Spinal cord injuries, heart disease, and osteoporosis afflict millions every year, and their current treatments are limited and temporary. Using varied approaches from materials science and biology, this proposal aims to develop longer lasting cell replacement therapies that can be applied to repair damaged areas of the body, ranging from nervous and cardiac tissues to bone. Specifically, we will develop novel, bioactive, injectable synthetic materials to enhance the therapeutic efficacy neural stem cell transplants.
The successful integration of stem cells into such therapies will hinge upon three critical steps: first, stem cells expansion in number; second, differentiation into a specific cell type or collection of cell types; and third, promotion of their functional integration into surrounding tissue. Precisely controlling each of these steps is essential to maximize therapeutic efficacy, as well as to minimize potential side effects that can occur when the cells numbers and types are not properly controlled. We have already developed a synthetic platform for reproducible control of several steps, and plan to expand into animal work with further collaboration among biology and physical sciences departments. The result will be a technology platform that can be applied in the future to numerous stem cell populations, tissues, and diseases.
We propose to use optical tweezers to investigate the folding and unfolding of metabolite-sensing (riboswitch) RNAs under mechanical tension. Riboswitches are messenger RNA sequences which regulate gene expression in response to binding of small molecules. They are found in both bacteria and higher organisms and regulate important metabolic pathways. Artificial riboswitches have been proposed for therapeutic use. Although certain broad features are known, the specific mechanism of these molecules activity remains unclear. Structures of some riboswitches have been determined by X-ray diffraction and NMR, but the conformational dynamics important for activity are not known. If we understood the kinetic behavior of RNA, we could predict how often and for how long the functionally critical states are present. Recent developments in manipulating single molecules make this possible. By attaching beads to the ends of an RNA molecule and capturing them in an optical trap, we can stretch the molecule with controlled force. This allows us to monitor folding in real time and study the role and lifetimes of intermediate states. By systematically studying the components of a riboswitch, we can not only learn how these important molecules work, but also make progress toward a general theory of RNA folding.
To better understand living organisms, scientists take pictures of cells and tissue using light microscopes and high-resolution digital cameras. The complex networks inside biological samples make extracting information about specific molecules difficult, so markers (also called biosensors) are often introduced into the sample before taking the picture to highlight features of interest. Biosensors work by directing a colored probe to a specific biomolecule using a chemical tag. When scientists want to generate images from deep inside tissue, they can apply magnetic resonance imaging (MRI), using radiowaves that penetrate tissue that is opaque to visible light. We have recently introduced a new type of MRI biosensor that is much more sensitive than previous ones. It works by using a xenon-carrying cage to tag a specific biomolecule with inert xenon atoms, whose MRI signal has been laser-enhanced. When inside the carrier molecule, this xenon can convey information to the MRI about its location and if it has tagged a biomolecule or not. We have teamed up with a biology expert to develop this biosensor technology for sensing specific types of tissue, like breast cancer, and specific biomolecules, like metabolites, inside of living organisms.
Currently, blood platelets can only be stored for 5 days as they cannot be refrigerated. There is experimental evidence that a simple sugar, trehalose, can solve this problem by stabilizing the lipid membranes of the platelets in the dry state. The mechanism of action of trehalose is of great interest for cell and tissue preservation. Preliminary evidence suggests that trehalose modifies the mixing behavior of the lipids in membranes. However, the biologically relevant scale (nanometers) with respect to mixing behavior and membrane structure has not yet been investigated. Our multidisciplinary environment will allow the trainee to examine the mechanism of action of trehalose, at the nanometer-scale, through parallel computer simulations and experiments. We will set achievable goals by having the trainee combine and learn existing technology from the investigator's labs. The trainee will be provided computer programs developed in Faller's group, training and access to AFM and optical techniques applied to lipid bilayers in Longo's lab, and training and access to blood platelets from Crowe and Tablin's lab. We will have a monthly team meeting to coordinate this effort and transfer the scientific knowledge gained directly to the practical application of cell preservation actively being pursued in the Biostabilization Center.
