NIH Director’s Pioneer Award

2006 Pioneer Award Recipient Abstracts

I propose to build Neurogrid, a specialized hardware platform that will perform cortex-scale emulations while offering software-like flexibility. Recent breakthroughs in brain mapping present an unprecedented opportunity to understand how the brain works, with profound implications for society. To understand the brain, we have to interpret these richly growing observations by modeling the brain, the only way to test our understanding— since building a real brain out of biological parts is currently infeasible. Neurogrid will emulate (simulate in real-time) one million neurons connected by six billion synapses—making it possible to model vertical, horizontal and top-down cortical interactions in biophysical detail. My ability to bring this endeavor to fruition and my commitment to biomedicine is evident in my past accomplishments. Over the past eight years, my lab has designed seven neuromorphic chips that model seven neural systems—retina, cochlea, cochlear nucleus, thalamus, hippocampus, visual cortex, and retinotectal development. To pursue such diverse projects, I established productive collaborations with six colleagues in Penn’s Neuroscience Department; our work was a Scientific American cover story (May 2005). The visual cortex chip illustrates the potential of Analog VLSI: Emulating 9,216 neurons, it is 2,765 times faster than the state-of-the-art. However, neither this chip nor the other six is programmable. Neurogrid will provide programmability by augmenting Analog VLSI with Digital VLSI, a mixed-mode approach that combines the best of both worlds. While including biophysical detail in a model provides contact with experiment, programmability supports replicating manipulations, performing controls, benchmarking models, and exploring mechanism. Realizing these two critical functions without sacrificing scale will make it possible to replicate tasks laboratory animals perform in biologically realistic models for the first time, which I will do in close collaboration with two neurophysiologists (Matthew Dalva Ph.D. and William Newsome Ph.D.).

The development of a scientific field is often punctuated by a period when diverse observations can be understood in terms of a set of unifying principles. This important moment in time occurs when a critical number (and type) of observations becomes available, and the mechanistic principles often result from an amalgamation of ideas drawn from different intellectual disciplines. This period also marks the emergence of predictive models. T lymphocytes (T cells) orchestrate (and can also misregulate) the adaptive immune response. I believe that T cell biology, especially T cell-mediated autoimmunity, is at a critical juncture where diverse data will soon be integrated in terms of overarching principles. Modern experiments are revealing, in unprecedented detail, the factors that are important in the emergence of T cell-mediated autoimmunity rather than tolerance to “self”. However, general mechanistic principles necessary for predictive models have proven elusive. This is because T cell-mediated autoimmunity is characterized by cooperative dynamic processes that occur over a spectrum of length and time scales. Phenomena occurring on large scales (tissues) influence cooperative molecular events in a single T cell which, in turn, influences the tissue environment. This complex hierarchical cooperativity makes it difficult to intuit underlying mechanisms from experimental observations alone. I propose to develop the principles governing T cell-mediated autoimmunity by parsing the pertinent cooperative dynamics which occur in a complex space of molecular and cellular variables by integrating three great advances of the twentieth century: statistical mechanics, computational technology, and genetic, biochemical, and imaging experiments. A key to success will be collaborations with experimental immunologists, and I have recently demonstrated how such synergies can be fruitful. If successful, the work that I envisage will provide the principles that could guide the development of therapies for diseases such as multiple sclerosis and diabetes which afflict millions of people.

Surprisingly little is known about how proteins fold in vivo, yet it is this process, and not the test-tube idealized folding reaction so intensively studied over the past several decades that are crucial to the fitness of an organism. The fidelity of folding and the stability of proteins in the cell are critical to their functions, their degradation, and their vulnerability to aggregation. Many diseases are now known to arise from defects in protein folding, either because of the loss or alteration of essential protein functions, or because of the build-up of toxic species such as aggregates. We believe that novel methods and creative collaborations will allow us to overcome the daunting technical obstacles that have impeded progress on protein folding in the cell. Focusing first on a small model protein for which we have detailed descriptions of folding in vitro will enable methods optimization. Folding in cellular conditions will be followed in systems of increasing complexity: bacterial protein expression, cell-free biosynthesis, and semi-permeabilized or intact eukaryotic cell expression. The new strategies will reveal how larger proteins of biomedical interest adopt their structures in their cellular context and how this process may go awry. Methodologically, we anticipate placing major reliance on spectroscopic methods, including fluorescence and nuclear magnetic resonance, and using novel labeling strategies to observe the protein under study in the complex cellular milieu. Complementary in-cell imaging methods will be used to insure that observed signals report on relevant phenomena and to reveal novel functionally significant spatial localization patterns. We anticipate that this research will lead to new paradigms for how amino acid sequences encode folding information and that the resulting enhanced understanding of folding in vivo will lead to new strategies for therapeutic intervention in misfolding and aggregation diseases.

