2012 BioMaP REU Research Projects

1. Immunomodulatory Nanovaccines against Infectious Diseases – Experimental

Mentor: Balaji Narasimhan

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 nanoparticles (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 nanoparticles. Our overall goal is to understand the cellular and molecular mechanisms by which these polymeric nano-adjuvants enhance and activate host immune responses. This work will be in collaboration with Professors Wannemuehler and Bellaire from the Veterinary Microbiology and Preventive Medicine department at ISU.

Example REU Project: Two REU students will learn to fabricate antigen-loaded nanoparticles (200-800 nm) using CPH:SA and CPTEG:CPH copolymers. The antigens of interest include ovalbumin, F1-V (plague antigen), influenza hemagglutinins, and rPA (recombinant protective antigen against anthrax). The students will learn to characterize these nanoparticles using electron microscopy and a Zetasizer (for size distribution). One REU student will study how the blank nanoparticles (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 nanoparticles with the appropriate controls. The immune response in these animals will be characterized using both antigen-specific antibody responses and T and/or B 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 to nanotechnology, materials science, biochemistry, animal studies, and applied immunology.

2. Nano-carriers for Reactions Catalyzed by Multi-Enzyme Complexes – Experimental

Mentors: Balaji Narasimhan and Surya Mallapragada

The Narasimhan and Mallapragada groups have designed and investigated novel active flexible and semi-flexible polymeric nano-carrier platforms to enable nanoscale spatial co-localization of multiple active enzymes (see Fig.). Several multi-enzyme complexes found in Nature are designed to ensure rapid transport of each intermediate in the reaction to the next neighboring active site, since the intermediates are often unstable. Thus, it is critical to molecularly co-localize these enzymes in nano-carriers so that the reactive intermediates can find the next active site for the desired products to be formed. Thus, the focus of this research is to create active nanostructured environments that can modify the direction of complex conversions by confinement of the active catalytic functionality within both spatial and temporal scales. We will investigate the biosynthesis of flavan-3-ol, whose production is mediated by two enzymes with a highly reactive intermediate. Flavan-3-ols, such as (-)-epicatechin, are flavonoid natural products with powerful antioxidant properties and are the major contributors to the cardioprotective and anticancer activity of various foods such as green tea and dark chocolate.

Example REU Project: The REU student will design and characterize novel nano-carrier platforms based on self-assembling ionic and degradable copolymers to co-localize and stabilize multiple enzymes with reactive intermediates. The ionic copolymers will be based on ethylene glycol and propylene glycol and will self-assemble into micelles. The degradable copolymers will be based on polyanhydrides and will be fabricated into nanoparticles. The student will synthesize these two platforms and characterize them using electron microscopy, light scattering, and NMR. Model enzymes from the flavan-3-ol pathway, including anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR), will be tethered to these platforms using well-known coupling chemistries. The activity of these enzymes will be measured using enzyme-linked immunosorbent assays and other bioactivity tests. The project integrates nanotechnology, materials science, and biochemistry.

3. Drug and Gene Delivery – Experimental

Mentor: Surya Mallapragada

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.

4. Aptamer-based Catalyst Design – Experimental and Computational

Mentors: Keith Woo and Monica Lamm

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. In this research, the application of computational and combinatorial techniques 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: From 1012 DNA variants, we have identified 14 catalytically active sequences. The primary research in this project will involve structural determination of these catalysts with computational approaches in collaboration with Monica Lamm. A new computational approach will be tested on structural motifs of oligomers with known structures. The REU student will also have the option to be involved in growing crystals of the active DNA sequences for x-ray structure studies. In working on this project, a student will become acquainted with a variety of techniques and approaches in chemical, biochemical and computational sciences.

5. Cellular Uptake of Engineered Nanomaterials – Computational

Mentor: Monica Lamm

With the rapid increase in production of nanomaterials and their inevitable discharge into the environment, it has become of paramount importance to develop simple and established rules for describing the impact of nanomaterials on living systems. Our primary objective in this research is to examine in-depth the impact that engineered nanomaterials have on mammalian and plant cells, with the ultimate goal of developing computational protocols for predicting the fate that nanoparticles will have in the ecosystem. We use a multiscale modeling approach for developing molecular simulations capable of modeling the interactions between nanoparticles and plant cell membranes. Our computations are validated by data from our experimental collaborator, Professor Pu-Chun Ke from the Biophysics Department at Clemson University.

