Robert D. Goldman - US grants

The structure and function of intermediate filaments (IF) are being investigated in a variety of cultured epithelial cells and fibroblasts. In the fibroblast IF system, two major structural proteins (54 kilodalton [desmin, skeletin], and 55 kilodalton [decamin, vimentin]) have been identified and extensively characterized using biochemical and immunological methods. This research program is concentrating on three classes of proteins which coisolate with native fibroblast IF and may represent true IF-associated proteins (IFAPs); one represented by a group of proteins in the 60 to 70 kilodalton molecular weight range and another consisting of a 300 kilodalton molecular weight protein. Three sets of monoclonal antibodies have been prepared which permit us to distinguish the 300 kilodalton, the 60 to 70 kilodalton and the 54/55 kilodalton structural proteins. Preliminary results employing these antibodies in light and electron microscopic studies suggest that the 60 to 70 kilodalton proteins are associated with the nuclear surface and may act to anchor or bind cytoplasmic IF. Recently we have obtained data which suggests that these proteins are very similar to the nuclear lamins. The 300 kilodalton protein appears to be an IF-IF crosslinking protein and recently we have managed to purify it using column chromatographic techniques. In epithelial cells, our efforts are focused on elucidating the function and biochemical properties of IF bundles and their interactions with desmosomes. Studies on desmosome assembly and dynamics are being carried out using cultures of mouse keratinocytes. In addition we are attempting to fractionate desmosomes and to carry out in vitro reconstructions of IF-desmosome complexes. Other studies focus on the relationship between the mitotic spindle and IF. The cell types used in these studies include BHK-21, HeLa, PtK1, mouse epidermal keratinocytes and transformed mouse epidermal cells. A multifaceted approach is taken in all studies and techniques including the isolation and biochemical-biophysical characterization of IF, in vitro polymerization assays, the use of monoclonal and polyclonal antibodies, immunofluorescence and other light optical methods, and all aspects of electron microscopy. The long-range goal of these studies is to determine whether or not IF are directly involved in forming connecting links between the nuclear surface and the plasma membrane. (L)

The studies outlined for this project are aimed at analyzing the changes in actomyosin-like contractile proteins which accompany the aging of non- muscle cell cultures. The goal of these studies is to determine whether or not these changes can help to account for the cessation of motile behavior and growth of normal cells which accompany senescence in vitro. Actin containing microfilaments and their associated proteins represent the major ultrastructural component of the cytoplasmic contractile machinery. Therefore their structure, organization, intracellular localization and function are emphasized throughout the proposed research. Attempts to correlate function with structure through the coordinated use of microscopical, physiological, biochemical and immunological techniques will be employed.

This award will support the travel of eight United States scientists to a workshop on the cytoskeleton and its formation and role in the growth, differentiation and structure of wool and hair. The workshop will be held in Adelaide, Australia, December 7-11, 1987. Dr. Robert D. Goldman, Northwestern University School of Medicine, and Dr. Tung-Tien H. Sun, New York University School of Medicine, are the U.S. organizers planning the workshop with Dr. George E. Rogers, University of Adelaide, and other Australian members of the Committee. The purpose of the workshop is to assess the use of hair and wool growth as a model system for studying the structure and function of the major intermediate filament (IF) proteins, in particular the keratins. Intermediate filament proteins are major components of the cytoskeletal system of all mammalian cell systems and keratins are the most well characterized of all of the intermediate filament proteins. Discussed at the workshop will be the structure and function of the cytoplasmic keratins and the newly described complexes of keratin-like proteins within the nuclear matrix, the lamins, as well as the microbiology of the keratin system including the characterization and regulation of the genes encoding these proteins. The result of the workshop will be an increased awareness of the scientific activities going on in the countries represented and greater possibilities for collaborative research between the participants. This meeting will bring together for the first time Australian keratin experts with investigators from the U.S. having expertise on the cellular and molecular aspects of the cytoskeleton of eukaryotic cells. The Australian Government Department of Science has already approved and funded support for the Australian participants. The NSF Program Director for Developmental Biology has recommended this activity at a high priority level.

