Michael B. Brenner - US grants

Research: Twenty-three new monoclonal antibodies (mAb) have been made which recognize the human T cell receptor. Different examples of these mAb are directed against: 1) all human T cell receptors; 2) one subset of T cell receptors on normal T cells; and 3) clonotypic epitopes on the HPB-ALL receptor molecule. These mAb will be used: 1) to isolate T cell receptors for biochemical analyses; 2) to identify T cell receptor epitopes which may be allotypic or represent conformational states; and 3) to isolate receptors from peripheral blood T cells of rheumatoid arthritis patients in order to immunize mice. MAb will be made from mice producing antibodies to rheumatoid arthritis T cell receptors in order to identify additional subsets of T cell receptors (other than those represented by the HPB-ALL receptor) and to investigate whether cross-reactive T cell receptor idiotypes (clonotypes) occur in rheumatoid arthritis. Cross-reactive idiotypes and T cell receptor subsets which are identified will be biochemically characterized. In addition, structural studies using chemical cross-linking will be performed to further delineate the arrangement of the 5 chains of the T cell receptor molecule: 1) on the cell surface; 2) within the plasma membrane; and 3) on the cytoplasmic face of the membrane. These studies may contribute to our understanding of the structure-function relationships for the receptor. For example, changes in chain association or conformation of the receptor which may occur upon T cell activation by antigen will be investigated. Candidate: The applicant is trained in clinical medicine having served as Chief Resident in Medicine at Vanderbilt University Hospital and as a clinical fellow in rheumatology at U.C.L.A. Research training began at the U.C.L.A.-affiliated Wadsworth Veterans Administration Hospital and has continued during the past 2 years with Dr. Jack Strominger, Professor of Biochemistry, Harvard University and Dana-Farber Cancer Institute. The applicant has recently been appointed to Asst. Prof. in Medicine at Harvard Medical School. Environment: The sponsor's laboratory is expert in biochemistry and molecular biology and the Rheumatology Division at Brigham and Women's Hospital offers an extensive clinical experience.

ABSTRACT: Using framework monoclonal antibodies (mAb) against the T cell receptor (TCR) alpha beta complex, we found that not all T cells in human peripheral blood were TCR alpha beta+. A population of TCR alpha beta- CD3+ cells was found to express a second T cell receptor composed of CD3, the protein product of the rearranging T gamma gene, and an incompletely characterized TCR delta subunit. This receptor is expressed on the surface of a novel population of human and murine peripheral blood T cells and occurs in specific forms based on the TCR gamma polypeptide size, C gamma I or C gamma 2 gene segment usage, and the presence or absence of disulfide linkage. We wish to elucidate the structure of the TCR gamma and delta proteins and genes. In particular, we will determine the peptide backbone size, number and type of carbohydrates, presence of variability in peptide maps and determined partial amino acid sequence for the TCR delta chain. We propose to clone the gene for this subunit by oligonucleotide hybridization (based on determined amino acid sequence) or by lambda gt11 expression screening (with specific anti-TCR delta sera) or by cross-hybridization with the murine X gene (Y-h Chien and M Davis). The deduced structure of cloned genes will be compared to the determined biochemical features and sequence obtained from purified TCR delta protein to establish the identity of the cloned genes. To study the function of TCR gamma delta lymphocytes, various stimulating antigens will be used in efforts to define the specificity (and possible MHC restriction) of these cells. In addition, their demonstrated ability to kill malignant cells (natural killer-like activity) will be investigated to determine if the TCR gamma delta recognizes tumor-specific antigens. The cloned TCR gamma and delta genes will then be transfected and expressed in order to determine the rules which govern pairing of the different TCR gamma and delta forms, and to reconstitute a functional receptor. The ultimate goal is to understand what functional role this new population of T cells plays in the immune system.