Example REU Project: In this project, the student will conduct computer simulations to provide a molecular description of the mechanism by which dendritic polymers and nanoparticles (see Fig.) are taken up by cell membrane lipid bilayers. Molecular dynamics simulations will be conducted to determine the predominate factor(s) by which these nanomaterials alter the structure of cell membranes. In particular, the student will study the effect of particle surface charge and aggregation on the structure of a model lipid bilayer. These modeling results will be combined with experimental cell membrane potential measurements from the Ke lab to construct a mechanistic picture of cell membrane disruption by dendritic polymers and nanoparticles.

6. Computation of Enzyme Structure and Function – Computational

Mentor: Peter Reilly

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. 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 specific enzymes. We have used quantum mechanical computation to determine the mechanisms of three enzymes, an endoglucanase that hydrolyzes cellulose, an α-1,2-mannosidase that cleaves terminal mannosyl residues from a oligosaccharide structure, and a β-xylosidase that attacks terminal xylosyl residues in short xylooligosaccharides. We are also data-mining amino acid sequences and 3-D structures of enzymes that are part of the fatty acid/polyketide synthesis cycle to create a major database that can be used for phylogenetic studies.

Example REU Project: The REU student will participate in one of two projects: 1) quantum mechanical computation to find the mechanism of a glycogen synthetase, which makes the animal polysaccharide glycogen from glucose and ATP, as it converts bound substrates through the transition state to bound products; or 2) advanced data-mining techniques to assemble thousands (!!) of sequences and 3-D structures of the same enzyme from already existing but scattered sources, and then determine how they are related to each other by these sequences and structures.

7. Competition between Soluble and Extracellular Matrix Signals during Cell Migration – Experimental

Mentor: Ian Schneider

During tumor metastasis, carcinoma cells migrate out of the tumor into the surrounding tissue. They do this by sensing gradients of soluble proteins such as epidermal growth factor (EGF) and organized fibers of insoluble extracellular matrix (ECM) proteins such as collagen. Both EGF and collagen induce underlying intracellular processes that critically regulate cell migration. One example of an 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 before pulling itself forward. Another example is to protease activity that allows cells to eat through dense ECM that surrounds normal tissue. We are interested in understanding how both EGF and collagen controls the remodeling of focal adhesions and the degradation of ECM during cell migration. The goal is to independently vary the spatial and temporal presentation of soluble proteins and ECM using engineering tools: soft lithography, electrospinning, epitaxial growth, magnetic matrix alignment and microfluidics. These tools will be used in conjunction with fluorescent biosensors and live-cell light microscopy techniques that allow for the visualization of focal adhesion dynamics, protease activity 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 in 3D collagen networks with varying degrees of fiber orientation. Gradients of EGF will be applied in different orientations with respect to the fiber alignment forcing the cell to choose between preferred directional cues. The REU student will learn how to align 3D collagen networks and culture cells in those networks. Additionally, they will analyze the migratory behavior of cells as well as the intracellular processes of adhesion turnover and localized protease activity. Cells will express green fluorescent protein tagged pavilion, a putative focal adhesion protein. Additionally, a quantum dot-based protease biosensor can be added to the 3D matrix to measure protease activity. This project will expose students to collagen fiber polymerization and alignment techniques, molecular and cellular biology and quantitative light microscopy.

8. Solvent Effects on Fractal Nanoparticle Diffusion – Computational

Mentor: Dennis Vigil

The formation of fractal clusters due to aggregation of compact spherical primary particles is an important process that occurs in many physical situations, such as during the synthesis of particulate material in aerosol and colloidal reactors. Because the resulting particle morphology and size distribution can have a strong impact on product quality, for example in biomedical drug delivery applications that may require specific particle size and shape, it is necessary to develop accurate models of particle aggregation for the purposes of prediction and control. The Vigil lab has been involved in the development of such models by understanding how diffusion of fractal clusters is influenced by the cluster size, fractal dimension, and by its interactions with solvent molecules.

Example REU Project: The REU student will use molecular dynamics simulations to delineate the how the solvent influences particle diffusion for solvaphilic and solvaphobic fractal clusters. The REU student working on this project will gain experience with molecular simulation methods and software, and high performance computing.

9. Production of Biofuels from Bio-Oil – Experimental

Mentor: Laura Jarboe

Biomass, as a temporary storage unit of sunlight-delivered energy and atmospheric CO2, has long served society’s energy needs in the form of food for people and livestock and fuel for burning. Given the increasing scarcity of fossil fuels and concerns about climate change, we now aim to use biomass as a source of carbon and energy for production of fuels and chemicals. Hybrid thermochemical processing converts biomass to bio-oil, syn-gas and char. We aim to use carbon-rich bio-oil as a substrate for the production of biorenewable fuels and chemicals by bacteria.

The goal of this project is to develop a bacterial strain that can rapidly metabolize bio-oil and is tolerant of inhibitory bio-oil components. This project could lead to the commercial utilization of bio-oil for biofuels production.

