Due to its biocompatibility, slow biodegradability, high strength, and low weight, there has been a recent
surge in the use of silk as a biomaterial for tissue engineering. Particularly, because of the robust mechanical
properties of silk (high strength and elasticity,) it has proven to be exceptionally successful for bone,
cartilage, and ligament tissue engineering. Unfortunately, however, usage has been limited to silkworm silk.
Spider silk fibers have much more impressive mechanical properties, but have so far evaded mass cultivation
or artificial production. Several recombinant spider silk proteins exist, but no one, to date, has been able to
recreate spider silk fibers with the same exceptional mechanical properties as those produced naturally. To
this end, we propose the development of a microfluidic device that mimics the advanced functionality of the
spider silk gland. Within the spider, fiber formation is an extremely judiciously and dynamically controlled
process involving complex fluid dynamics and biophysics. We hypothesize that by replicating the complexity
of the in vivo silk gland within an in vitro system, we be able to spin spider silk fibers from that have
comparable mechanical properties to their naturally spun counterparts for use as a high performance
biomaterial in tissue engineering scaffolds.
Cadherins are proteins that allow cells to adhere to each other. In addition to their vital function in processes
like the formation of tissues and organs, they also have been implied to play an important role in the
propagation of cancers. To better understand how cadherins glue cells to each other at one point, and allow
their disengagement at another, we will construct a new, ultrasensitive force microscope. This instrument will
be used to measure the difference in adhesion strengths between two types of cadherin molecules, E- and
N-cadherin. We will manipulate individual cells or microbeads using a micropipette and bring them into
repeated contact with a minuscule force-measuring elastic beam. By comparing the force needed to break
different types of cadherin-mediated attachments we will be able to determine the contributions of specific
parts of the adhesion molecules, as well as of other proteins that regulate the cadherin binding strengths. We
will further investigate cadherin-mediated adhesion between normal cells and cells affected by drugs or
genetic alteration. We are optimistic that this study will uncover fundamental properties of cadherins and
help us better understand normal development as well as pathological conditions related to cell-cell adhesion.
Cellular processes as varied as synaptic transmission and muscle contraction depend on both the molecular
properties and the nanoscale localization of membrane ion channels. However, whereas existing
electrophysiological techniques can measure the current flow through single ion channels with great fidelity,
they do not provide spatial information. To circumvent this limitation, we will utilize a technique we pioneered
based on total internal reflection microscopy (TIRFM) to monitor the individual gating of calcium-permeable
channels with millisecond resolution, while simultaneously mapping channel locations with nanometer
precision. We aim to confirm and extend our preliminary studies suggesting that the nanoscale organization of
channels is an important regulator of their activity; quantify the distance scales over which spatial correlates
of channel gating operate; and discriminate among possible underlying mechanisms, including the role of
lipid rafts, in modulating channel function and localization. The results will contribute to an understanding of
both the normal mechanisms of cellular calcium signaling, and how it is disrupted in diseases ranging from
heart failure to Alzheimer's.
One of the most important goals of biomedical research is to identify the genetic factors that help determine
who is at high risk or low risk for the common diseases that plague humankind. The Human Genome Project
and similar endeavors have created a scientific revolution such that this goal may soon be achieved for
several such diseases, including diabetes, arthritis, and many forms of heart disease. It is proving much
more difficult to understand how genetic factors predispose some people to devastating diseases of the brain,
particularly those that affect human characteristics such as speech or the capacity for abstract thoughts. Our
inability to delineate clinical features that unambiguously define such diseases limits progress in identifying
their genetic basis. The training program proposed here makes use of exceptional data that provide detailed
information on measures of a very wide range of brain-related attributes for sets of many thousands of
people, for whom state of the art genetic studies are also being carried out. We will analyze these data using
novel methodologies being developed for this project, all of which require collaboration between biologists
and quantitative scientists. The collaborations in this project thus provide an ideal interdisciplinary training
experience.