The program described in this application is aimed at determining the causative factors involved in the blistering skin disease pemphigus. These studies are based on our initial findings that pemphigus patients contain circulating autoantibodies directed against components associated with the desmosomes of stratified epithelial tissues. The specific aims of this proposal are to continue these studies by determining the precise cellular sites recognized by these autoantibodies using morphological, biochemical, immunochemical and physiological methods. The morphological methods which we will use will include immunofluorescence, immunogold localization for ultrastructural studies, and double label immunofluorescence techniques. These will be applied to both thin cryostat sections of various tissues including human biopsy material and cultured keratinocytes. The biochemical analyses will involve the isolation of desmosomes. These desmosomes will be used as substrates for the 'Western' immunoblotting procedure which will permit the identification of pemphigus autoantibodies which recognize desmosome associated components. Affinity procedures will be used to purify these antibodies from human serum samples and the resulting monospecific antibodies will, in turn be used to determine if these autoantibodies are factors involved in the loss of cell cohesion in the disease. Other studies will be aimed at determining whether or not there are different autoantibodies involved in the major variants of the disease, pemphigus vulgaris and pemphigus foliaceus. Attempts will be made to prepare rabbit antibodies against desmosome associated proteins recognized by autoantibodies in the two variants of the disease and determine whether or not these can mimic pemphigus autoantibodies. Finally we will attempt to prepare monoclonal human antibodies by fusing peripheral blood lymphocytes from blood samples of patients with pemphigus to a human-mouse heteromyeloma line. The long term goal of these studies is to determine the causative factors in pemphigus and to provide new insights into the cellular/molecular basis of the disease. Ultimately this information may be important in determining the therapeutic regimens of pemphigus patients.

We propose to continue studying the intermediate filament (IF) based cytoskeletal system in mammalian cells. Emphasis will be placed on the regulation of the organization of IF and their interactions with the nuclear surface. The long term goal of these studies is to determine the possible functions of the IF system and its interaction with the nucleus, now known to contain another IF protein family system known as the nuclear lamins. The experimental plan involves detailed morphological studies on the coordinated activities of the cytoplasmic IF and nuclear surface systems throughout the cell cycle using immunocytochemical techniques, biochemical and physiological analyses, the effects of phosphorylation on the cytoplasmic IF system, structural studies of the nuclear lamin paracrystals, and the use of microinjection of derivatized IF proteins. The IF system is thought to be involved in various physiological activities, including nuclear-cytoplasmic interactions, signal processing between the cell surface and the nucleus, etc. With regard to disease processes, this system is frequently found in an altered state in many diseases including cancer, Alzheimers disease, liver diseases and several other neurological disorders.

The purpose of this grant application is to obtain funds for a Zeiss Laser Scan Microscope (LSM) equipped with a Zeiss VIDAS Image Processing System. The LSM provides both confocal optics for fluorescence analysis and laser scan transmitted capabilities for phase contrast and DIC observations. This item of equipment will be used by three PHS extramural awardees who are all members of the Department of Cell Biology and Anatomy at Northwestern University Medical School. All three of these primary users of the LSM currently require extensive use of standard immunofluorescence microscopy for many of the specific aims of their funded projects. The confocal mode of the LSM is designed to optimize resolution of immunofluorescence preparations with the highest numerical aperture infinity corrected lenses. It achieves this through the use of a laser scanning system capable of resolving thin focal planes within microscopic specimens of varying thickness including single cells in culture, as well as in cells in aggregates and in tissue sections. This focussing achievement in turn permits users to carry out 3-dimensional reconstructions from multiple sequential images obtained through the use of a stage equipped with an integrated Z-scanner motor drive. These images are stored digitally and 3-D reconstructions are achieved with the VIDAS system. These 3-D analyses are impossible with conventional fluorescence optics. The projects of the three major investigators are: (1) Determination of the structure and function of intermediate filament systems (Goldman). (2) The regulation of the organization and function of cytoskeletal elements in Dictyostelium discoideum (Fukui). (3) The structure, function and assembly of hemidesmosomes and desmosomes in mammalian cells and tissues (Jones). In this proposal we emphasize the benefits of the LSM for all these investigators. The LSM is a relatively new research tool which will revolutionize many areas of structural biology, especially those dealing with subcellular structure and function.