Understanding protein function on a genome-wide scale is a main goal of biology and will dramatically affect the progress of medicine. Unraveling protein-protein interactions (PPI) is an important way to help achieve this goal since protein function frequently depends on PPI. Furthermore, PPI underlie a wide range of physiological and disease processes. Despite advances in high-throughput technologies, large-scale PPI mapping remains a daunting task due to the huge demand of time and resources. Current whole-proteome platforms (e.g., yeast two-hybrid or proteome microarrays) screen one "bait" protein each time and obtain on average 5 positive signals, which is extremely inefficient compared to the capacity of the platforms themselves at thousands of proteins. Screening multiple baits together in an experiment seems a natural solution to improve efficiency, if the relationship between preys and the multiply loaded baits can be resolved. We propose a simple strategy for efficient PPI mapping that solves the tracking problem in a fundamentally different way. This new method has the potential to accelerate protein interaction mapping, as it can reduce the number of experiments by one order of magnitude as compared to existing methods. Successful completion of this project will have important impact on biotechnology and medicine.
Development of nano-electronic sensor arrays with a highly controllable architecture that are capable of identifying hundred of analytes with high sensitivity and selectivity has gained considerable interest in the area of proteomics, disease diagnostics, homeland security, and environmental monitoring, enabling simultaneous detection of a complex mixture of analytes at sub-ppb concentrations. Although current nano-sensor arrays have revolutionized our ability to provide label-free, real-time, sensitive and selective detection, they have low throughput and limited controllability and are unattractive for high-density sensors array. More importantly, surface modifications, typically required to incorporate bioreceptors, have to be performed post-synthesis and post-assembly, limiting our ability to individually address each nanostructures with the desired bioreceptor.
Electropolymerization of conducting-polymers is a promising and versatile alternative for fabricating nanowire sensor arrays with the required controllability. The benign conditions of electropolymerization enable the sequential deposition of nanowires with embedded bioreceptors, providing a revolutionary route to create truly high-density and individually addressable nano-sensor arrays. Our ultimate goal is to develop a novel technique for the fabrication of high-density biosensor arrays based on biologically-functionalized conducting-polymer nanowires and to investigate techniques to tailor-make the sensitivity, selectivity and response time of the sensors for the development of protein/antibody array for label-free and rapid detection.
The emergence of high-throughput technologies in recent years has enabled the study of cells as systems. In silico models provide a way to represent these data sets in a concise and analyzable format, where biological functions can be simulated and tested. A model of the mitochondrial metabolism has been constructed based on the proteome and legacy data on this organelle. We propose to develop a general algorithm to integrate isotopomer data for flux analysis. We will evaluate the models predictions with published data and apply the model to design effective experiments using stable isotope tracers in cultured cells. Successful completion of this project will allow one to systematically reconstruct a metabolic network and apply isotopomer data, in addition to conventional measurements of extracellular fluxes, to reliably calculate steady state intracellular fluxes in absence of kinetic parameters. This work will be carried out by the nominated fellow under the guidance of two faculty members, Dr. Palsson and Dr. Lee. Dr. Palsson, a pioneer in metabolic network reconstruction, will be the advisor in the area of mathematical modeling. Dr. Lee, an expert in isotopic study of TCA cycle metabolism, will provide training in the area of mass spectrometry analysis and experimental methods.
Many molecular and cellular changes occur in the course of disease. It has been difficult to distinguish between changes that are a cause rather than an effect of the disease. For example, in the course of Huntingtons disease, mutant protein forms aggregates. The role of these aggregates in Huntingtons disease has been controversial. We developed an automated robotic imaging and analysis platform to follow individual cells and the processes occurring within them. Using this platform, we have recently determined that large aggregates help cells cope with mutant protein. Here, we propose using our robotic platform in combination with biophysics and biostatistics to relate changes occurring in Huntingtons disease within individual cells and to the fates of those same cells. Using this approach, we can identify early molecular and cellular changes that are the most important to target in the course of disease.
Living cells have a remarkable ability to detect and process environmental signals and to respond to them appropriately. These responses are mediated by networks of signal transduction proteins that act as biochemical circuits. If we fully understood how these cellular circuits work, it might be possible to engineer them in order to endow cells with useful, highly specific biosensor function. For example, it might be possible to engineer microbes that function as sensitive but inexpensive sensors for particular harmful chemicals. It might be possible to engineer cells to replace pancreatic beta-cells, which detect blood sugar levels and respond by releasing the appropriate amount of insulin. To generate such precision engineered biological systems, we first need to understand how to modulate the quantitative input/output behavior of signaling circuits with the precision and flexibility of an electrical engineer. Here we propose to explore the engineerability of a natural signaling pathway in the model microorganism yeast. We will introduce synthetic feedback loops into the pathway in order to attempt to systematically reprogram the circuits input/output behavior. What we learn from this study will help lay the groundwork for precision engineering of cellular circuits as an important tool in biotechnology.