This proposal synthesizes materials science, bioengineering, and life sciences into an integrative program for
interdisciplinary training in optimizing novel implant materials. At the present time, our understanding of the
factors that are required for optimal bone/biomaterial integration (osseointegration) is limited. An innovative
approach that we propose is the use of new nanotechnology materials, specifically, nanotubes. This training
program aims to provide an in vitro and in vivo understanding of bone healing and the mechanisms whereby
novel titanium dioxide nanotubes enhance bone growth, as our preliminary research indicates. We explore
how chemical and structural nanotube engineering (Aims 1 & 4) influences in vitro osteoblast function (Aim
2) and in vivo bone-implant osseointegration (Aim 3). The primary sponsor, Dr. Sungho Jin, is Director of the
Materials Science Program at UCSD and a recognized specialist in nanomaterials. The co-sponsor, Dr. Lars
Bjursten, M.D./Ph.D., is a physician-scientist and orthopedic implant expert. The trainees UCSD
Bioengineering background forms a bridge between sponsors expertise, also allowing for teaching
opportunities and industry internships in applied biotechnology. This program provides the cross-disciplinary,
multi-scale analysis tools needed to gain the technical and collaborative skill-set for translational biomaterials
research.
Sepsis, the leading cause of death in hospitalized patients, is when the immune system overreacts to the
presence of an infection. This overreaction causes the immune system to attack the body, resulting in organ
failure and death. Scientific research suggests that mast cells, the same cells that are responsible for
anaphylactic shock and allergies may play a role in sepsis. In addition to releasing the chemicals that result
in allergic symptoms (itchy, watery eyes, etc) mast cells are also among the cells responsible for raising the
alarm that there is an infection in the body. Sepsis arises when the bodys response to that alarm is
overwhelming and system-wide as opposed to local. We have developed a chemical that should stop mast
cells from releasing all the chemical signals that call other immune cells, and activate the immune system to
dangerous levels. We are confident that silencing the alarm that leads to an overwhelming immune response
will keep the immune system from turning on the body. In short, we seek to prevent mast cells from pulling
the fire alarm in a crowded room. We hope that this novel approach will lead to advancements in treatment
options for sepsis.
Biotechnology is accelerated by the discovery and engineering of enzymes which provide new approaches to
manipulate biological molecules. DNA methyltransferases, highly specific biocatalysts, are ubiquitous in all
kingdoms of life and have proven to be highly useful for genetic manipulation techniques. These enzymes
naturally add methyl groups to a DNA molecule at a very precise spot based upon the sequence of the DNA.
We have used these enzymes to deliver different molecules, such as fluorescent dyes or nanoparticles, to
DNA. This ability may be useful to track DNA inside a living cell or to make new molecular-scale structures,
such as nanowires for use in miniature biomedical sensors that travel the bloodstream and monitor health.
One limitation to these efforts is the lack of enzymes that recognize diverse DNA sequences, or enzymes
which can be used and thus limit the amount of functionalization that can take place on a single DNA strand.
Our goal is to create an enzyme that is well established to add functional groups to DNA and expand the
number of DNA sites it can recognize by a combination of rational design and directed evolution using
high-throughput screening technology.
Precise gene activity is important for development and differentiation. Active and inactive genes are regulated
by enhancer and silencer elements in the context of packaged in open and closed chromatin domains
respectively. However regulatory elements within domains need to be corralled to prevent mis-regulation of
genes in adjacent domains. Insulator elements are partly responsible for this. In this proposal we hypothesize
that specific tRNA genes function as insulators in human cells just as they do in yeast cells. We have used
computational tools to identify human tDNAs. We will validate the ability of these genes to function as
insulators in human cells and characterize the epigenetic structure and regulation of neighboring genes by
these insulators. By examining the sequences of these insulators and the sequences flanking them, we will
develop computational prediction methods for insulator elements. Insulators are beginning to be used in gene
therapy. Another goal of this project is to create gene therapy vectors capable of isolating a therapeutic gene
from its surroundings using tDNA insulators. Our results will allow us to design an insulated gene therapy
vector for therapeutic purposes and gain a better understanding of how the human genome is organized.