We propose to continue studying the intermediate filament (IF) based cytoskeletal system in mammalian cells. Emphasis will be placed on the regulation of the organization of IF and their interactions with the nuclear surface. The long term goal of these studies is to determine the possible functions of the IF system and its interaction with the nucleus, now known to contain another IF protein family system known as the nuclear lamins. The experimental plan involves detailed morphological studies on the coordinated activities of the cytoplasmic IF and nuclear surface systems throughout the cell cycle using immunocytochemical techniques, biochemical and physiological analyses, the effects of phosphorylation on the cytoplasmic IF system, structural studies of the nuclear lamin paracrystals, and the use of microinjection of derivatized IF proteins. The IF system is thought to be involved in various physiological activities, including nuclear-cytoplasmic interactions, signal processing between the cell surface and the nucleus, etc. With regard to disease processes, this system is frequently found in an altered state in many diseases including cancer, Alzheimers disease, liver diseases and several other neurological disorders.

The overall goals of this proposal are to determine the dynamic properties of the intermediate filament (IF) cytoskeletal networks in fibroblasts and epithelial cells as well as to determine the nature of the interaction between IF and the cell surface. IF dynamics will be studied in mitotic cells in which the IF system is depolymerized and in daughter cells in which IF are repolymerized. Specifically, studies of the molecular basis of the extensive remodeling of Type III IF networks containing vimentin or vimentin/desmin will be undertaken. The explosive depolymerization which typifies early-mid prometaphase and the rapid reassembly of IF which takes place during anaphase-telophase can be explained, for the most part, by the hyperphosphorylation of IF structural proteins. Two mitotic kinases have been found in BHK cells which are involved in the phosphorylation reaction. We are undertaking an extensive analysis of one of these kinases termed vimentin protein kinase (VPK). VPK is a complex of three proteins: a 65kD component, a 110kD component, and p34cdc2 which is the catalytic subunit of maturation promoting factor (MPF) and is known to play a major role in triggering mitosis. We plan to carry out experiments aimed at determining the functional significance of the specific site(s) phosphorylated by VPK and other kinases which act on Type HI IF proteins using a variety of cell physiological (e.g., microinjection), morphological (immunofluorescence, confocal, and electron microscopy), biochemical and molecular biological (the use of bacterially expressed proteins and transient transfection of cultured mammalian cells) techniques. We will also attempt to study the interphase dynamic properties of IF. For these studies we will utilize the microinjection of derivatized IF proteins such as biotinylated vimentin and keratin into live cells and to determine their fate in situ using confocal and electron microscopy. Similar approaches using x-rhodamine labelled IF proteins will be used to determine whether or not a steady state or dynamic equilibrium exists in interphase cells through the use of fluorescence recovery after photobleaching (FRAP) experiments. Studies are also described which are aimed at determining the biochemical basis of the binding of IF to cell surface associated desmosomes in epithelial cells. These studies involve the isolation and biochemical characterization of bovine tongue desmosome components and the determination of how keratin polypeptides bind to them. The information derived from our investigations should shed new light on the properties and functions of IF in mammalian cells. A better understanding of IF structure and function is basic to understanding the pathogenesis of a variety of diseases in which changes in IF occur, such as Alzheimer's disease, Parkinson's disease, various cancers, and alcoholic cirrhosis.