The nominated candidate for this GREAT fellowship is Rashda K. Khan, a second year Ph.D. student in the Department of Chemistry and Biochemistry at UCSB. In her first year of graduate studies, she took the opportunity of taking classes from various disciplines: Materials Science, Chemistry and Biology. She is now finished with her coursework. Rashda K. Khan continues to attend excellent research seminars that enlighten her with multidiscipline both within the UC system and around the country. Ms. Khan is carrying out independent research in both Stucky and Waite's laboratories. She will continue to participate in both weekly group meetings. Ms. Khan expects to advance to Ph.D. candidacy by July 2005. As her graduate career begins to progress she is practicing experimental design, execution, written and oral communication; all are important for a future career in industry, academia or government.
The novel and interdisciplinary training experience at UCSB will integrate chemistry, biology and materials in the outlined project of Nereis jaws. This research will distill a set of biomimetic rules derived from the analyses of jaws, allowing for novel material compositions and novel robust lightweight material designs, thus positively impacting the biotechnology industry.
The ability to observe, analyze and manipulate individual biological molecules such as DNA and proteins will allow us to better understand human diseases and to design highly sensitive analytical instruments that can work with very small amounts of sample material. This development requires research and training that is increasingly interdisciplinary and unites biology with other areas such as engineering, microfabrication and nano-science.
The first component of this project is the development of a novel bio-photonic sensor device in which single molecules can be studied with optical methods. It requires a gate to introduce molecules one by one into the optical sample volume, and the capability to collect light emitted from the molecules through the liquid solution in which the molecules are transported. To accomplish this, we combine nanopore gates with hollow-core optical waveguides. Many of these resulting sensors are placed in parallel on a chip using silicon fabrication technology. As a result, they are compact, robust, highly sensitive and very fast.
The second component is a multidisciplinary education of a graduate student in electrical engineering, microfabrication, and molecular biology. This broad and unique skill set is required for a successful career at the interface of the biology, physics and engineering.
DNA fingerprint analysis represents one of the most common tests performed by crime laboratories. With the increasing backlog of casework DNA samples in virtually all states and internationally, the need for higher speed, higher throughput, more reliable, more sensitive and less laborious methods for genetic and forensic studies are immediate. Polymerase chain reaction (PCR)-based short tandem repeat (STR) assays using capillary electrophoresis (CE) is the method of choice for genetic fingerprinting owning to the highly discriminating DNA profiles generated among individuals. However, this method is slow, cumbersome and costly using the current technology. To address these problems, a microfabricated capillary array electrophoresis (muCAE)-based device with integrated nanoliter-scale thermal cycling and sample processing is proposed for STR analysis. The integrated muCAE device can be applied to rapid genetic and forensic analysis of multiple samples in a highly parallel fashion and also can be adapted for a small portable device for point-of-care medical diagnostics and mass disaster forensic investigation. There will be two stages in the project: 1) the establishment of the optimal PCR and separation conditions for multiplex STR amplification and electrophoretic analysis using energy-transfer (ET) cassettes to label STR primers for simple fluorescence-dye labeling process and 2) the design and integration of PCR amplification and sample processing into a muCAE device. The expertise in microfabrication and microfluidics in Professor Mathies' laboratory together with the in-depth knowledge of genetics and forensics in Professor Sensabaugh's research group provide a powerful platform for performing this project. This effort will also be aided by collaboration with the Virginia Division of Forensic Science (VDFS) and Palm Beach Sheriff Office (PBSO) for device and assay validation. These two goals will require the knowledge, expertise and collaboration in the fields of genetics, biochemistry, microfluidics, microfabrication and engineering. Independent research activities will be conducted with regular discussion of research progress with my faculty sponsor, Professor Mathies and presentation to his entire research group. Co-sponsor Professor Sensabaugh will provide guidance in genetics and forensics. Forensic scientists from VDFS and PBSO will critically evaluate the technologies. The results of this project will be actively disseminated through relevant publications and conferences concerning the development of analytical chemistry, genetic analysis devices and methods and microanalytical devices. In addition, work of this project regarding the evaluation of forensic typing will be published in forensic journals such as Journal of Forensic Science and through presentation at the Promega Conference. This unique project requires a truly cross-disciplinary experience in biotechnology and advances the current limits of forensic science and human identification to the next level.
