Research projects for Summer 2009
1. Immune Cell Activation by Polymer Nanoparticles (Narasimhan (ISU) and Lapizco (ITESM)) – Experimental
Narasimhan has designed novel biodegradable amphiphilic polyanhydrides with the ability to enhance the immune response and stabilize protein antigens. These capabilities have important implications for the design of single dose vaccines for diseases such as cancer, HIV, anthrax, plague, tetanus, and diphtheria. We have fabricated nanoparticles 
(based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)3,6-dioxaoctane (CPTEG) and demonstrated the stability and immunogenicity of antigens released from these nanoparticles. Our overall goal is to understand the cellular and molecular mechanisms by which these polymeric nano-adjuvants enhance and activate host immune responses. Lapizco has developed dielectrophoresis (DEP)-based methods for the separation and concentration of cells, proteins, and nanoparticles. She has designed a new class of micro-devices called insulator-based DEPs (iDEP). In this device, non-uniform electric fields are created with an array of insulators instead of electrodes (see Fig.). Separation of the particles takes place inside micro-channels within the insulating structures. Insulating materials such as polymers have excellent malleability and because they can be mass-replicated, provide for high-throughput and large-volume devices. Below, we describe a collaborative project that will provide a stimulating research experience.
Example REU Project: Students will prepare 100-500 nm polyanhydride nanoparticles loaded with FITC and based on copolymers of SA, CPH, and CPTEG. These nanospheres will be incubated with the J774.1A macrophage cell line to ascertain adjuvanticity (i.e., activated macrophages) and tested for uptake and cellular activation using flow cytometry and cytokine profiles. Students will use lithography at the ISU Keck Laboratory (consisting of class 10/100 clean rooms) to fabricate micro-devices of glass, polyimide, or poly(methyl methacrylate). These devices will contain micro-channels and an array of cylindrical posts inside the micro-channels, whose surface and geometry will affect the dielectrophoretic response obtained from the cells. This response will be used to distinguish the cells that have taken up the nanospheres. These micro-devices and the cells will be analyzed by iDEP at ITESM using synchronized video microscopy. By working on this project, the students will learn: (a) fabrication of nanospheres; (b) fabrication of patterned substrates; and (c) cell screening.
2. Immunomodulatory Vaccines (Narasimhan and Wannemuehler) – Experimental
We have designed novel biodegradable amphiphilic polyanhydrides that have the ability to enhance the immune response and stabilize protein antigens. These capabilities have important implications for the design of single dose vaccines for diseases ranging from cancer and HIV to anthrax and plague to tetanus and diphtheria. We have fabricated nanospheres (see Fig.) based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)3,6-dioxaoctane (CPTEG) and demonstrated the stability and immunogenicity of antigens released from these nanospheres (4-6). Our overall goal is to understand the cellular and molecular mechanisms by which these polymeric nano-adjuvants enhance and activate host immune responses.
Example REU Project: REU students will learn to fabricate antigen-loaded nanospheres (200-800 nm) using CPH:SA and CPTEG:CPH copolymers. The antigens of interest include ovalbumin, tetanus toxoid, F1-V antigen, pertussis toxoid, and rPA (recombinant protective antigen against anthrax). The students will learn to characterize these nanospheres using electron microscopy and a Zetasizer (for size distribution). One REU student will study how the blank nanospheres (i.e., no antigen) are uptaken by antigen presenting cells of the immune system using confocal laser scanning microscopy. The second REU student will immunize mice with these antigen-loaded nanospheres with the appropriate controls. The immune response in these animals will be characterized using both antigen-specific antibody responses and T cell proliferative responses. Together, all these studies will provide molecular and cellular information about how polymer chemistry enhances and activates host immune responses. This project will expose the students and the teacher to nanotechnology, biochemistry, animal studies, and applied immunology.