We propose to develop a new form of microscopy for bio-imaging that can remove the image degradation
that occurs as light travels through thick biological specimens. Similar to the image degradation we see as we
look at stars whose light has passed through the earths atmosphere -- a phenomenon that causes stars to
appear to twinkle -- the light passing through a thick biological specimen can be refracted by local changes in
the optical properties of the specimen. This phenomenon is problematic, because it effectively blocks our view
of important structures and processes deep inside living tissues. Therefore, it is important to find methods
that will compensate for the changes and allow deeper imaging through thick tissues, making it possible for
biologists to image deeply within living tissue, rather than the current technique of sectioning fixed tissue.
Once developed, this technology will be used to image in real time the self renewal and differentiation
lineages in living, mammalian embryonic stem cells. This is a highly interdisciplinary project involving cell
biologists, engineers and astronomers.
We seek to develop methods for understanding how genes operate within a special class of microbes called
the Archaea. This group of organisms are similar in appearance to bacteria but are believed to be much
closer cousins of eukaryotes, the branch of life inhabited by humans. Diverse archaeal species are useful for
production of energy sources such as hydrogen, methane or ethanol. Some are important contributors to
maintaining balance in our biosphere by fixing nitrogen and carbon. Other species have provided important
tools for biotechnology. Archaea are able to thrive in the most extreme conditions of temperature, pressure,
acidity, and high salt. Collectively, they provide insight into the range of possibilities for life. We will employ
two newly discovered mechanisms, one from bacteria and one from eukaryotes, to create a new tool for
exploring archaeal gene function. CRISPR arrays and associated proteins act as a bacterial immune system
that can inactivate genes from invading viruses. These arrays are stored in many bacterial and most archaeal
chromosomes and contain fragments of invading viral genes. Some Archaea may have a second system,
RNAi, that silences genes using antisense RNA and a special protein, Argonaute. This system has been
invaluable for eukaryotic research.
This training experience will provide a foundation in several disciplines and laboratory skills for advanced careers in the
interplay of engineering and neuroscience. Training included rotations in the Brain Machine Interface Systems Laboratory
as well as the Cognition and Action Laboratory. With Brain Machine Interface (BMI) research bridging engineering and
cognition, a number of supporting courses included: Topics in Theoretical and Computational Neuroscience, covering
analytical methods in neuroscience, and Neural Computation, and Brain-Machine Interface Systems. In addition, t
Advances in Computational Motor Control symposium at the 2006 SFN conference provided exposure to the motor
control community. The seminar The Cerebellum and basal ganglia - movement control and execution from UC Berkel
Neuroscience Program provided an introduction to human psychophysics experiments and FMRI studies related to motor
control. Finally, this training experience will include directed research in performing neural recordings to investigate
using brain signals to control BMI prosthetic devices under conditions simulating real world force perturbations. The
long-term goal of this work is to produce a brain-controlled prosthesis capable of reproducing the wide range of functions
performed by the human upper limb, so that patients can enact voluntary motor actions using only their thoughts.
Mechanics how soft a cell is, and how much force it can exert on its environment are very important in determining the
cell s fate. For example, cancer cells are characteristically unresponsive to the stiffness of their surroundings and
therefore grow uncontrollably. The mechanical properties of a cell are thought to arise from the actin cytoskeleton,
which forms a large part of the cell s internal scaffold. Actin filament networks are particularly important because in
addition to supporting and distributing forces produced by molecular motors and other cells, they can also produce
protrusive force by polymerizing. To understand how the architecture of these networks determines their mechanical
properties, we propose to work from the bottom up, measuring the mechanical properties of small network units, which
are Y-shaped branches. Using computer modeling, we will then use these measurements to predict how entire networks
will respond to force. This will provide cell biologists and clinical researchers an important tool or addressing such
questions as: How do cells move through tissues? How can they can squeeze through tiny blood vessels and why, in
some diseases, can they not? And how do they measure the stiffness of their surroundings?