We propose to study the clonal heterogeneity of T lymphocytes in autoimmune diseases, particularly in rheumatoid arthritis. This will be carried out through an analysis of the T cell receptors (TCR). Framework monoclonal antibodies (mAb) against the TCR delta chain (beta F1) and against the TCR beta chain (anti-TCR delta 1) will be used to study the TCR alpha beta and TCR gamma delta proteins from rheumatoid arthritis T cells. The T cells will include the activated T cells from peripheral blood, as well as T cells from synovial fluid and synovial tissue. TCR protein will be characterized by two dimensional gel analyses. TCR gene rearrangements will be examined in Southern blotting analyses. TCR protein will then be isolated for frequently immunization of mice in the production of mAb that may recognize frequently used or cross-reactive TCR gene segment encoded determinants. Complementary used or cross-reactive TCR gene segment encoded determinants. DNA clones corresponding to these gene segments will be cloned and sequenced. Analysis on a more limited basis will be carried out in type 1 diabetes and systemic lupus erythematosus. The framework mAb against the TCR will also be evaluated as diagnostic tools for distinguishing malignant versus benign T- lineage proliferations, based on the discordant expression of CD3 and TCR proteins in neoplasia. All analyses performed will include both receptors, TCR alpha beta and TCR gamma delta. We hope that the proposed studies will help us to understand the role of T cells in autoimmune diseases and that T cell receptors may prove useful in the diagnosis of T-lineage neoplasms.

This project addresses several problems of current scientific and technological importance, from fluid mechanics, materials science, and biology. Specifically, the first project includes a mathematical study of the events that occur before a liquid droplet splashes on either solid or liquid substrates. This is a problem of immense technological importance, with applications ranging from the design of ink jet printers to the entrainment of carbon dioxide at the ocean surface. Nonetheless, basic features of the process are still not understood, and our mathematical models aim to resolve the critical issue. The second topic is a mathematical study of shadow lithography, an emerging method for creating arrays of small nanoscopic structures and new optical materials. We aim to develop a complete mathematical description of patterns that can form using this method, leading to a logical dictionary of structures that can be made. The third project aims to better understand the development of biofilms, bacterial colonies which strongly adhere to surfaces. These have been implicated as the cause of infection and decay both in medicine and technology. The biological transitions that lead to biofilm development are not well understood. We will develop a mathematical model of these transitions in a model organism. This has the potential to lead to new fundamental insights into biofilm development and how to control it. The broader impact centers on both personnel development of graduate students and postdocs, and the development of educational materials for teaching science and mathematics through cooking. Our teaching initiatives on science and cooking have reached nearly 100,000 people through an online class, and we aim to extend the reach and content to secondary education.

The study of the splashing of droplets will start from a description of incompressible potential flow in the liquid, coupled to the compressible dynamics in the surrounding air. The dynamics in the air is well captured by a lubrication approximation. Previous work has modeled the initial dynamics in the liquid phase using potential flow. However, both theoretical estimates and experiments indicate that viscous forces become important in the liquid in the later stages; we will extend the mathematical description to include this, using a boundary layer description of the viscous effects in the liquid. The study of shadow lithography uses methods in geometry to completely characterize the shadows made by the array of spheres. Mathematical characterization of structures will be made with both two-dimensional masks of spheres and will also extend current experimental methods to three dimensional masks of opaque spheres in a transparent matrix. The latter could lead to new method for fabrication complex devices. The study of biofilms use a new experimental method for whole film imaging of gene expression in Bacillus subtilis, which gives an unprecedented look at the spatiotemporal dynamics of how changes occur in a biofilm. The project will construct mathematical models of the nutrient field in the experiment and then investigate whether the nutrient field itself is sufficient to explain the transitions, or whether signaling molecules are also required. Predictions will be tested in experiments and may lead to understanding of how development unfolds in Bacillus.

Abstract Not Provided.

Project 9: Inter-module integration - plasticity and robustness in brain and behavior (Hofmann) (#51-55) We explored the molecular basis of neural and behavioral plasticity in the African cichlid fish Astatotilapia burtoni. The males make regular transitions between territorial ("macho") and non-territorial ("wimp") forms, and we used a combination of behavioral observation and microarray analysis to map the genetic modules responsible for these dramatic behavioral and physiological transitions. We made a cDNA microarray containing 18,000 features from an estimated 8,000 genes21 and made arrays, DMA sequences, and the associated database freely available (http://cichlid.biosci.utexas.edu/html/cichlid genomics.html). an effort that sparked an NHGRI-approved initiative to sequence four cichlid genomes. Whole-brain expression profiling in individuals of known social status led us to perform detailed analysis on small groups of cells microdissected from the preoptic area of the hypothalamus. Examining gene expression in territorial and non-territorial males and correlating it with behavioral and physiological markers showed that genes can be grouped into a small number of modules, clusters where gene expression is highly correlated, and that expression of the modules correlates strongly with phenotypic traits. The relationships between the modules are interesting and complex. For example, aggressive and sexual behavior drive expression in opposite directions, echoing a theme from classical ethology, but both types of changes are inhibited by the acute stress response. We focused on the role of somatostatin and showed that this ancient peptide has a role in regulating aggressive behavior22. The cichlid fish have speciated explosively, and many different traits have appeared independently in separate lineages. We correlated ecological, behavioral, and neuroanatomical data for a single closely related clade (the Ectodini) and reached the conclusion that selection can act independently and rapidly on different regions of the brain, a finding that has direct relevance for the recent evolution of the human brain23. Hans Hofmann, the PI was a Bauer Fellow and is now Assistant Professor of Integrative Biology at UT-Austin.