3. Neural Tissue Engineering (Mallapragada) – Experimental
The Mallapragada group has designed micropatterned polymer substrates to control neuronal outgrowth and promote peripheral nerve regeneration. Studies are currently underway to use these well-defined substrates to investigate neural stem cell differentiation. Current knowledge suggests that the pattern formation and differentiation of stem cells into various cell types that occurs during development of the brain is guided by a variety of signals including preformed distributions of extracellular matrix proteins and soluble cues. A thorough understanding of this differentiation is important not only to answer fundamental questions in biology related to development of the elaborate organization of the mammalian brain, but also to control the differentiation of neural stem cells in a tissue engineered scaffold to promote regeneration.
Example REU Project: We have developed polymer substrates with micropatterns of various proteins using photolithography techniques. The REU student will investigate the growth and differentiation of adult hippocampal progenitor cells (AHPCs from rats that the Mallapragada group regularly works with) on these micropatterned substrates. These studies will be used to test the hypothesis that unique combinations of signals from preformed distributions of extracellular matrix proteins will affect the fate of neural stem cells. The cell culture system will involve AHPCs growing on a polymer substrate micropatterned with proteins such as laminin and fibroblast growth factor (FGF). The pattern sizes and order of exposure of the cells to laminin and FGF will be interchanged and the migration of the cells and their differentiation will be monitored using morphological characterization. The cells will be stained for microtubule associated protein (MAP2) and β-tubulin (TUJ1), Receptor interacting protein (RIP) and glial fibrillary acidic protein (GFAP) to investigate whether they adopt neuronal or glial fates (see Fig.). This work is part of a larger project involving collaboration between Mallapragada’s group with researchers in biochemistry and genetics to use engineering techniques to answer some basic questions in biology. Through this experience, the student will learn 1) cell culture and sterile technique 2) staining techniques 3) working in an interdisciplinary environment integrating engineering and biological approaches.
4. Drug and Gene Delivery (Mallapragada) – Experimental 
The Mallapragada group has designed and synthesized novel smart bioinspired multi-block copolymers that exhibit pH and temperature sensitivity. These polymers are ionic and undergo thermoreversible gelation at body temperatures. Above a critical gelation temperature and polymer concentration, the micelles formed by the multi-block copolymers described above self-assemble to form macroscale thermoreversible physical gels. Since the critical gelation temperatures are close to physiological temperatures, these polymers can be used as injectable delivery devices and have significant advantages over other crosslinked stimuli-sensitive hydrogels that have been investigated. These physical gels eventually dissolve in the body. These polymers are ideal candidates for self-regulating systems for drug delivery. These cationic polymers exhibit complexation with DNA and serve as excellent injectable controlled gene delivery vectors, with selective transfection in fast growing cells such as cancer cells as opposed to normal cells (see Fig.).
Example REU Project: The REU student will investigate transfection of reporter gene in co-cultures of cancer and normal cells using the novel pentablock copolymer. Furthermore, this transgene system is also serum resistant and able to be injectable for sustained release, which makes it promising as an ideal sustained transgene vector. The student will investigate transfection using both dissolved vector as well as high enough concentrations where the vector forms polyplex gels which will dissolve slowly to release polyplexes. Through this experience, the student will learn 1) cell culture and sterile technique 2) transfection techniques 3) working in an interdisciplinary environment integrating engineering and biological approaches.
5. Robust Fluorescent Markers for Intracellular Delivery and Tracking (Clapp) – Experimental
Colloidal quantum dots (QDs) are nanometer-sized semiconductor crystals that exhibit many desirable and unique optical properties. Two obvious advantages of QDs are the ability to precisely tune the emission wavelength and a strong resistance to photobleaching. By tailoring the ligands bound to their surface, QDs can be delivered inside cells
and used to monitor native processes using fluorescence microscopy. Because they are larger than organic dyes, QDs can be functionalized to have a combination of ligands and biomolecules bound to their surface. This enables not only biocompatibility, but also the opportunity to target specific structures, pathways, and processes within living cells.