We propose a multidisciplinary training program that will enable Michael Plesha, a doctoral candidate in Chemical
Engineering with a Designated Emphasis in Biotechnology, to establish himself as a scientific leader in the emerging area
of plant-based production systems with broad potential applications ranging from accelerated vaccine production to
biofuels. Michael is part of a multidisciplinary research team comprised of engineers, plant scientists and plant
virologists working on the development and analysis of a novel plant expression system recently developed by the UC
Davis team. He will not only receive scientific training in the fundamental aspects of plant molecular biology, virology
and bioprocess engineering but will also contribute to the improved fundamental understanding of new protein
expression systems that could provide a safer and/or more economical route for the production of enzymes, therapeutic
proteins, antitoxins, vaccines and biofuels. This type of integrated research and training program provides an ideal
educational environment for preparing biotechnologists of the future who are capable of working as a team on an
inherently complex research problem and who can think beyond their own discipline.
Protein separation plays a critical part in biotechnology, including, separation of therapeutic proteins, food processing,
blood plasma fractionation and proteomics. Safety concerns require high purity for therapeutic proteins. Conical
nanoporous membranes represent a new class of filters that exploit electrostatic and size effects for high transport rates
and vastly improved selectivity. Recent studies have indicated that for small ions the conical pore membranes give high
selectivity in ion separation. We will build conical nanoporous membranes made out of thin silicon and test them for
protein separation. The proposed work is collaboration between scientists at LLNL and UC Davis. The graduate student
will fabricate and characterize the membranes at LLNL and conduct experimental and theoretical studies at UC Davis on
protein transport in the silicon membranes. The proposed research will result in fundamental molecular level knowledge
on protein transport through conical nanopores and thus directly impacts the practice of membrane protein separation
and purification. The knowledge gained from the project on selective molecular transport has general implications in
nanoscale fluid transport and have potential impact on related fields such as nanofluidic devices, drug and biomolecule
separations, biomimetic devices and sensors.
Neuronal communication within the brain is mediated by numerous chemical
transmitters, the activity of which ultimately governs behavior. Endeavors to
understand such processes would be greatly facilitated by means with which to
measure the release of transmitters in localized regions of the brain. We are
developing implantable microelectrodes capable of detecting changes in the
concentration of multiple transmitters in brain tissue, simultaneously and at high
speed, thereby permitting correlation of neurotransmitter release with behavior. Thes
devices incorporate enzymes that catalyze the breakdown of specific
neurotransmitters, producing a by-product that can be detected electrochemically at
the surface of miniature platinum sites on the implantable device.
Our goals are to develop the technology while applying it to animal models of human
behavior. In particular, we are interested in elucidating the neurochemical and
neuroanatomical underpinnings of addictive behavior and will use the sensors to
simultaneously measure the release of two major neurotransmitters implicated in dru
addiction, glutamate and dopamine, in specific areas of the rodent brain during
performance of behavioral tasks modeling the addicted state. Ultimately, these
biosensors could be used in humans to provide online monitoring of the effectiveness
of therapeutic treatments, thereby enabling continuous autoregulation of drug dosing.
Over the past decade, tools available to geneticists have predominately limited their research to rare mutations with
strong genetic effect, for example mutations leading to cystic fibrosis. California companies have developed key genetic
technology to allow researchers to identify common genetic variants within the population that genetically predispose
individuals to complex disorders, such as Alzheimer s Disease, Autism, and Diabetes. These studies, referred to as
genome-wide SNP association studies, are built on assaying hundreds of thousands of single base changes everywhere in
the genome. Unfortunately, these studies are unattainable for most researchers due to the cost of genotyping hundreds
or thousands of case and control samples. To reduce the overall cost of GWA studies and expand the types of disorders
approachable, a pooling-based GWA study can be completed from ten to thirty thousand dollars; whereas individual
genotyping may require millions of dollars. Computational analysis tools will be developed to accomplish pooling-based
GWA studies and apply these methodologies to the study of complex diseases. Collaboration between the Human
Genetics (Dr. Stan Nelson) and Computer Science Department (Graduate Student Nils Homer) at UCLA as well as the
Translational Genomics Research Institute (Dr. David Craig) is proposed in order to develop these analysis methods.