A fundamental problem in cell and organism biology is to understand how intracellular structures are properly scaled to carry out their essential functions. I propose to explore the phenomenon of organelle size, documenting the changes that occur during development and tumorigenesis, and investigating the underlying molecular mechanisms. My laboratory will undertake a systematic analysis of cell and organelle scaling during embryonic development, evaluating nuclei, spindles and other compartments in Xenopus laevis, as the ~1 millimeter diameter egg rapidly cleaves to form smaller blastomeres, which by the 15th division are reduced to 40 microns across. Which structures have constant dimensions, and which change their size as cells become smaller? Other model organisms and cancer cells will be compared to generate a survey of organelle scaling in normal and abnormal cell growth states. Cytoplasmic egg and embryo extracts of X. laevis and the related, smaller frog X. tropicalis will be used to monitor nuclear, spindle and cellular compartment scaling in vitro. This approach is prompted by our observation that meiotic extracts prepared from X. tropicalis eggs generate spindles that are ~30% shorter than those in X. laevis reactions using the same chromosome source, and mixing experiments have revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. We will determine which organelles in addition to the spindle are scaled in X. laevis and X. tropicalis extracts, and use activity-based assays to identify the factors responsible for the observed differences. Candidate factors will be tested for their roles in organelle scaling during development and cancer progression, and computational approaches applied to model our observations. These studies will provide novel insight into how cell/organelle scaling contributes to intracellular morphogenesis and cell division, processes essential for viability and development, and defective in human diseases including cancer.

The high error rate of the RNA-dependent RNA polymerases responsible for RNA viral genome replication presents an enormous challenge to successful drug therapy due to the high probability of mutations that can confer resistance to any antiviral pharmaceutical. Usually, the only solutions presented are multi-drug therapy, choosing a host-encoded target, or choosing the drug target such that resistant viruses are predicted to display only limited fitness; this latter option has not proved particularly successful due to the large amount of sequence space that can be explored by these highly mutable genomes.

My laboratory has devoted considerable time to understanding the transmission genetics of positive-strand RNA viruses. Recently, we have consolidated this research to show that, for poliovirus, choices of drug target can be made so that drug-sensitive genomes dominantly inhibit the outgrowth of drug-resistant genomes. The ability of relatively unfit viruses to inhibit the growth of viruses with increased fitness derives from the intracellular amplification of positive-strand RNA viral genomes, their translation into large polyproteins and the higher-order oligomerization of several of their protein products. Here, I propose to use this understanding to identify “dominant drug targets” for other positive-strand viruses such as rhinoviruses, coxsackieviruses, hepatitis C virus, Dengue virus and West Nile virus, informed by analogy with poliovirus and tested by direct genetic and biochemical investigation. This new paradigm will facilitate the development of therapeutics for which there is reduced danger of outgrowth of the inevitable drug-resistant genomes. My thesis is that theoretical and experimental understanding of the unusual genetics of intracellular viral growth can lead to the identification of “Achilles’ heels” for each targeted positivestand RNA virus, and possibly for other intracellular pathogens as well.