Single cell analysis plays an important role in the study of cell metabolism and protein expression. The ability to manipulate single cells is highly sought after in the biomedical and biological communities. The nominated student, Pei Yu "Eric" Chiou, has recently invented a revolutionary tool for single cell manipulation. This new tool, called optoelectronic tweezers (OET), enables trapping, moving, and sorting of cells at single cell level using a very low power optical beam (~ 1000 times lower than that of conventional optical tweezers). In this project, we propose to develop a programmable OET for parallel manipulation of single cells. Cell sorting, screening, separation, and parallel addressing of single cells will be accomplished by programming a digital light projector similar to that used for PC presentation. This will greatly increase the throughput of single cell analysis. There are three specific Aims for the proposed research. In Aim 1, we will develop an automatic cell manipulator by integrating OET with computer vision and programmable OET with Digital Micromirror Device (DMD) spatial light modulator. After successful development of the OET tool, we will focus on two biomedical applications. Aim 2 will focus on the implementation of micro-Fluorescence Activated Cell Sorter (FACS) using parallel OET cell cage array. Aim 3 will explore the use of OET array for organizing cells in an array to study the radiation effect (gamma rays and UV light) on cells. This project will be jointly supervised by Dr. Ming Wu, Professor of Electrical Engineeringat UCLA and a Fellow of IEEE, and Dr. Edward McCabe, Professor and Executive Chair of Pediatrics at UCLA and Physician-in-Chief in Mattel s Children s Hospital. Professor James Liao of Chemical Engineering and Professor Bruce Dunn of Materials Science and Engineering, UCLA, will also serve as mentors for Mr. Chiou. Their recommendation letters are attached in the Appendix. The proposed project will provide cross-disciplinary training in physics, chemistry, engineering, and biology for the nominated graduate student.
We propose to study protein-DNA and protein-membrane interactions in microarray format using label-free optical scanning microscopy developed by Prof. Zhu and James Landry (the nominee). In this method, we detect changes in molecular density and conformation of macromolecules as a result of their binding to surface-immobilized target DNA or membranes by following corresponding observable changes in the microscope such as in thickness, refractive index, and optical extinction coefficient. The microscope is coupled with flow cells that (a) contain DNA microarrays or membrane immobilized on glass or mica, and (b) enable optical access and introduction of fluids for reaction. Through a set of experimental studies of (1) protein-DNA interactions in DNA repair and replication, and (2) protein-membrane interactions in protein binding on functionalized lipid bilayers, we expect to train an exceptional physics graduate student, Mr. James Landry, into a truly interdisciplinary scientist whose expertise will abridge those of two, by tradition, vastly separated disciplines with respective backgrounds and scientific approaches.
Through this UC-GREAT program, Mr. Landry will combine the expertise in surface chemistry and optical physics from Prof. Zhu's group and the know-how in microarray fabrication from Prof. Gregg,s group with molecular biochemistry techniques from Dr. Cosmans group. Specifically, he will (1) learn to synthesize unmodified and modified DNA oligomers, and to overexpress and purify proteins in Dr. Cosman's laboratory; (2) fabricate microarrays of immobilized DNA oligomers in Prof. Gregg's laboratory; and (3) obtain and analyze the label-free, in-situ optical measurements of protein binding reactions with DNA microarrays and lipid bilayers in Prof. Zhu's laboratory. He will also aid Prof. Zhu in building a new high scan speed optical microscope for versatile, real-time imaging. In addition he will attend biophysics and biochemistry conferences and seminars regularly to communicate his research and interact with others in the field. This process will empower him to pursue a productive career in the cross fields of life science and physical science.