Human embryonic stem cells (hESCs) have the unique ability to grow indefinitely in an undifferentiated state
(proliferation, maintenance, self renewal) or adopt new cellular fates (differentiation) depending on the physiochemical
environment in which they are cultured. Manipulation of factors within this environment is central for the production of
defined cell types for uses in the engineering of specific tissues to treat diseases. However, current approaches to study
factors affecting stem cell fate are limited in their capability to assess the multitude of parameters to which hESCs
respond.
The proposed research will develop a platform for the simultaneous screening of thousands of physiochemical
parameters. This platform is based on a microarray technology that uses robotics to position thousands of microspots
composed of individual and combinations of proteins in a precise pattern on a glass slide surface. Additionally, hESCs will
be subjected to mechanical forces imposed by fluid flow. Results from these studies will lead to the definition of the
optimal parameters for the control of differentiation of hESCs into specific cell types.
The proposed interdisciplinary project is a collaborative effort synergizing the expertise of Dr. Shu Chien (UCSD
Bioengineering) and Dr. Karl Willert (UCSD Cellular & Molecular Medicine).
The spatial resolution of the light microscope can be greatly extended using structured illumination light combined with
nonlinear phenomena. In fact, there is no hard limit to the resolution that is theoretically possible using this concept. We
have already demonstrated 50 nm resolution on test samples using one form of this general method in which fluorescent
saturation provides the required nonlinearity. Another, highly promising version of the same idea derives the nonlinearity
from the ability of certain proteins, especially one called Dronpa, to have their fluorescence switched on and off by light;
this carries the promise of combining nano-scale microscopic resolution with the labeling power of genetically encodable
markers, potentially in living cells. We propose to demonstrate, develop and apply these concepts to cellular biology
problems including the structural characterization of the bacterial cytoskeleton, and the molecular organization of
neuronal pre-synaptic terminals.
The process of programmed cell death, or apoptosis, is crucial for development and survival of multicellular organisms.
Destruction of unneeded, damaged or virus-infected cells is carried out by a set of proteins called caspases, which
destroy or alter other proteins by cutting them. We hypothesize that caspases act as demolition experts, targeting key
molecular structures so the cell is destroyed in safely and efficiently. Until now, comprehensive caspase target
identification has been hampered by the difficulty of distinguishing cut proteins from the sea of uncut proteins in a cell.
Here we describe a technology recently developed in the Wells lab for overcoming this problem. An engineered enzyme
called subtiligase recognizes newly-exposed ends of cut proteins and labels them with a molecular handle called biotin.
The protein pieces can then easily be pulled out of the complex solution and identified using mass spectrometry
technology. We propose use of this system for detecting caspase targets on a proteomic scale. We believe identifying
caspase targets will aid in understanding cell death and may lead to discovery of new drug targets. In addition, the
technology developed here will be generally applicable for studying the hundreds of proteases known to play important
roles in biology.
Industrial processes used to create mineral materials such as specialty glasses and semiconductors usually involve high
temperatures and pressures or caustic chemical environments. In contrast, many biological organisms are able to
synthesize comparable mineral-based materials from water at mild temperatures and pressures. Recent investigations
conducted on the bio-glass skeleton of a marine sponge have revealed unique enzymes called silicateins, that participate
in this biologically mediated glass formation. The studies outlined here propose to recapitulate, in a laboratory setting,
the essential evolutionary processes responsible for the development of these bio-glass-forming systems, and
manipulate that evolution to obtain enzyme variants that produce specific crystal forms of silica and other minerals.