Example REU project: Students will study bacterial (E. coli) inhibition by bio-oil. Researchers will obtain fresh fractionated bio-oil samples and monitor the effect of this bio-oil on bacterial growth. Students will use sterile technique to prepare media and monitor bacterial. Students will also obtain and analyze bacterial RNA in order to identify genes with altered expression in the presence of bio-oil, providing insight into the mechanism of bacterial inhibition.

10. Mechanisms of Fast Pyrolysis – Experimental

Mentor: Robert Brown

Fast pyrolysis is the thermal decomposition of biomass into liquid (bio-oil), gas (syngas), and solid (bio-char). The bio-oil can be upgraded to transportation fuels, the syngas can be burned for process heat, and the bio-char, once considered a low-value byproduct, is receiving wide attention for its potential as a carbon sequestration agent and soil amendment (for example, see http://www.biochar-international.org/). Fast pyrolysis is receiving increasing attention as an attractive pathway to drop-in biofuels, which can be directly substituted for petroleum-based gasoline and diesel fuel without expensive changes to the existing transportation infrastructure.

The partition between solid, liquid, and gas and the composition of the bio-oil depends upon the kind of biomass, the presence of inorganic compounds, heating rate, and reactor configuration. As traditionally produced, bio-oil contains hundreds of organic compounds including lignin-derived oligomers. Recent work at ISU indicates that under the correct conditions, pyrolysis can be controlled to produce mostly monomeric products via depolymerization of both holocellulose and lignin in plant materials. These monomers would generally be easier to recover from bio-oil and upgraded to desired products than traditional bio-oil. So different are the products of this process from those of fast pyrolysis that we dub it “selective thermal depolymerization” (STD). We are conducting fundamental investigations of the reaction networks that described STD.

Example REU Project: The REU student will conduct experiments to understand the mechanisms by which carbohydrate and lignin are thermally depolymerized and subsequently repolymerized to form polysaccharides and polyphenols in bio-oil. Experiments will be performed in “micropyrolyzers” that have been modified to encourage gas-phase reactions that occur subsequently to thermal depolymerization of biomass feedstock. The REU student will be responsible for designing and overseeing construction of a “secondary reaction” test section between the micropyrolyzer and the gas chromatograph to which it is normally directly attached. This will allow new kinds of experiments in STD that have never been previously performed, providing better understanding of the mechanisms that control product distribution. The REU student will work with a team of graduate students and professional staff who are conducting experiments with other micropyrolyzer systems in our laboratories.

11. The Artificial Pancreas Project – Experimental and Computational

Mentor: Derrick K. Rollins, Sr.

Type 1 diabetics often experience extreme variations in glucose concentrations which can have long- and short-term adverse effects such as seizures, coma and organ degeneration. Studies have established that there is a need to maintain the glucose levels within a normal range to avoid complications caused by diabetes. However, initial attempts to regulate blood glucose levels using insulin infusion, multiple injections or their combinations have had limited success as they lack the ability to decide the appropriate rate and/or amount of insulin infusion based on the current metabolic state of the body. An “artificial pancreas” consisting of a continuous glucose monitor, an insulin infusion pump, and a control algorithm has the potential to improve glucose regulation by intelligently deciding the proper amount of insulin delivery at the proper time.

In this research, we are developing patient-specific dynamic, block-oriented, multivariable models that can predict how various disturbances (inputs) such as food consumption, activity and stress affect glucose concentrations. The modeling methodology chosen has the ability to determine causality even from correlated inputs, which invariably results in free-living real subject studies. Since glucose levels are highly dependent on the metabolic and physiologic states of the body, the models includes several non-invasive variables that are related to activity, stress, food consumption, etc. as inputs to the model so that the affect of their interactions can be explicitly taken into account.

Example of REU Project: The student will help with assist in our inpatient and outpatient clinical trials in data collection, processing, modeling and analysis. This work is supported by the Juvenile Diabetes Research Foundation (JDRF) and the National Institute of Health (NIH). Our partners on engineers, doctors and nurses at the Illinois Institute of Technology (IIT), the University of Ill at Chicago, and the University of Chicago Medical Center. Students on this project will have an opportunity to analyze and develop models using real subject data and could assist with our data collection with the patients in a hospital setting. Student may also have an opportunity to assist with the development of a new non-invasive continuous-time glucose monitor that uses data from an armband as shown with several sensors to measure activity and stress.