This proposal seeks support for a talented graduate student, Debasish Sen, to pursue thesis research on quantum dots as a unique platform of flexible probe design to modulate the immune response. Quantum dots are crystalline spheres of 3 to 6 nanometer diameter that exhibit very bright, photostable fluorescence with tunable narrow bandwidth emission characteristics. Containing a CdSe core, Qdot" particles are coated with a mixed hydrophobic/hydrophilic polymer, making them suitable for work with living cells, and can be conjugated with streptavidin, forming the basis for specific biological labeling. Preliminary studies carried out by the fellowship candidate have demonstrated that quantum dots are taken up avidly by dendritic cells and can be imaged through the endocytic pathway. We will use two-photon microscopy to investigate the dynamics of quantum-dot labeled dendritic cells as they traffic through the body and present antigen to lymphocytes inside the lymph node. The collaborative nature of this project, involving an immunologist and a neurobiologist at UCI, both with strong biophysical training and inclination, will provide expertise to carry out an ambitious series of experiments. Collaboration with Mark Ellisman's group at UCSD will provide complementary expertise in electron microscopy. With both in vitro and in vivo experimentation, the project will provide excellent training in immunology, multi-photon imaging microscopy and quantitative analysis, and probe design with single-particle detection. Laboratory research will be supplemented by coursework in cell biology, immunology, and microscopy. The candidate will attend seminars in the home department and in the monthly Immunology seminar series. He will also participate in the active journal clubs in the home department and in the Center for Immunology. This graduate training experience will provide excellent preparation for continued research in immunology with emphasis on applying discoveries in biophysics and biotechnology to biomedical problems that hold therapeutic promise.
Targeted drug delivery to treat diseases is advantageous to reduce both drug dosage and collateral damage to other tissues. When applied to cancer therapy, the targeted delivery of cytotoxic agents to tumor vasculature has shown a therapeutic benefit1. To increase the quantity of therapeutic agent delivered to the disease site, we propose to amplify the targeting signal, with a scheme similar to the aggregation of platelets at a clot. Our goal is to deliver nanoparticle conjugates to the extracellular matrix of tumors, and then locally heat the targeted area by inductively coupling RF energy to the magnetic nanoparticle core. We propose that this local heating will denature collagen proteins, exposing cryptic binding sites and recruiting additional particles from the bloodstream. The technology we plan to develop constitutes the design of "mutlifunctional" nanoparticles that can diagnose and treat disease in a minimally invasive manner. Our ultimate goal is to design these particles to recognize the target, bind to that site, exponentially accumulate, and then release their therapeutic payload. The merging of sponsors' expertise in nanomaterials design, tumor biology and surface chemistry with the fellow's background in electrical engineering and nanoparticle bioconjugation will facilitate the development of this novel treatment modality. Drs. Bhatia, Ruoslahti, and Sailor have a record of collaboration, including co-mentoring of students, co-authoring high impact publications2-5, and receiving shared funding. Austin Derfus, the nominated fellow, will benefit from monthly meetings with the three scientists and will be trained in the skill sets held by each of their laboratories. In addition to training the fellow for a scientific career, technology developed from this research has the potential to improve clinical medicine and generate revenue for the California economy.
Micro RNAs (miRNAs) are 21-23nt single stranded RNAs that are processed from 60-80nt stem-loop precursors. These small regulatory sequences pepper the genome and have been shown, in some cases, to regulate gene expression by inhibiting translation of mRNAs to which they are partially complementary. More than 250 miRNAs have been identified in numerous species ranging from C. elegans to humans and recent computational approaches have predicted many putative miRNA targets, the vast majority of which require experimental validation. Despite the significant advances in target identification, further advances are still required. Since miRNAs can regulate genes that are in control of fundamental life processes such as development, fat metabolism, stress, apoptosis, and hematopoiesis, the genomic target sites of these small regulatory RNAs are of great interest. We propose to complement the current methods using a combination of experimental and computational methods to identify genomic miRNA target sites with high sensitivity and specificity. The experimental approach utilizes an immunoprecipitation strategy to selectively enrich for target mRNAs, followed by microarray identification. The computational approach will enable us to define groups of genes that are targets of specific miRNAs, define the requirements for target sites for each miRNA, and potentially find sequence motifs involved in miRNA-mediated gene regulation. Finally, we will also examine the relationship between a miRNA's complementarity to its target mRNAs and the number of target sites per miRNA per target gene to assess how miRNAs affect gene suppression, providing new insights into the organization of gene regulatory networks.