Example REU Project: The REU students will learn and refine inorganic synthetic techniques for generating high quality core-shell QDs and related techniques (fluorescence spectroscopy, TEM) for characterization. They will synthesize organic ligands for rendering nanocrystals water soluble and biocompatible and perform necessary ligand exchange steps. QDs will be added to the growth media of rat astrocytes to encourage uptake across the cell membrane via endocytosis. Total internal reflection fluorescence microscopy (TIRFM) will be used to image individual QDs as they move within the cell. TIRFM ensures that only a thin layer of the cell is illuminated which increases the brightness within the region and helps to eliminate background fluorescence. The cells will be stimulated with Ca2+ or ATP to encourage vesicles containing QDs to release their contents to the exterior across the cell membrane. A series of images are collected shortly after stimulation to determine the extent and location of release. These experiments are compared with control experiments using organic dyes to elucidate the release mechanism and whether the vesicles are fully collapsed or retained following a pore-opening event. This project combines aspects of synthetic chemistry, nanotechnology, and cell biology.
6. Aptamer-based Catalyst Design (Woo and Lamm) – Experimental and Computational
Catalysis is one of the most important applications in industry in terms of manufacturing bulk materials as well as producing pharmaceuticals. Economically, the application of catalysis adds more than $500 B per year to US commerce. Moreover, worldwide sales of catalysts amount to over $25B, with a $10.2B market share in the US. Although catalytic science has made tremendous advances in developing practical catalysts, it is still difficult and 
arduous to rationally design and improve a new catalyst using first principles. The collective interactions in the active site of a catalyst are extremely intricate and it is not always clear how to organize molecular features to effectively promote the transformation of one or more reactants into products. The intrinsic complexities of many dependent variables have previously forced the use of enormously time-consuming empirical methods to identify and optimize ligands and metal complexes for catalysis. A key challenge in catalysis involves accelerating the creation and optimization of efficient and selective catalytic materials from many possible compositions and structures. In the past decade, combinatorial approaches have been advanced that illustrate new potential for rapidly producing catalyst innovations (1). In this research, the application of combinatorial techniques and molecular evolution will be applied to homogeneous catalysis based on DNA aptamers (short segments of single stranded DNA) generated by the selection scheme shown in the Fig.
Example REU Project: The student will be involved in experimentally testing a family of evolved catalysts for coupling two small molecules. Activities, yields and selectivities will be determined by HPLC. In working on this project, a student will become acquainted with a variety of techniques and approaches in chemical, biochemical, and catalytic sciences. Preliminary structural determination of the most effective catalysts will be undertaken with computational approaches using homology modeling in collaboration with Monica Lamm. Database searching will be undertaken to find structural motifs of oligomers with similar sequences.
7. Molecular Modeling of Peptide Mimics (Lamm) – Computational
Peptide mimics are low molecular weight sequences of amino acids that reproduce the key structure or function of a larger protein. In this project, we are using molecular simulation to design a peptide mimic for the protein thrombin. A small thrombin mimic is needed for a specific biomaterials application where a thrombin-DNA aptamer complex is used to link a synthetic block copolymer to a fusion protein. The approximate molecular weights of thrombin and the fusion protein are 30 kDa and 6 kDa, respectively. Hence, it is desirable to design a miniature peptide that mimics the thrombin binding domain to reduce the size of the linking complex relative to the fusion protein and thereby improve linking efficiency. Selecting an appropriate peptide sequence from thrombin is not straightforward in this instance 
because the binding domain is not a linear sequence of residues. Rather, it is a groove that includes a single helix and non-helical side-chains from other parts of the protein.
Example REU Project: In this project, the REU student will conduct molecular dynamics simulations to study the binding domain of the protein thrombin. The information learned about the binding domain will then be used to generate several lower molecular weight peptide prototypes. The student will evaluate these peptide prototypes for their ability to bind the thrombin DNA aptamer (see Fig.). The REU student working on this project will gain experience with molecular simulation methods and software, and high performance computing.