TECHNICAL SUMMARY: The goal of this research project is to explore new methods to alter electronic band structure and therefore the optical properties of semiconductor materials so as to transform them into more efficient solar energy converters. The electronic band structure will be modified by optical hyperdoping via femtosecond laser irradiation in the presence of various gases. This process leads to higher levels of doping than are possible using other methods. To better understand and optimize the dynamics of optical hyperdoping, the team will develop new mathematical tools for modeling the hyperdoping process, focusing on the quantum mechanics associated with the unusual band structure, as well as methods for understanding and controlling the non-equilibrium process itself. The multidisciplinary team of PIs will integrate expertise in mathematics and continuum modeling of optical hyperdoping, theoretical chemistry and modeling at the quantum level, materials science of optoelectronic materials, and chemistry of surfaces and interfaces to make a concerted attack on creating and understanding new materials with transformative potential and broader impacts.
NON-TECHNICAL SUMMARY: Meeting the challenge of harvesting solar energy with Earth-abundant materials such as Si and TiO2 require transformative approaches to increase efficiency, lower manufacturing cost, and reduce material requirements. While these materials have been widely studied, a multidisciplinary team from Harvard University brings a new approach to modifying the properties of semiconductors so as to open the door to more efficient solar cells. In addition, the multidisciplinary nature of the research provides unique training for students and postdocs involved in the project, with a synergistic experimental program combining materials science and chemistry, intimately coupled to a theoretical program combining mathematical analysis of the materials processes and quantum mechanical calculations of the band structure.
This project is co-funded by the Divisions of Chemistry, Materials Research, and Mathematical Sciences.

NON-TECHNICAL SUMMARY
Creating materials and devices that can assemble themselves has long been a holy grail in materials science, since it would introduce qualitatively new ways of making nano-scale structures and materials. The field of DNA nanotechnology has exploded with striking advances in creating robust and dynamic materials made entirely out of DNA, but the uses of DNA as a material on its own are much more limited than if it could be combined with other materials. A potentially vast synergy lies in the combination of colloidal assembly and DNA nanotechnology. Instead of mediating the interactions between particles by using DNA linkers (the current state of the art), one could control these interactions using more complex and potentially dynamic DNA nanostructures in solution. This project seeks to develop a fundamental understanding of how (dynamic) DNA nanostructures can control and program colloidal self-assembly. By putting this concept on a strong fundamental footing, it will be possible to exploit it to maximum effect through the design of DNA reaction networks that solve essential challenges to making colloidal self-assembly a practical materials fabrication platform. The set of possible interactions between building blocks is so large that this design space can only be systematically explored with a combined attack from theory, numerical simulation, and experiment. The project aims both to discover the fundamental principles underlying DNA particle-nanostructure interactions and to create new materials from plasmonic molecules to metafluids with immediate technological impact. The project will also involve Harvard undergraduates in a design curriculum for inner city middle schools, in a summer software development project through Harvard's Institute for Applied Computational Science, and in teams that participate in the International Genetically Engineered Machines competition.

TECHNICAL SUMMARY
The investigators will work to understand and harness the different ways of making novel periodic and aperiodic colloidal structures out of particles and DNA nanostructures. Several types of DNA nanostructures can be used to control colloidal self-assembly. The simplest type of mediating DNA nanostructure involves single strands of DNA (ssDNA) in solution. Preliminary experiments show that free DNA strands give an unprecedented level of control over assembly and melting through strand displacement reactions. The investigators will also develop mediating DNA nanostructures that give an effective valence to inter-particle interactions, even when the particles are uniformly coated with ssDNA. Valence creates many new directions for robust self-assembly, from building large structures to designing structures that form spontaneously in a bath at finite concentration. Finally, they will investigate how DNA hairpins can lead to non-equilibrium interactions. This opens up new vistas for theory and experiments, including the design of self-replicating colloidal clusters and the development of kinetic proofreading schemes for significantly increasing the fidelity of the interactions over the equilibrium limit. For each type of DNA-mediated interaction, the team will use theory and simulation--which in some cases requires developing new methods--to enumerate the possibilities of what can be assembled. These will be used in conjunction with experiment to realize the assemblies described in the simulations, and these experiments will inform refinements of theory and simulations. Ultimately, they will arrive at a holistic view of what can be assembled with each mediating nanostructure.