Kinase-mediated protein phosphorylation is a key regulator of nearly every cellular signaling pathway. Thus, the ability to map phosphorylation pathways is critical to understanding cell biology. Historically this process has been slow, and has of necessity relied largely on the performance of pair-wise examinations of interactions between individual kinases and candidate substrates. Given the very large number of protein kinases, and the fact that many likely have numerous substrates, methods to accelerate this process are badly needed. We have previously reported the development of a method for kinase-specific labeling of substrates using ATP analogs that are poor substrates for wild-type kinases, but which are efficiently used by engineered kinases bearing an altered ATP binding site. This method has been used to identify novel substrates of several widely divergent kinases; however identification still relies on laborious conventional purification methods subsequent to labeling. Herein we describe the development of a method for the simultaneous purification of entire sets of kinase specific substrate proteins. An ATP analog specific for engineered kinases and bearing a terminal thiophosphate group is used to specifically thiophosphorylate the substrates of a kinase of interest in a cell extract. The extract proteins are selectively protected at cysteine residues and then passed over an iodoacetyl-functionalized resin, covalently trapping the thiophosphorylated substrates in the solid phase. After washing, the substrates are released by specific cleavage of the thiophosphate linkage yielding a pool of purified substrates, which may then be analyzed by mass spectrometry.
The proposed course of research will provide a broadly interdisciplinary training environment for the nominated fellow. Completion of this project will involve the application of methods in synthetic chemistry, biochemistry and molecular biology, cell biology, bioinformatics, and mass spectrometry.
Oral drug delivery, although attractive compared to injections, has been difficult to utilize for the administration of peptides and proteins due to poor epithelial permeability and proteolytic degradation within the gastrointestinal tract. We plan to develop a novel method for the oral delivery of peptides and proteins. In this study, we will utilize mucoadhesive intestinal patches to deliver therapeutic doses of insulin into systemic circulation. Our preliminary results indicate that the patches adhere securely to the intestine and that insulin patches with doses in the range of 1-10 U/kg induce dose-dependent hypoglycemia. The objective of the proposed studies is to understand the mechanisms of oral insulin delivery using intestinal patches and develop the technology so that it can be tested in diabetic volunteers. With the proposed research, intestinal patches could not only offer a novel methodology for the oral delivery of insulin, but for various other macromolecules, including growth hormones, heparin, and vaccines, as well. A significant emphasis will also be placed on the training activities of the nominee in engineering and life science. The nominee will gain proficiency in her laboratory skills in the Mitragotri Laboratory and affiliated facilities at UCSB, including the Materials Research Laboratory, California Nanosystems Institute, and Institute of Collaborative Biotechnologies. Moreover, she will gain clinical experience through her work with Dr. Lois Jovanovic, director and chief scientific officer of the Sansum Medical Research Institute. The nominee will also enhance her skills through supplementary coursework. Already proficient in the areas of chemical engineering, she will obtain further education in human biology in the context of the field of drug delivery. This will be achieved by taking courses on human immunology and pharmacology in the Department of Molecular, Cellular, and Developmental Biology at UCSB. The nominee will enhance her professional skills in oral and written presentation as well as in the supervision of undergraduate students.
The designated recipient for this GREAT award is August Estabrook, a Ph.D. candidate in Chemistry and Biochemistry at UCSB. Mr. Estabrook is a second year student, has finished his coursework and exams and thus advanced to candidacy, and is engaged in carrying out independent research involving the research groups of Professors Norbert Reich (Chemistry/Biochemistry) and Andrew Cleland (Physics).
The proposed project brings together molecular biology, biochemistry, nanoparticle synthesis, nanoscale electrode construction, and single molecule electronics. Mr. Estabrook's part in this effort is largely focused on the first Aim involving the design, construction, and incorporation of various nucleic acids and proteins into nano-scale constructs that will be the foundation of single molecule electrical devices. He is also developing new approaches to attach nanoparticles to nucleic acids and modify nanoparticles for improved electronic characteristics. The second Aim proposes to use the engineered nucleic acid and protein assemblies in conjunction with various nanoparticle attachment strategies to probe simple molecular electronic configurations. Mr. Estabrook works closely with students in Physics in both the fabrication and testing of these simple circuits. The extremely interdisciplinary nature of the overall project demands that Mr. Estabrook continue to foster intellectual connections with his coworkers engaged in the electronic device construction and conductance measurements.