8. Thermal Depolymerization of Biomass (B. Shanks and Brown) – Experimental
Fast pyrolysis can be used to thermally depolymerize biomass largely into a liquid fraction. While this phenomenon has been known for many years, the complexity of the chemistry associated with the thermal process has limited research effectively to empirical efforts. We are using model compounds in a micropyrolyzer coupled to a GC/mass spectrometer to probe more fundamentally the depolymerization reaction. Through pyrolyzing glucose, cellobiose, and cellulose, which have 0, 2, and many glucose units connected by β-1,4 glycosidic bonds, we have been able to
demonstrate the depolymerization reaction mechanism shown in the accompanying figure. Further insight into the pyrolytic cleavage of glycosidic bonds has been gleamed by examining compounds having α-1,4, α-1,6, or β-1,3 glycosidic bonds. Future work will focus on hemicellulose and lignin model compounds, interaction effects between cellulose, hemicellulose, and lignin, and the catalytic impact of naturally occurring alkali species on biomass depolymerization. Our overall goal is to develop a more fundamental model for the thermal depolymerization of biomass, which will support the desire to intentionally manipulate the product slate from this important reaction system.
Example REU Project: A REU student will learn to operate the micropyrolyzer analytical system, which includes a GC/mass spectrometer for quantifying complex reaction product mixtures. Model compounds for cellulose and starch will be impregnated with alkali species at levels present in biomass to evaluate the catalytic effect of this species on the thermal depolymerization reaction. Of particular importance will be the competition of the glycosidic bond cleavage reaction with the thermal decomposition reaction of the monosaccharides. These studies will provide fundamental information of the reaction system that can then be translated to real biomass feedstocks. This project will expose the student to biochemistry, reaction mechanisms, catalysis, and thermal systems.
9. Identification of Important RNOS Response Genes in Escherichia coli (Jarboe and Rollins) – Experimental
Bacterial species that attempt to colonize a mammalian host are exposed to the reactive nitrogen oxide species (RNOS) produced by the host immune system. We have previously measured the transcriptomic response of Escherichia coli to the RNOS nitric oxide (NO) and S-nitrosoglutathione (GSNO). By incorporating the known regulatory connections in E. coli, we were able to assemble a partial response network to each of these compounds.
However, much of the regulatory network structure in E. coli is as yet unknown. For this reason, many of the genes perturbed in response to NO or GSNO were not included in the response network despite their potential importance.
Therefore, we have used a PCA-based method to identify the most important genes composing the NO- and GSNO-specific gene signatures. While many of the genes identified in this analysis are part of the previously-identified response networks, many of these genes have not been previously linked to RNOS exposure and have little or no functional data available. Thus, examination of these genes and their function presents the opportunity to identify new RNOS-response network elements and potential drug targets. Here we propose to test the contribution of these genes to the NO- and GSNO-response in E. coli.
Example REU Project: An REU student will clone a gene of interest so that its expression can be exogenously controlled. The cloning process involves standard molecular biology techniques such primer design, PCR and gel electrophoresis and will be repeated for many genes. A high school student will then measure the growth of these clones following exposure to RNOS. Genes that are beneficial to the bacterial response will confer increased tolerance of these compounds. Genes that are found to be beneficial will be subjected to further phenotypic and genetic analysis by the REU student.
10. Metabolic Engineering of Terpenoid Indole Alkaloid Pathway (J. Shanks) – Experimental
The periwinkle Catharanthus roseus synthesize several valuable terpenoid indole alkaloids (TIAs), most notably the anticancer drugs vinblastine (Velbe®) and vincristine (Oncovin®), but in extremely low levels. We have engineered
several Catharanthus roseus hairy root cultures to overproduce the TIAs at levels feasible for commercial production in bioreactors (1-5). However, targeted production of the most valuable compounds has not been reached. Future successful engineering of these pathways will require tools such as flux analysis (6), expression profiling of target genes by Q-RT PCR (5), and metabolite profiling via Ion Trap MS/MS analysis (see Fig.) to better understand the bottlenecks within the TIA pathways.
Example REU Project: Two REU students will learn to characterize the metabolism of hairy root lines that are engineered to overexpress specific TIA genes, D4H: desacetoxyvindoline 4-hydroxylase and 16-OMT: 16-hydroxytabersonine-16-O-methyltransferase, that are in the tabersonine to vindoline branch, a key branch to the production of vinblastine. Each REU student will perform a temporal study of a particular hairy root line: i.e. how the specific gene alters the alkaloid profile, by growing hairy roots under induced and uninduced (control) conditions. Both standard metabolite analysis via HPLC and more sophisticated metabolite profiling using Ion Trap MS/MS analysis will be performed to characterize metabolites. Since both students will be working on genes in the same branch, they will be able to directly compare their alkaloid profiles and aid in an improved understanding of the biochemistry and regulation of this branch. This project will expose the students to biochemistry, plant tissue culture, separations and analytical chemistry.