The Team Research in Computational and Applied Mathematics (TRiCAM) REU program aims to give students an experience in real-world collaborative problem solving, challenging them to apply mathematics and computation to tackle team projects posed by Harvard faculty and industrial partners. Projects will involve the application of computational and mathematical tools such as machine learning, data analysis, and numerical simulation to solve problems in fields such as geoscience, medicine, materials science, and the social sciences. Topics will be chosen to appeal to a wide population of students who are at early stages of their academic development, and who have limited awareness of the vast range of potential career paths in applied mathematics and computational science. The program's team-based approach to research will teach students an appreciation for scientific collaboration, prepare them for future employment, and provide opportunities to practice conflict resolution skills. Ultimately the program will help develop a new generation of collaborative scientists and engineers who are excited about applying computation and mathematics to solve interdisciplinary, real-world problems.

This REU project will support four teams of undergraduate students for ten weeks each summer as they gain both the mathematical, computational, and statistical skills necessary to tackle a research problem, and real-world experience of working in a team-based collaborative environment. The summer will be divided into four phases: a two-week orientation where teams formulate a statement of work for summer in response to the problem posed by the faculty or industrial sponsor, two three-week work periods, the first resulting in a midterm report submitted to the sponsor, and the second focused on responding to feedback from the midterm report, and a one-week completion phase where teams prepare final reports and presentations to both the sponsors and the larger Harvard REU community. Students will be selected for teams based on their individual academic strengths and for their potential fit as a member of a team and for a particular project. The program will focus on students for whom this will be an early, even perhaps a first, experience with research, by targeted recruiting at historically black colleges and non-research institutions.

Non-technical Description: Manufacturing of complex objects is the key engine of technological progress. Learning to build smart, digital, and mechanically functional objects at the microscale could be as revolutionary as human-scale manufacturing. This grant will support research to develop a new way of meeting this challenging goal. It combines two technologies: modern magnetic information storage, which can create tiny magnets in any pattern desired, and ultrathin flexible materials that can bend in response to tiny forces. These will be combined with the design principles of colloidal systems, polymer physics, and molecular biology to create intelligent, functional objects, machines, and materials. The pieces will interact in a way analogous to the way DNA bases bind together, with magnets playing the role of the base pairs, and the thin materials playing the role of the DNA backbone. The magnetic information will determine how multiple strands connect and form complex structures and micron sized machines that can be controlled with external magnetic fields. These materials will ultimately have fundamental impacts on micro-engineering, with a range of potential applications, from materials to medicine. As such, this research will promote the progress of science and ultimately benefit the US economy and society. This research borrows concepts from a variety of fields - a multi-disciplinary approach that will help broaden participation of underrepresented groups in research and positively impact engineering and science education. For example, macroscopic analogs will be adopted into lending kits that will be used to explain the basic principles behind base paring in DNA and its assembly into DNA origami structures to impoverished communities in upper Appalachia.


Technical Description: This grant will support research aimed at building a new platform for self-assembly that uses panels with magnetic handshakes - microscopic patterns of magnetic dipoles - that enable panels to bond together using specific, intelligent interactions analogous to Watson-Crick base pairs in DNA. By fabricating these panels on nm thin elastic strands grown via atomic layer deposition, the panel sequence will determine how multiple strands connect to one another and form complex untethered structures and micron sized machines that can be manipulated with external magnetic fields. These handshake panels will be programmed using either magneto-optic recording (micron scale) or commercial scanned magnetic write head (50 nm scale). The resulting magnetic colloids, strands, or nets will be released from the substrate into solution, and allowed to bend, move, and assemble according to their designed interactions. The approach of the grant is to integrate design, macroscale models, advanced simulations, and experiment, to master the programmed self-assembly of these magnetic handshake materials. Overall, this strategy both takes advantage of the complementary binding principle behind current state of the art 3D DNA based assembly and overcomes many of its limitations, including vastly expanding the range of operating parameters such as temperature, solvent, etc. The resulting structures can be fully integrated with other lithographic elements (electronics, optics, etc.) and will have broad applications in sensing, actuation, and microrobotics at the cellular scale.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.