Mr. Estabrook will continue to present his results via several venues, including our weekly meetings involving all members in both groups engaged in the bioelectronic effort (undergraduates Eran Levy and Tara Holstein, graduate students Gary Braun, Stephanie Wilkinson, David Wood and Professor Andrew Cleland), monthly participation in our "Biomolecular Materials Seminar" series for post docs and graduate students in all departments at UCSB, our weekly "Literature in Biomolecular Materials" series, and relevant conferences (e.g., the Veeco/UCSB conference to which Mr. Estabrook was invited to speak at in 2003). Mr. Estabrook is obtaining training as a research mentor in his supervisory role involving undergraduates engaged on projects directly related to his own research. Thus, Tara Holstein has worked under Mr. Estabrook's direct supervision for the last 12 months in constructing various DNA cruciforms; she is graduating June 2004. Eran Levy (UCSB electrical engineering major, UC LEADS participant), will start working with Mr. Estabrook this Spring quarter.
The capability to amplify through polymerase chain reaction (PCR) technology has caused a revolution in biotechnology. It has provided the means to detect genetic mutations and pathogenic organisms. In this work, we propose to address an equally fundamental need the capability to sort, that is, to separate and isolate particular molecules, viruses, bacteria and other cells, from a large background of complex mixtures, at very high throughput, purity and efficiency. This technology is the prerequisite to many promising applications in biological, pharmaceutical and medical fields that span from stem cell research to cell based therapies. As a part of this training program, we propose to combine a novel technique of molecular and cellular labeling with Microelectromechanical Systems (MEMS) technology to create a disposable, massively parallel, rare-cell sorting system. The separation mechanism will be based on dielectrophoresis (DEP) using inhomogeneous AC electrical fields. The technical goals of the training program are two fold; Firstly, we will demonstrate the principle of specifically labeling cells with dielectric particles with a pre-engineered dielectrophoretic response. This way, we may have complete control over the separation forces acting on the specifically labeled cells so that we may obtain an effective means to separate the labeled and unlabeled cells in inhomogeneous AC electrical fields. We will first simulate the electro- ydrodynamic fields inside the separation chamber to understand design principles and optimize the hydrodynamic-dielectrophoretic forces. We will consider such factors as shear stress on the cells and other factors that may affect cell viability. Secondly, using the dielectric labeling paradigm above, we will combine massive parallelism and multistaged design to create an integrated microfluidic devices that is capable of sorting the labeled and unlabeled cells with a dramatic performance improvement over current sorting technologies in the most significant aspects - throughput, purity and cell recovery. The ultimate goal for this research is to develop advanced tools to sort very rare cells for the purposes of diagnosis, treatment and fundamental understanding of cancers. The nature of this work is truly multi-disciplinary, and provides an uncommon opportunity to bring together many facets of engineering with life sciences; we will be engaged in problems that involve live cells, their surface markers as well as their manipulation through electric field driven mechanical motions in fluids. Almost all underlying infrastructure is in place and we have brought together expertise and collaborations to make this project successful. We believe this is an ideal training ground for the fellowship candidate.
We propose a research and training project to develop new statistical and computational methods for the detection of functional elements in the human genome. These methods will be based on the analysis of multiple, aligned mammalian genomes using phylogenetic hidden Markov models (phylo-HMMs), which describe molecular evolution as a stochastic process in the dimensions of both space (changes from one position in a genome to the next) and time (changes to the nucleotide at each position over evolutionary time). Besides being useful in the detection of functional elements, these models will be helpful in furthering our understanding of mammalian evolution. Newly developed methods will be applied to the complete human genome and the aligned genomes of other mammals, and results will be made available to researchers in the public and private sectors via the UCSC Genome Browser, which has become an important resource for genomics researchers around the world. Wet-laboratory experiments will be undertaken to validate a subset of novel, predicted elements. The proposed project will form the bulk of the nominated fellow's Ph.D. dissertation, and is highly complementary to other projects of the faculty sponsors. In addition, the project has potential to reveal novel functional elements and produce new analytical methods that can directly benefit the biotechnology industry and, ultimately, the economy of the State of California.