The objectives of this research are to determine the basic mechanisms that regulate the interactions between cytoskeletal keratin intermediate filaments and the cell surface associated adhesion junctions, desmosomes and hemidesmosomes, of gingival epithelia cells. The long term goal of the proposed studies is to gain insights into the molecular bases of the epithelial cell migration that represents a hallmark of periodontal disease. The specific aims include determining how the intermediate filament associated protein, IFAP300, connects desmosomes and hemidesmosomes to intermediate filaments. To this end, IFAP300 will be cloned and sequenced. Once the complete cDNA sequence is available, transient transfection will be used to determine the physiological functions of this important cross-bridging protein. Other aims are to determine in vitro the molecular interactions among keratins, IFAP300 and the major cytoplasmic plaque proteins of the desmosome (desmoplakin) and the hemidesmosome (BP230). In vivo studies will be targeted at determining the dynamic properties of keratin-intermediate filaments in living cells using microscopic imaging techniques.

The goals of these studies are to determine the structure and function of intermediate filaments and their associated proteins in mammalian cells. Intermediate filaments are major cytoskeletal structures and their constituent proteins vary in a cell type specific fashion. The specific aims of the proposed research are to determine the mechanisms underlying the assembly of intermediate filament networks, the interactions of intermediate filaments with microtubules, and the regulation of their dynamic and motile properties. The techniques employed in these studies include light and electron microscopy, single cell microinjection methods, biochemical characterization of proteins, and the cloning and transfection of wild type and mutant cDNAs in cultured mammalian cells. The results of these studies are related to human health as it has been demonstrated that there are many diseases linked to mutations in the structural proteins comprising intermediate filaments and there are other diseases in which abnormal accumulations of intermediate filaments are a pathological hallmark of disease. The latter include the formation of Mallory bodies in alcoholic liver disease and Lewy bodies in Parkinson's disease.

DESCRIPTION: (provided by applicant) The overall goal of this research proposal is to determine the functional significance of the changes in the intermediate filament (IF) cytoskeletal system that accompany the epithelial-mesenchymal transition (EMT). The EMT takes place when normal oral epithelial cells become malignant and metastasize. The EMT involves both changes in cell shape and motility. Specifically, it has been shown that one of the hallmarks of the conversion of normal epithelial cells into metastatic cells is the aberrant expression of vimentin, a Type III IF protein. These cells normally express only IF Types I and II, the keratins. We propose to carry out studies aimed at shedding new light on the functional significance of the induction of vimentin IF expression in the conversion of the normal cuboidal shape of oral epithelial cells into a mesenchymal morphology. In addition, we will attempt to determine whether vimentin expression plays a role in the increased locomotory activity of metastatic epithelial cells. The specific aims include: 1) determining the role of vimentin IF expression in the conversion of oral epithelial cells into mesenchymal cells using a variety of microscopic and molecular methodologies; 2) determining whether vimentin IF expression adds a new dimension to the cytoskeletal "crosstalk" in oral epithelial cells; 3) determining the role of plectin in establishing the molecular cross talk between IF and microtubules induced by vimentin expression in oral epithelial cells; 4) a continuation of our studies on the dynamic properties of keratin IF/tonofibrils in normal oral epithelial cells, with emphasis on alterations in their properties upon the introduction of vimentin IF.

Patients with acute lung injury are often placed on positive-pressure mechanical ventilation to improve gas exchange. However, mechanical ventilation may generate large shear forces during the cyclic closure and reopening of fluid filled alveoli, while the relatively spared, air-filled alveoli are cyclically overdistended. This may cause or worsen ventilator induced lung injury which may have a negative impact in patients with acute lung injury. In alveolar epithelial cells keratin intermediate filaments (IF) are the major structural proteins. Keratin IF are known to play an important role in maintaining the mechanical integrity of epithelial cells, and in vitro they are able to withstand a wide range of strain conditions without alterations in their structural integrity. In response to shear stress in vivo, IF have been shown to undergo adaptive changes in response to shear stress. This application proposes to study the response of keratin IFs to cyclic stretch, and cyclic and continuous shear stress in alveolar epithelial cells, and determine the effect of these changes in keratin IF on alveolar epithelial function via three interrelated specific aims. Specific aim#1. To determine whether cyclic stretch and/or shear stress-induced changes in keratin IFs alter alveolar fluid reabsorption in wild-type and keratin 8 knockout mice. Specific aim #2. To determine whether cyclic shear stress and/or stretch-induced changes (disassembly) in keratin IFs are mediated by protein kinase C-dependent phosphorylation in alveolar epithelial cells. Specific aim #3. To determine whether cyclic stretch and/or shear stress causes changes in ubiquitination and the regulated degradation of keratin IF networks by the ubiquitin-proteasome pathway in alveolar epithelial cells leading to lung cell injury. Completion of these studies will provide new insights into mechanisms responsible for cyclic stretch-and shear stress-induced alveolar epithelial lung injury.

