David S. Talaga - US grants

nucleic acid structure; DNA; temperature jump; infrared spectrometry;

DESCRIPTION (provided by applicant): Single molecule (SM) measurements are rapidly becoming commonplace in research laboratories around the world and are contributing to many areas of investigation because of their ability to provide insight into phenomena that were previously intractable because of the ensemble averaging present in bulk measurements. In particular the dynamics of conformationally heterogeneous systems are benefiting from single-molecule studies. Protein folding and conformational dynamics, enzymology, ribozyme function, bacterial light harvesting, and protein-nucleic acid interactions are just a few examples of complex systems that have benefited from the application of SM techniques. However, the impact of SM results has been mitigated by the lack of uniform data analysis and interpretation. The proposed research focuses on SM fluorescence measurements and how to place the experimental design, analysis, and expectations onto solid statistical and theoretical ground. Three specific aims are proposed: 1. Use information theory to determine the fundamental limits of SM experiments. 2. Develop statistically rigorous analysis methods based on hidden Markov models. 3. Implement methods as user-oriented additions to common data analysis packages. The significance to health of this research is through its contribution to the many ongoing SM investigations into biological systems. SM measurements are revolutionizing our approach to many problems in chemical biology, yet they are still being interpreted and designed based on assumptions that are only valid for ensemble measurements of bulk samples. This can result in collection of data that cannot be adequately interpreted using traditional methods. A consistent theoretical framework for SM measurements would be a significant step forward for the field. Aim 1 will provide a theoretical framework that can be used for experimental design as it provides the limit of the measurement's ability to make inferences about the properties of the system. It will also provide the benchmark (the Cram[unreadable]r-Rao bound) by which to judge data reduction methods. Aim 2 develops the algorithms and core codes to implement statistically rigorous methods of data analysis that allow unbiased estimation of system parameters with accuracy approaching the Cram[unreadable]r-Rao bound including meaning uncertainty estimates. Aim 3 provides useable tools for experimental design and analysis to allow other investigators to exploit these methods for their own research.

0609000
Moghe

This proposal was received in response to Nanoscale Science and Engineering initiative, NSF 05-610, category NIRT. The objectives of this research are (1a) to design, fabricate, and characterize nanoparticles functionalized with matrix protein fragments, and (1b) to elucidate the role of nanoparticle size, ligand sequence and ligand loading on cell motility and matrix assembly; (2) to identify the key molecular signaling pathways that mediate nanoparticle internalization and increased cell activation; and (3) to develop imaging modalities to quantify nanoparticle trafficking and internalization dynamics. The approach involves the functionalization of albumin-derived nanoparticles with various fragments of fibronectin (ligand). The first phase of the study will examine how optimal configurations of substrates based on the ligand-nanoparticles can significantly alter the morphology and dynamics (motility, matrix assembly) of skin derived cells (keratinocytes, fibroblasts). The second phase will focus on identifying the underlying biologic mechanisms using protein and gene level signaling assays. The third phase will probe the nature and kinetics of nanoparticle-cell interactions using high resolution, two-photon microscopy and a magnetically responsive biosensor platform with nanoscale fidelity.

This research can help design improved nanomaterials for potential cell targeting applications in wound healing, tissue engineering, drug delivery, and cancer therapy.
The nanoparticles designed here are biodegradable, can be targeted to cells, and can be customized to cell functions by altering their size. Smaller nanoparticles can stimulate increased motility in epidermal cells (relevant for skin healing in burns, ulcers) while larger nanoparticles promote skin contractility and matrix assembly (relevant to wound repair). This project extends its outreach through the wide diversity network (graduate, postdoc, undergraduate) at Rutgers and beyond, and resonates with three new integrative graduate courses at Rutgers on engineering of cellular biointerfaces with biomaterials, a graduate training program on biointerfaces, and an international research collaboration.

? DESCRIPTION (provided by applicant): Aggregation and fibrillization of ?-synuclein has been implicated in the progression of Parkinson's Disease, which currently has no cure. This research project is focussed on the molecular steps for initiation of ?-synuclein aggregation and fibrillization. The project builds on the PI's discovery that ?-synuclein appears resistant to aggregation except when exposed to non-fluid hydrophobic interfaces or to preformed seeds. The research uses chemically functionalized silica substrates to measure ?-synuclein conformation at the interface, and uses identically functionalized mixing balls to assay for the interfacial influence on aggregation and fibrillization. This platform allows systematic evaluation of the relationship between the physical and chemical properties of the interface, the conformational changes in ?-synuclein, and the initiation of aggregation and fibrillization. The research will identify the physical and chemical properties of interface and the changes in ?-synuclein structure that lead to aggregation and fibrillization. The project then builds on the identification of that first step of aggregation and fibrillization to develop a fluorescence assayfor the changes in ?-synuclein that occur during the first step(s). The assay is intended to enable single-molecule sensitivity. The assay will be used to learn more about the first steps of ?-synuclein aggregation and fibrillization. To evaluate the hypothesis that ?-synuclein resists fibrillization without a nucleation partner, samples free from nucleating interfaces will be evaluated to try to find conditions for fibrillization that do not include nucleating parnters. Fially the fluorescence assays developed to probe the first steps of aggregation leading to fibrillization will be used to evaluate macromolecular binding partners. The macromolecules will be classified using the assay according to the type of structure they induce in ?-synuclein and how they inhibit or promote aggregation and fibrillization. These assays will evaluated for their future utiity as discovery reagents for ?-synuclein binding partners in cell lysates.