 

12. Polymer Properties that Selectively Target Tumor-Associated Macrophages – Experimental

Mentor: Kaitlin Bratlie

We are interested in understanding what material parameters can be exploited to discriminately deliver drugs to tumor-associated macrophages (TAMs). TAMs promote tumor growth through the release of angiogenic – blood vessel forming – molecules. These growth factors provide nutrients to the tumor and enable metastasis. TAMs are alternatively activated macrophages that can be reprogrammed to classically activated macrophages, which kill neoplastic cells through the release of cytotoxic molecules, such as tumor necrosis factor. Our goal is to determine what polymer properties will allow for select targeting of these alternatively activated macrophages. We will examine how the cytokine expression of classically and alternatively activated macrophages changes in response to different functional groups. We also will monitor phagocytosis through production of reactive oxygen species (ROS). The internalization velocity will also be explored using light microscopy.

Example REU Project: One to two REU student(s) will learn how to polarize macrophages into classically and alternatively activated cells. The macrophages will be cultured with different size, shape, and functionalized polystyrene microparticles. The degradation of the polystyrene particles will be monitored via a superoxide anion assay in which the ROS produced during phagocytosis will react with luminol to produce chemiluminescent light, which will be measure with a plate reader. The cytokines expressed will also be studied through colorimetric enzyme linked immunosorbant assays (ELISA) to quantitatively detect the presence of antigen. Finally, the internalization velocity of the different particles will be tested through light microscopy. The ultimate goal for this project is to examine all of the data generated through these assays and determine how the particle parameters influence the two different macrophage polarizations. This study will lay the groundwork for future studies in targeted drug delivery. The student(s) will learn 1) sterile cell culture technique, 2) scientific communication skills, 3) how to process data, and 4) how to perform biochemical assays.

13. Bacteriophages on Surfaces used for Detection and Protection – Experimental

Mentor: Rebecca Cademartiri

Bacteriophages – also known as phages ‑ are viruses that attack and sometimes destroy bacteria. They can be used as an alternative to standard antibiotics in a variety of systems (drinking water, food, animals and humans).
While the behavior of phages towards bacteria is well studied, there is no detailed understanding of their interaction with the surface of materials. Recent progress (e.g. in drug delivery, tissue engineering) has shown that materials are becoming increasingly important in medicine, biodetection, filtration and sensing. Unraveling the interaction of bacteriophages with surfaces will be necessary to take full advantage of them in medical or environmental applications.
In the R. Cademartiri lab we are investigating the interaction of phages with materials that are relevant to medical (e.g. drug delivery) and industrial applications (e.g. filters, membranes). An initial screen of the interaction of phages with materials (e.g. polymers, oxides, metals) with different surface chemistries will lead to the formulation of heuristic rules about the bioconjugation of phages to materials. In a second step we will use this new understanding of material-phage interactions to design specific bacteriophage-material conjugates for applications in e.g. drug delivery, wound healing, filtration and detection.

Example REU project: The REU student will investigate the binding properties of bacteriophages to various materials. The student will use sterile microbiological techniques to grow bacteria and phages, and determine the activity of the phages on the various surfaces. The student will also use standard techniques in material science to modify the chemistry of the surface of the materials. The student will test the effectiveness of the surface modifications by quantifying the number of phages bound to the surface and by demining the chemical composition of the surface. This project combines sterile microbial, material science and analytical techniques.

PHOTO: (Left) antibacterial properties of surfaces coated with bacteriophages. (Right) Bacteriophages on silica particles. Scale bar: 100 nm.

14. Optimization of aptamer structure and function for sensor applications – Experimental or Computational

Mentors: Monica Lamm and Marit Nilsen-Hamilton

Aptamers are short nucleic acids that fold to specifically recognize other molecules. They are compatible with many sensor platforms and are stable to dehydration, high temperatures and long storage.  Despite their obvious advantages as sensors for molecular identification, aptamer technology has not been widely translated into clinical or other practical applications.  This is particularly true of aptamers that recognize small molecules as there are very few that recognize most medical drugs, drugs of substance abuse, environmental contaminants and other important chemicals.  Obtaining an aptamer today involves fishing from a combinatorial pool of oligonucleotides that represents a miniscule portion of the structural landscape for nucleic acids.  For aptamers to fulfill their clear promise as superior detectors, it is important to develop a reliable and rapid means of optimizing the identification of new aptamers and their optimization for application. Our goal is to develop a computational approach to consistently and accurately predict aptamers that can be validated in the laboratory.

Example REU project: Two students will work together, one in a wet laboratory and one in a computational laboratory to model an RNA aptamer and its ligand. The modelling work will involve changing the sequence of the known aptamer and predicting, by molecular dynamics simulation, the minimized structure.  The experimental work will involve synthesizing the aptamer and variants in the laboratory and testing their binding affinities for the ligand using isothermal titration calorimetry.  Results obtained by each of the students will inform the experiment/computational progress of the other.