DESCRIPTION (provided by applicant): Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare human disease with characteristics of premature aging that include loss of subcutaneous fat, wrinkled skin, loss of hair, arteriosclerosis, and difficulty in moving joints. About 90% of progeria patients die at an early age from progressive arteriosclerosis. HGPS is caused by mutations in human lamin A (hLA), a protein component of the nuclear lamina. The long-term objective of the proposed research is to determine the molecular basis by which mutations in the hLA gene alter nuclear function to cause these premature aging defects. It is our hypothesis that nucleoplasmic lamin structures, in addition to those in the lamina, form a nucleoskeletal system that provides the infrastructure required for crucial nuclear functions, including DNA replication, transcription, chromatin organization, nucleocytoplasmic transport and nuclear disassembly, assembly and shape. Understanding how these functions are altered by HGPS mutations will shed light on the mechanisms responsible for the multiple age-related disorders seen in patients with progeria, including cardiomyopathies and strokes. To this end, two laboratories with considerable expertise in lamin genetics, structure, function and nuclear transport will collaborate to address the following specific aims: 1) Characterize the effects of HGPS hLA mutations on nuclear structure and organization by the coordinated use of biochemical and microscopic methods. 2) Characterize the effects of HGPS hLA mutations on nuclear functions: DNA replication and cell division using HGPS patient cells and cell-free preparations of Xenopus nuclei. 3) Characterize the effects of HGPS hLA mutations on nuclear functions: nuclear import and export, nuclear pore complex structure and nuclear envelope permeability. 4) The use of human and animal cell models to test the effects of hLA mutations on mesenchymal cell types most affected in HGPS. These collective studies will provide important insights into how hLA mutations cause the defects seen in progeria.

This project will address some fundamental questions about the biology of human embryonic stem cells. Our overall, long-term goal is that the answers to these questions should help to hasten the development of novel stem-cell based strategies for treating human diseases. We will pursue the following aims. First, we will evaluate a range of antibodies directed against cytoplasmic intermediate filament (IF) proteins as markers to help identify specific classes of progenitor cells in cultures of human embryonic stem cells (hESC). We will use these antibodies to track the changing patterns of IF protein expression as cells progress to terminal differentiation. Second, we will use small interfering RNAs (siRNAs) to down regulate specific IF proteins to help determine the functional significance of the different IF protein expression profiles that emerge during development. Third, we will make a detailed study of the structure and function of nestin, a type IV IF protein widely-used as a neural progenitor cell marker, focussing on our recent finding that its C terminus interacts with the insulin degrading enzyme and may regulate its activity. The experiments will make use of an extensive collection of mono- and polyclonal antibodies raised against cytoskeletal IF proteins that we have developed and tested in our laboratory over the years. We will employ high resolution, confocal fluorescence microscopy to study fixed, immunostained cells. Time lapse studies will be made of living cells expressing various GFP-IF protein constructs and the dynamic properties of IF of varying protein composition will be studied using fluorescence recovery after photobleaching (FRAP). Public health relevance of this research: Human embryonic stem cells have the potential to give rise to all cell types of the human body, and thus have great utility for treating diseases such as Parkinsons' Disease and diabetes, as well as spinal cord injuries. The proposed research will advance our understanding of the cell biological events that take place as stem cells change into more highly differentiated cell types. This information is crucial for guiding cells down the right pathway so that they can eventually be used to treat patients.