This project will develop a chemistry-based approach to monitoring and manipulating the immune system. If successful, it will provide a diagnostic platform of extraordinary utility as well as lead compounds for drugs to inhibit specific immune responses without general immunosuppression. We will employ microarrays comprised of thousands of peptide-like compounds called peptoids and hybridize serum samples to these arrays, resulting in the binding of thousands of proteins and several cell types to the immobilized peptoids. The hybridization pattern of interest will be visualized using an appropriate fluorescently labeled antibody, for example a labeled anti-IgG antibody will "light up" the pattern of binding of all IgG antibodies. Each antibody will evince a different pattern of binding to the array based on the spectrum of affinities of that molecule for the thousands of peptoids on the array.  The weighted sum of these binding signatures is expected to provide a "superpattern" that should provide a sensitive readout of the population of circulating antibodies. By comparison of appropriate patients and matched control samples, we should be able to identify features in the superpatterns that are common to the disease of interest, reflecting capture of autoantibodies that are amplified in an autoimmune disease, cancer, a particular infection, etc. The same type of experiment could be done to identify disease state-associated T cells. The most informative peptoids could be used as pseudo biomarkers for diagnosis. Finally, the peptoids identified as good pseudo biomarkers for the disease state will be examined as possible therapeutic agents in various autoimmune conditions, the idea being that these peptoids may capture antibodies or T cells that are not only indicative of the disease, but may be causative as well. This approach holds out the possibility of discovering reagents capable of blocking specific immune responses without global suppression of the immune system.

During hibernation, certain mammals can undergo a physiological state analogous to reversible suspended animation, with severe hypothermia and a core body temperature (CBT) close to 0oC. In non-hibernators including the human, this degree of hypothermia is fatal. It is well established that cells under hypoxia survive longer in hypothermic conditions due to slowing of metabolism, a feature with many clinical applications. Due to the risk of organ failure, clinical application of hypothermia is limited to a CBT of 32-34oC. Even at this temperature, the beneficial effect of controlled hypothermia is significant. The laboratory mouse is a non-hibernator but under caloric restriction it can undergo torpor (hibernating-like) behavior with a CBT of 31oC or below. Primates such as Malagasy lemurs can undergo torpor, suggesting that the basic mechanism for such behavior may be preserved in humans. We have recently identified endogenous 5’-adenosine monophosphate (5’-AMP) as a mediator of torpor behavior in mice. Our studies revealed that torpor behavior is linked to the regulation of blood glucose by 5’-AMP, an important allosteric regulator of several rate-limiting enzymes involved in glucose homeostasis. Our finding raised the possibility that non-hibernators can also achieve a state of suspended animation observed only in hibernating mammals. Using the physiological variables, 5’- AMP, environmental temperature and glucose, we can induce, sustain and rescue mice in suspended animation. Shivering, a sign of thermo-regulatory defense is blocked by 5’-AMP, allowing rapid cooling of CBT to 17oC or below, causing the animal to enter suspended animation. Recovery from a suspended animation state of up to 10 hours is spontaneous but was enhanced by glucose. Our goal is to bring this technology into two areas. 1) To explore the physiological limits of suspended animation in non-hibernating mammals. 2) To extend findings from the laboratory mouse to other non-hibernating mammals along the evolutionary chain.

Despite the phenomenal success of antibiotics and vaccines, infectious diseases remain one of the leading causes of death worldwide. The emergence of multidrug-resistant bacteria has created a situation in which there are few or no options for treating certain infections. The intrinsic limitations of existing vaccines (cost, safety, shelf-life, etc.) and the current trend in major pharmaceutical companies to abandon antibiotic and vaccine development programs create an alarming situation with potentially catastrophic public health consequences. The radical solution for this global biomedical problem and the goal of the proposed research is the development of conceptually new approaches to prevent and treat infectious diseases. One general strategy is to create a new type of fully synthetic antimicrobials for which it would be intrinsically difficult for bacteria to develop resistance. At least one prototype, a chemical platform based on 5-aminonaphthalenesulfonamides (ANSA), has been designed in our laboratory as a proof-of-principle. Unlike the majority of antibiotics that target individual macromolecules, ANSA compounds irreversibly damage various proteins at once, thus rapidly killing a target cell. This process requires nitric oxide (NO). Endogenous NO is sufficient to render bacteria susceptible to ANSA, and exogenous NO (e.g. from macrophages) further stimulates the bactericidal effect. The combinatorial selection of the most potent ANSA and related compounds will be carried out to define the specificity for different pathogenic bacterial species. The second general approach involves creating probiotic-based vaccines. Probiotics are live bacteria that colonize or temporarily survive in the host, while conferring beneficial effect on its health. Probiotics that stably and controllably secrete or display antigens for various pathogens could effectively serve as self-sustaining vaccines. As a proof-of-principle we will engineer probiotic strains that upon oral administration to mice will induce a proper immune response rendering animals resistant to otherwise lethal infections.