State-of-the-art methods in biotechnology will be used in conjunction with the analytical methods of materials science to
evolve such functionally specific silicatein mutants, thereby creating new artificial biomineralizing systems that may b
studied in the laboratory. The unique interdisciplinary training at the core of this project will equip the student trainee for
a career at the interface between the biotechnology and semiconductor-based industries that have been mainstays of the
California economy. This program thus will serve as a prototype for future training to help ensure the future economic
health of California.
Self-healing materials are a group of materials that, when extreme stresses break internal bonds and deform it, the
material can reform the broken bonds and regain its original shape and mechanical properties after the stress is
removed, with no outside aid. There are numerous uses for self-healing materials including, but not limited to,
biomedical applications, construction, aerospace, and robotics. Many naturally occurring materials show self-healing
properties, some that are more impressive than what is currently synthesized in labs. An example of this is WECB (whelk
egg capsule biopolymer), produced by a group of sea snails, genus Busycon (the whelks), to protect their eggs. This
material is composed of protein, but does not contain cells, is elastic, and shows remarkable self-healing properties. By
studying the chemistry of the individual molecular components, and how they are processed and arranged into the
macroscopic material, it will be possible to create novel materials that have similar mechanical properties, but will be
better suited for use in industry. A variety of techniques from different fields will be used to study this material including
biochemistry, molecular biology, materials science, and engineering. Research of this kind will lead to the next
generation of high-performance materials.
Cells are organized in a modular fashion. Each central function (e.g. division, motility, intracellular signaling) is performed by complex assemblies of interacting components that behave as self-contained molecular machines. In many of these machines, charged polymers such as DNA, RNA, and proteins, are translocated through pores during the machine s operating cycle. Polymer translocation takes place in the vast majority of cellular processes, including protein synthesis, DNA transcription, and protein degradation. The proteasome, a molecular complex which degrades proteins, rips folded proteins apart and passes the unstructured amino acid chain through a 1.3 nm diameter aperture [1] for proteolysis. The ring-shaped translocase FtsK passes 2nm-diameter DNA through its 3nm channel, stripping off DNA binding proteins as it goes. By combining single molecule biophysics with microfabrication and technology we will develop a model system to mechanically pull individual biopolymers through artificial nanopores. This system applies the same local shearing forces applied by molecular machines. We will answer questions such as: how do biopolymers respond to local application of mechanical forces? What forces are required to shear bound proteins from DNA? What are the kinetics of mechanical shearing by small apertures? These are fundamental biological questions inaccessible to any other experimental technique.
We are interested in using a new tool for direct manipulation of single molecules, optical tweezers, to explore the protein folding process. Proteins are the main working agents within a cell: synthesized as a single strand of amino acids, which folds into a much more compact structure, their function is determined by both their shape and composition. Traditional measurements in solution use high temperature or harsh chemicals to destabilize proteins and probe their folding behavior. These experiments, though powerful, suffer from the limitation that the molecules observed number in the quadrillions, obscuring many intriguing but infrequent variations.
Viral infection, nutrient delivery, and neurological function are all possible because of a single biological mechanism: fusion of one lipid membrane to another. Also, filling membrane pouches with potent pharmaceuticals and selectively fusing them to cells promises to be an efficient vehicle for drug-delivery. While some conditions that promote fusion have been identified, its physical process is not understood; consequently membrane fusion is not being actively used as the biotechnology tool it could be. Our hypothesis is that the macroscopic mechanical energy of the membrane plays a governing role in facilitating fusion. Two recent cross-disciplinary advances enable our investigation. First, we have recently developed curvature-controlled platforms that support phospholipid tension-tunable membranes. Second, we have coupled these membrane constructs to optical detection devices. We will introduce vesicles of controlled sizes to these curved membranes and count fusion events as a function of curvature and environmental parameters. These experiments could establish a model for membrane fusion that would be instrumental in explaining how fusion-enabling proteins function, as well as provide design rules for liposome formulations for targeted drug delivery.