11. Biomass Gasification for Biofuels Production (Brown) – Experimental
Gasification is the thermal decomposition of biomass in the absence of oxygen to yield syngas, a mixture of carbon monoxide and hydrogen that can be catalytically upgraded to synthetic fuels. Among the major challenges of this technology is controlling contaminants in the syngas, which includes tar (condensable hydrocarbons), hydrogen sulfide, ammonia, and hydrogen chloride. Because of the difficulty of on-line measurement of these contaminants, the formation and fate of these contaminants is not well understood, which has slowed the development of technologies to control them. These contaminants can foul or poison the catalysts used to synthesize biofuels from syngas. Brown’s research team has recently acquired a mass spectrometer able to accept syngas samples at temperatures as high as 300C, which allows on-line measurement of syngas constituents that would normally condense before entering conventional gas analysis equipment. These species include many of the contaminants that have traditionally defied on-line measurements.
Example REU Project: During the project, the student will learn the operating principles of a gasification reactor and how to make measurements with the on-line mass spectrometer. An experimental program will be devised to evaluate the effect of gasification operating conditions on the formation and fate of sulfur and nitrogen contaminants from a model biomass feedstock.
12. Computation of Enzyme Structure and Function (Reilly) – Computational 
The Reilly group has performed extensive computational analysis of amino acid sequences of the same enzyme produced by different organisms and of 3-D enzyme structures obtained by crystallography. The crystallography experiments to determine enzyme structure and function are difficult and time-consuming, and computation, which is relatively fast and quite accurate, can greatly expand what is known about the specific enzyme. We have used automated docking with glucoamylase, β-amylase, α-1,2-mannosidase, phospholipase D (see Fig.), several cellulases, and the non-enzymatic mannose-binding protein C.
Example REU Project: The student will study hydrolases, which cleave the glycosidic bonds between sugar residues. She/he will perform two activities: a) Use existing algorithms to align amino acid sequences obtained by different research groups and found in databases. Such alignments will distinguish different subfamilies within an enzyme family, such as glucoamylase, and from this the student can infer that structures and properties of the different forms of the same enzyme produced by different organisms are closer or more distant from each other. b) Use automated docking with the algorithm AutoDock of different ligands, either already known substrates, potential substrates, or inhibitors, into the active sites of enzymes with known structures. This allows the student to understand enzyme function supplement knowledge gained by experimentation.
13. Dynamic Intracellular Processes During Cell Migration (Schneider) – Experimental
During tumor metastasis, carcinoma cells migrate out of the tumor into the surrounding tissue. They do this by integrating intracellular signals from both soluble proteins such as epidermal growth factor (EGF) and insoluble extracellular matrix (ECM) proteins such as collagen. The way in which each of these proteins are spatially presented determine the migratory behavior of the cell, however EGF and collagen induce overlapping intracellular processes. These overlapping intracellular processes complicate the understanding of cell migration in physiological and pathological contexts. One example of an overlapping intracellular process controlled by EGF and collagen is the formation and remodeling of focal adhesions. Focal adhesions are 
intracellular macromolecular protein complexes that facilitate adhesion to ECM and allow the cell to generate traction, propelling itself forward. In order to understand how both EGF and collagen controls the remodeling of focal adhesions and cell migration, one must be able to independently vary the spatial and temporal presentation of these signals. Research in the past several years has yielded soft lithography techniques for microcontact printing of ECM protein and microfluidic devices for the spatial control over soluble protein. These tools will be used in conjunction with live-cell light microscopy techniques that allow for the visualization of focal adhesion dynamics and cell migration. Understanding how EGF and collagen cooperatively or antagonistically affect dynamic intracellular processes during cell migration will lead to a better understanding of cancer metastasis.