My long term goal is to understand the effects of macromolecular crowding on biochemical processes by acquiring atomic-level information on proteins under actual biological conditions. To this end, my group has pioneered in-cell NMR. We have shown how to assess structure, quantify dynamics, and measure stability under the crowded conditions found in living Escherichia coli cells using this powerful new technique. Now, we want to take the next step. With support from a Pioneer Award, we will focus on using in-cell NMR in eukaryotic cells to study two key proteins in neurodegenerative diseases, the intrinsically disordered proteins, α-synuclein and tau. These proteins are excellent candidates not only because of their disease relevance, but also because we know that macromolecular crowding has extremely large effects on the properties of disordered proteins. Our understanding of protein structure and function has grown enormously in the last 100 years. We have progressed from pondering what role, if any, polypeptides play in the cell, to unraveling, at the atomic level, the mechanisms of enzymes and the molecular bases of protein-protein interactions vital to understanding human disease. Our accumulating wealth of knowledge has largely come from in vitro studies performed under conditions far different from those found in biology. For example, most biochemical examinations of protein behavior are performed at concentrations in the μg-to-mg-per-mL range, but the insides of cells, where most proteins perform their work, have protein concentrations of >300 mg per mL. Thus, our knowledge comes from data acquired under conditions that are far from physiological relevant, and theory predicts these differences can have extremely large effects on biophysical parameters. Moving beyond the test tube by performing truly in vivo studies in living eukaryotic cells by using NMR spectroscopy is the next frontier in protein chemistry.

In our effort to understand human biology and human individuality we emphasize the importance of our genetic blueprint, i.e., the human genome, and our environment. However, our view of self tends to ignore our microbial inhabitants, which outnumber our own cells by ten-fold. A growing body of evidence implicates these microbial inhabitants in a wide array of activities critical to human well-being, as well as to disease. Early molecular explorations of diversity within the human indigenous microbiota demonstrate vast populations of uncultivated and uncharacterized organisms, previously-unrecognized potential function, and significant variation between different human hosts. It is time to embrace a more extended view of self, one that emphasizes our mutualistic and symbiotic relationships with our microbiota, and one that considers the net human-microbe “metagenome”. The first phase of the proposed work will entail a detailed molecular survey of the human indigenous microbiota. The second phase will examine variability in patterns of microbial diversity, as a function of human individuality (including genetics), age and time (microbial succession), space (biogeography within the host landscape), and human diet. A third phase will focus on the effects of perturbation, e.g. Antibiotics, on the structure of human indigenous microbial communities (community robustness), as well as on the relationships between patterns of microbial diversity and mucosal health and disease. These efforts will have profound and practical implications for human biology and for the promotion of health. The goals of this work are a more complete understanding and definition of human health based on indigenous microbial community profiles, the identification of microbial community “signatures” that predict the development or course of local disease, and strategies for the maintenance or restoration of health that involve well-informed manipulations of the human microbiota.

