presentation at the 1998 Biophysical Society Annual Meeting.)

Another research project involved computer simulations of the ordering of phospholipid molecules in condensed phases; this work has appeared in Langmuir. Madison Compton (Physics '99), Nicholas Wilder and I worked on this project this research in collaboration with Lyle Roelofs. Click here to see our abstract on this work for the American Physical Society's March Meeting.

With research students Kathryn Long, Nicholas Wilder and M. Irfan Safdar , I have also completed a study of how the exact lipid composition of model pulmonary surfactant films influence such properties as the onset of the gas-liquid transition(which may influence how readily the films spread in vivo) and the exact phase behavior in the monolayer, and how this might relate to the phenomenon of "squeeze-out" of different lipid species, long thought to have an important influence on the stability of the layers at high pressures. This work appeared in a poster presentation at the 1996 Biophysical Society.

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