Example REU Project: Carcinoma cells will be seeded on different collagen-patterned substrates inducing a polarized cell morphology. Two REU students will learn how to pattern substrates and culture cells on those patterned substrates. One of them will analyze the migratory behavior of cells on patterned substrates alone. The other student will use these patterned substrates in conjunction with microfluidic chambers to analyze focal adhesion dynamics and cell migration behavior with different collagen patterns and in different EGF gradients. Cell migration will be assessed using transmitted light microscopy and focal adhesion remodeling will be assessed using total internal reflection fluorescence microscopy in cells expressing green fluorescent protein tagged paxillin, a putative focal adhesion protein. This project will expose the students to soft lithography techniques, molecular and cellular biology and quantitative light microscopy.
14. Recovery of High-Value Biological Products (Glatz (ISU) and Rito (ITESM)) – Experimental
The Glatz and Rito laboratories both work to develop optimal conditions for the recovery of macromolecular
products (e.g. proteins, aroma compounds, colorants, inclusion bodies, virus like particles) of biological expression hosts and bioprocessing streams using aqueous two-phase (ATP) partitioning in PEG-salt and PEG-dextran systems. Intensive study of the behavior of specific proteins, previously well characterized in conventional analyses and purifications, have helped to establish ground rules to predict molecular partition in novel systems based upon quantifying physical and biochemical parameters of system components. Each is interested in further steps for product recovery from these systems and reuse of phase components, which will be the focus of their joint project.
Example REU Project: The two labs have considerable expertise in precipitation, ultrafiltration and chromatography to serve in subsequent process steps. Each lab also has product proteins (Fig. 5) of particular interest: high-value colored proteins (c-phycocyanin and B-phycoerytrin) from microbial sources and recombinant pharmaceutical/industrial enzymes from corn. The student project will be to take the source material and partitioning step from one lab into the other lab where they will evaluate the subsequent downstream step. That step will be either precipitation (ISU), where the phase system polymers and salts might aid in reducing solubility or ultrafiltration (ITESM), where differences in size of phase components and the product will be exploited. Back extraction with ATPS and expanded bed adsorption also will be evaluated. Students will obtain protein extracts, follow recovery by enzymatic or ELISA assays, and interpret results in terms of solubility, partition coefficient, or retention time, depending on the separation method.
15. Targeted Drug Delivery Modeling (Vigil in collaboration with Prof. Ananth Annapragada, University of Texas School of Health Information Sciences) – Computational
One promising method for increasing the selectivity of drug delivery to target cells has been to attach ligand molecules to the surface of the drug carrier (using a flexible polymer tether) that bind to specific receptors prevalent on the target cells. However, when only a single receptor is targeted, off-target uptake of drug is still significant. However, because diseased cells often over-express multiple receptor types, the attachment of multiple types of ligands onto drug carriers could potentially lead to more highly selective targeting. REU students have developed over the past three summers a Monte-Carlo computer simulation code that can simulate carrier-cell interactions with the goal of predicting the number and type of ligands that optimize the rate and selectivity of drug delivery to target cells. This stochastic simulation incorporates many physical and biological details including accurate models for the tether length probability distribution, accounting for spatial relationships between all attached liposomes and surface receptors, the physical distribution of receptors on the cell surface (blotches as well as singlets), as well as down-regulation of endocytosis (via reduced folated re-expression) triggered by the cumulative number of folate molecules uptaken. While considerable progress has been made, several important outstanding issues remain including validating the code against experimental data obtained in the Annapragada lab, incorporating mobility of surface receptors, and optimization of the total number and distribution of ligands for maximal targeting efficiency.
Example REU Project: An REU student will spend two to three weeks developing a complete and detailed understanding of the current C++ simulation code and how to compile and use it. They will also carry out limiting-case scenario simulations in order to validate it against known analytical results. Next, they will carry out simulations to validate the current down regulation model for liposome uptake against experimental data obtained in Annapragada’s lab. Depending upon the success of the current model, they may develop alternative models. Once the down regulation model has been validated, the student will carry out a full set (varying ligand numbers and types) of simulations to determine the conditions that lead to optimal targeting of liposomes to cancer cells in a dual ligand system (folate and transferrin).
