Michael J. Levene - US grants

DESCRIPTION (provided by applicant): PROJECT SUMMARY We propose the development of fluorescence fluctuation spectroscopy (FFS) for the evaluation of von Willebrand Factor (vWF) multimers as a more accurate and reproducible technique in von Willebrand Disease (vWD) diagnosis, classification, and monitoring, for elucidating specifics about the pathogenetic mechanism of thrombotic thrombocytopenia purpura (TTP), and as a new tool for assessment of coagulation or bleeding risk in a variety of common systemic conditions. The developed approach will have broad applicability to the measurement of oligomerization, including the study of amyloid and prion diseases. VWF is a blood protein that is critical for proper clotting and exists in multimers composed of between 2 and 80 monomers. Deficiencies in the function and distribution of vWF multimers lead to vWD, the most prevalent group of inherited coagulation disorders. Knowledge of the concentration and size distribution of vWF multimers would aid in the clinical subtyping and management of vWD and would be valuable as a diagnostic and research tool in TTP. Traditional testing parameters for vWF testing and current gel-based methods for multimer measurement suffer from significant problems of inter-laboratory variability and low reproducibility. The multimer process is also laborious, technically challenging, and radiation dependent, so it is typically only available from reference laboratories. FFS includes several techniques with single-molecule sensitivity, including fluorescence correlation spectroscopy (FCS) and photon counting histograms (PCH). FFS monitors fluctuations in the number of freely- diffusing particles within small confocal observation volumes - smaller than the largest vWF multimers. We will optimize the observation volume in our instrument for use with a large range of particle sizes using fluorescence beads. Maximum entropy regularization has been used for multi-parameter FCS (MEMFCS) fits;we will develop our own MEMFCS algorithm to improve accuracy and reproducibility, and will apply a similar approach to develop the first PCH fits to broad distributions. The FFS applicability to vWF measurement will be demonstrated on samples from normal healthy controls, Type I vWD, Type IIA vWD, and TTP patients. Clustering of vWF distribution amplitude, mean, width, kurtosis and skew will be used to establish diagnostic relevance. Present day methods for predicting clinical behavior from patients with disordered vWF activity are inadequate. Our ability to examine the physiology of vWF in different settings is limited by the challenges that multimeric testing by traditional methods present. We are adapting the techniques of single molecule analysis from the field of biophysics to address these shortcomings in an effort to produce a practical tool for the routine measurement of vWF multimers in the many diseases with abnormal coagulation. PUBLIC HEALTH RELEVANCE: Relevance Statement The development of a simple-to-use, more accurate, and reproducible method for measuring the distribution of sizes of the blood protein von Willebrand Factor will increase our understanding and improve diagnosis and treatment of von Willebrand Disease (the most common inheritable bleeding disorder), thrombotic thrombocytopenia purpura (a serious condition of abnormal clotting), and the broad range of common systemic diseases that are associated with abnormalities in coagulation.

Major questions in experimental Neuroscience, ranging from how neuronal computation works at the level of micro-circuitry to the effects of thousands of different genes on basic anatomy, are best studied optically. Yet, current optics devices can only reach ~5% of the intact mouse brain. The devices developed in this project will open up the remaining 95% of the intact mouse brain to optical microscopy through novel invasive micro-optics. The devices will be capable of penetrating the brain to gain access to deep brain regions, while evaluating and minimizing tissue damage to ensure the validity of experiments. The micro-optics will take the form of needle-like lenses that can access deep, sub-cortical brain structures and micro-prisms that can image entire cortical columns, the fundamental processing unit in cortex. These systems will also be combined to form micro-periscopes, for side-on viewing of deep structures. Taken together, these devices will fundamentally change the limits of experimental Neuroscience in living animals. In addition, they will also find application in a wide range of non-Neuroscience fields, including, for example, the monitoring of tumor growth and the investigation of the stem cell niche, or even in plant systems. The anticipated low-price of the micro-optics will facilitate rapid dissemination of this technology to the research community. The dissemination will be aided by classes on how to assemble and use micro-optics, offered as part of the free Yale Microscopy Workshop and Symposium. This symposium occurs every summer and is open to the broader research community outside of Yale. In addition, each summer, two or more high school students will be funded to join the Yale Engineering and Science Summer Internship (YESSI), run by the primary investigator. This program runs a competition amongst interested graduate students to mentor high school students, who also compete to enter the program. The graduate students receive valuable experience as mentors, while the high school students get exposed to cutting edge research.More information may be found at http://www.eng.yale.edu/levenelab/