Recent studies have focused attention on the role of mitogenic niches in regulating stem cell self–renewal, and have emphasized the importance of proteoglycans in forming such microenvironments. However, more than 50 years after the genetic code was deciphered, we do not know whether sugar chains on proteoglycans encode biologically important information. The potential complexity of such a “glyco code” is enormous, but little is known about the features of the proteoglycans involved in mitogenic regulation, or the signaling mechanisms required for stem cell renewal. To decipher whether there is a glyco code we will identify proteoglycans that specify stem cell renewal in the mammalian brain. We will rely on a genetic approach to proteoglycan biology and on newly developing innovations in mass spectrometry that allow large scale analysis of the sugar composition of proteoglycans. We will generate mutated growth factors capable of binding to cognate receptors, but unable to bind proteoglycans. We will ascertain whether individual proteoglycans modulate the signaling pathway and the biological response elicited by a growth factor, perhaps by influencing the location, presentation, or oligomerization of the factor. We will begin by focusing on Sonic Hedgehog (Shh), which is mitogenic for stem cells in the cerebellum, cortex, and in diverse cancers. We will identify proteoglycans required for Shh- mediated proliferation using a new assay for mitogenic niches, and we will determine how a glyco code might regulate stem cell propagation. As the work progresses we will extend our studies to define proteoglycan structures that modulate the response of stem cells to additional agents such as EGF or FGF family members. These studies will contribute to the identification of a glyco-code, and will determine mechanisms that maintain “stemness”. Such studies can lead to enhanced therapies for disorders from Alzheimers disease to cancers to stroke.

The research proposed has two complementary goals. The first goal is to elucidate the biological properties of adult stem cells (ASCs) that renew human tissues. The second is to translate human ASC discoveries into advances in cellular medicine. For decades, lack of critical knowledge about ASCs has been a formidable wall at the frontier of ASC research and cellular medicine. Three intractable research challenges form this wall. Methods for exact identification of ASCs are lacking, ASCs are difficult to produce in large quantities, and, therefore, important biological features of ASCs remain obscure. To address these challenges, during the past 14 years as a principal investigator, the applicant undertook research with genetically-engineered mouse cells that modeled essential unique properties of ASCs. These properties are asymmetric self-renewal and immortal DNA strand co-segregation, two defining features of ASCs. A series of reports from the applicant’s laboratory established the relevance of discoveries made with the mouse ASC models to rodent ASCs in vivo. These discoveries established an innovative foundation for identifying ASCs, expanding ASCs in culture, and investigating ASC cellular and molecular properties. With NDPA support, the applicant will change the direction of his research program, forged with rodent ASC studies, to focus on human ASCs and their conversion into embryonic stem cell (ESC)-like cells. Human ASCs responsible for liver, hematopoietic cells, pancreas, and hair will be the focus of research with the goal of developing both pre-clinical and clinical applications for cellular medicine. The applicant’s research program enjoys collaborations with several laboratories from diverse scientific disciplines and technological fields, including mathematics, chromosome biology, mass spectrometry, bioinformatics, genomics, electrical engineering, computer sciences, and the cell products industry. These interactions will complement research to expand selected human ASCs, develop tools for their exact identification, and advance ASCs as effective cellular medicines for human disease.

Nanostructures have received extensive attention for their promising applications in electronics, photonics, and information storage. I believe these minuscule structures also hold great potential for advancing biomedical research. In particular, I have always wanted to harness the power of nanostructures to radically change the way cell behavior is probed and regulated. Here I propose to develop the next generation of toolset for studying and manipulating cell activity by bringing together three classes of complementary nanostructures: gold nanocages capable of absorbing near infrared light and effectively converting it to heat; smart polymers capable of changing conformation in response to small variation in temperature; and enzymes. The stimuli-responsive polymer will be covalently attached to a specific position near the active site of the enzyme; the resultant unit will be conjugated to the surface of gold nanocage. When the nanocage is struck with a pulsed laser, the polymer conformation will be quickly and reversibly switched between the extended and collapsed states, turning on and off the enzyme. To demonstrate the biological importance of such hybrid nanostructures, I will initially apply them to manipulate cell behavior such as apoptosis. A variety of trapping techniques will also be adapted to control the spatial position of the hybrid nanostructure inside and outside an individual cell. For the first time, I will be able to ascertain the minimum number of active enzymes required to initiate apoptosis, and whether and how the spatial location of the enzyme affects apoptosis signaling. Once it has been demonstrated for apoptosis, the concept will be extended to develop similar hybrid nanostructures for reading and controlling other cellular processes and signaling pathways. Such a toolset based on spatially and temporally addressable nanostructures is complementary to many other bioimaging techniques under development, and will find broad use in studying complex biological systems.

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