Cell based therapies offer much promise for the treatment of nervous system diseases and conditions that require tissue regeneration, such as Parkinson s disease or spinal chord injuries. The direct delivery of cells to the target site is less than optimal, because few cells survive over the long term. Typically, delivering the cells in a polymer gel material offers better results, because the gel can be decorated with important biochemical molecules that signal the cells to attach, proliferate and develop into mature tissue. For nerve tissue culture the gel material must be porous to provide sufficient room for the nerve cells to extend processes and make contacts with other cells. In this work, we will prepare polymeric molecules that can self-assemble into the desired porous gel structures. Biochemical molecules will be attached to the polymers, so that the cells will receive biochemical cues to grow and differentiate. When completed, the project will generate a new approach to tissue engineering and potentially a therapy for treating nervous system diseases and conditions that require tissue regeneration. The student working on this project will work between groups that have expertise in synthesizing polymers and gel materials and in biological engineering and tissue culture.
Mitosis is the process by which the duplicated genetic material is segregated, thereby ensuring that each cell gets a full set of genetic instructions. This process is important since mitotic dysfunction can lead to cancer and birth defects. We want to know how multiple mitotic force generators and regulatory molecules comprising the mitotic machinery coordinate the transitions between sequential stages of mitosis. To accomplish this we will first identify the key genes involved in mitosis in the fruit fly genome. We will combine molecular biology techniques of gene function silencing with high-throughput automated microscopy. To deal with the very large amount of data produced in this phase of the project, we will develop and utilize computer vision techniques to automatically identify dividing cells. To complement this large scale screen we will perform a more focused analysis that will characterize in detail all the genes that are found to be important for mitosis. To understand how these genes and their products interact to coordinate the events of mitosis, we will use computational tools to systematically test literally millions of mathematical models of mitosis and we will thereby determine which properties and components of the mitotic network play key roles.
Pulsed laser microbeams have been used in biology for over 35 years. Although laser microbeams offer a the wide range of cellular manipulation capabilities, most studies utilizing microbeams typically employ only a narrow range of laser parameters tailored to a specific task. In the future, laser microbeams may become more attractive for utilization in biological studies if a wide, and rapidly interchangeable, set of laser-cell interactions can be provided on a single platform. Furthermore, relatively few studies have investigated the basic processes governing the interactions of highly focused pulsed laser beams with cells. A better understanding of the processes underlying the laser-cell interactions that pulsed laser microbeams offer will prove critical in determining the parameters necessary to achieve specific cellular capabilities.
This proposal aims to engineer an experimental system capable of performing a wide variety of cellular manipulations within a single platform through investigation of critical laser parameters governing these processes. We will demonstrate this system s capabilities by providing rapidly interchangeable laser microbeam capabilities for optoporation, injection and lysis of single cells for single cell bioanalytics. We will utilize the system to provide a unique, novel approach for the measurement of analytes with rapid reaction kinetics inside single cells.
In the post-genomic era, the next challenge is nanoscale imaging. Technological advances are setting the stage as more molecular tools are becoming available. However, the main obstacle is the resolution of the light microscope, which is limited by diffraction to ~200 nm lateral and ~500 nm axial. Breaking the diffraction limit will usher in a new wave of important scientific investigations on subcellular biological complexes. We propose to create a microscope (4Pi-Stimulated Emission Depletion, 4Pi-STED) with a 3-D resolution (x, y and z directions) of 10-20 nm to determine the nature of biological complexes. Because this type of microscope is nonexistent in the U.S., the success of this project will mark a major breakthrough in nanoscale bio-imaging and its medical applications. The nominated student, Margaret C. Chiang, will carry out this project as her Ph.D. thesis research under Professor Jia-Ming Liu from UCLA s Electrical Engineering Department with the joint mentorship of Professor Enrico Stefani from the Division of Molecular Medicine in UCLA s Department of Anesthesiology. This project is feasible due to the non-overlapping, but complementary, expertise of Professor Stefani in molecular studies and confocal microscopy and of Professor Liu in nonlinear optics and ultrafast laser technology.
The action of turning genes on and off is performed by the interaction of a large number of proteins at regulatory sequences contained in our DNA. In order to fully understand how these molecular switches control our genes, we must identify the components of the protein machineries that bind them. Current techniques for doing so are limited because they ask the question from the perspective of the individual proteins (where do I bind?) rather than from the perspective of the regulatory sequence (what binds to me?). Thus, we propose to reverse this paradigm by developing a new technology that will allow us to take a molecular snapshot of the proteins present at a regulatory sequence at a given time. In this way, we can identify all of the proteins that cooperate to control a gene, including those that have not been previously identified. In conclusion, our new technology will greatly advance our understanding of one of the most fundamental processes in biology by focusing on the forest rather than a single tree at gene regulatory sequences.
Knowledge from diverse fields, including cell biology and chemical genomics, will be applied to the problem of beta-cell replacement therapy for diabetes. Beta-cells, which secrete insulin in response to blood sugar level, are absent or functionally deficient in diabetics. Increasing the number of beta-cells by promoting them to divide, either in the patient or in culture, could remedy this problem. We will investigate the mechanisms that control beta-cell replication using high-throughput screening technology to discover small molecules that induce beta-cells to proliferate. This technology, the same as that used by pharmaceutical companies to find new drugs, is available in the Burnham Institute Screening Center, led by Dr. Mercola. Thousands of compounds will be tested for their ability to affect beta-cell replication in a human beta-cell line developed in Dr. Levine s laboratory. Positive compounds will be selected using a sophisticated image analysis process developed by Dr. Jeff Price. A large dataset of the effect of the compounds on the proteins and genes in the cell line will be inputted into algorithms developed by Dr. Subramaniam to create a model of the signaling pathways controlling pancreatic beta-cell replication. Compounds discovered from this research may serve as the basis for new diabetes treatments.
Adult heart cells are too fragile to transplant, but immature, fetal cardiomyocytes grafted into injured animal heart muscle improves heart function and minimizes long-term disease. To extend these studies to humans will require a renewable source of human heart muscle cells and improvement in the technology to ensure the incorporation of cardiomyocytes into the damaged heart. Specifically, we propose to create a three-dimensional (3D), in-vitro model of cardiac tissue, using human embryonic stem cells, to identify the physical factors that regulate myocyte maturation and the phenotype of cardiac muscle. To study the integration and function of replacement heart muscle cells in as natural an in-vitro environment as possible, we will culture the engineered tissue under mechanical strain using a novel culture platform. We will use this system to characterize cellular interactions and metabolic conditions that are needed for cardiomyocyte maturation and the creation of a robust muscle tissue. Exploring these inputs will lay the foundation for new therapies and drug discovery techniques. This work will be conducted under the guidance of Drs. McCulloch and Mercola, leaders in their respective fields of cardiac biomechanics and tissue engineering and cardiac stem cell biology.
The synovial fluid of human joints normally functions as a biomechanical lubricant, providing low-friction and low-wear properties through the combined contributions of several molecules. These lubricants are secreted by cells in tissues lining joints, including chondrocytes in articular cartilage and synoviocytes in synovium. Lubrication failure contributes to erosion of the cartilage in arthritis and to deterioration of biological and synthetic grafts used to treat damaged cartilage. Chemical and mechanical factors are known to regulate chondrocyte and synoviocyte secretion of lubricants into synovial fluid. However, in the complex environment of living joints, there exists a need for a technology platform to analyze lubricant regulation and function. Thus, the aim of this proposal is to develop a biotechnology platform to nurture native knee joints, where stimuli mimicking health or disease can be applied and to demonstrate the usefulness of the technology for translational applications, as follows. First, cartilage and synovium joint components will be incubated together to generate a fluid that like native synovial fluid is effective in lubrication. Second, tests will be performed to determine whether lubrication is adequate for therapies with drugs, devices, biomaterials, and engineered tissues. Future applications of the bioreactor platform are envisioned for orthopaedic and regeneration industries.
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.