Patients with acute lung injury are often placed on positive-pressure mechanical ventilation to improve gas exchange. However, mechanical ventilation may generate large shear forces during the cyclic closure and reopening of fluid filled alveoli, while the relatively spared, air-filled alveoli are cyclically overdistended. This may cause or worsen ventilator induced lung injury which may have a negative impact in patients with acute lung injury. In alveolar epithelial cells keratin intermediate filaments (IF) are the major structural proteins. Keratin IF are known to play an important role in maintaining the mechanical integrity of epithelial cells, and in vitro they are able to withstand a wide range of strain conditions without alterations in their structural integrity. In response to shear stress in vivo, IF have been shown to undergo adaptive changes in response to shear stress. This application proposes to study the response of keratin IFs to cyclic stretch, and cyclic and continuous shear stress in alveolar epithelial cells, and determine the effect of these changes in keratin IF on alveolar epithelial function via three interrelated specific aims. Specific aim#1. To determine whether cyclic stretch and/or shear stress-induced changes in keratin IFs alter alveolar fluid reabsorption in wild-type and keratin 8 knockout mice. Specific aim #2. To determine whether cyclic shear stress and/or stretch-induced changes (disassembly) in keratin IFs are mediated by protein kinase C-dependent phosphorylation in alveolar epithelial cells. Specific aim #3. To determine whether cyclic stretch and/or shear stress causes changes in ubiquitination and the regulated degradation of keratin IF networks by the ubiquitin-proteasome pathway in alveolar epithelial cells leading to lung cell injury. Completion of these studies will provide new insights into mechanisms responsible for cyclic stretch-and shear stress-induced alveolar epithelial lung injury.

In this Program Project Grant our overarching hypothesis is that alterations in the assembly states and mechanical properties of cytoskeletal IF, specifically the type III IF composed of vimentin (VIF), play important roles in regulating the micromechanical properties of cells in response to mechano- and chemosignalling. These studies are critically important as IF are major elements of the cytoskeletal system of mammalian cells and yet their specific functions in cell motility remain unknown. The 6 Project Leaders, and their research aims are as follows: R. Goldman, Northwestern University, will determine how the vimentin IF (VIF) system changes in response to mechanical and chemical signals, and will purify and characterize the vimentin precursors that form as VIF assemble and disassemble. V. Gelfand, Northwestern University will determine the relationship between VIF with microtubules and actin filaments, identify the molecular motors responsible for translocating VIF precursors, and investigate how VIF affect cytoskeletal dynamics and microtubule (MT) dynamics. G. Danuser, Harvard University, will test the hypotheses that VIF network formation involves a spatially distributed assembly line, that this process involves molecular motors, and that VIF network assembly modulates assembly of MT and microfilaments (MF). D. Weitz, Harvard University, will determine the micromechanical properties of in vitro asembled VIF networks prepared from purified vimentin, native VIF networks isolated from cells and in living cells. P. Janmey, University of Pennsylvannia, will determine how the VIF network contributes to the micromechanical properties of living cells and how this changes in response to substrate stiffness and external forces. P. Burkhard, University of Connecticut, will use biophysical techniques and X-ray crystallography to determine the structure and biophysical properties of the vimentin dimer and study the formation of VIF assembly intermediates. In all of these studies, the regulatory function of different posttranslational phosphorylation events will be examined.

? DESCRIPTION (provided by applicant): Aging involves the time-dependent accumulation of damage at the cellular level. While much is known about telomere shortening, genomic instability, oxidative stress, and mitochondrial malfunction, virtually nothing is known about age-dependent changes in cytoskeletal protein structure and function. An important protein modification associated with aging is the cumulative, non-enzymatic addition of sugar residues to proteins (i.e. glycation). Glycation is also a significant factor in diabetes, due to elevated glucose levels. It has recently been discovered that vimentin, a cytoskeletal intermediate filament (IF) protein, is a major target of glycation. The overarching goal of this proposal is to determine how glycation alters the normal structure and function of vimentin IF (VIF).This is of great importance to understanding the cellular basis of aging, as it is well known that IF are major factors in determining the mechanical properties of cells and tissues. It is also known that changes in the mechanical properties of cells accompany aging. In the exploratory (R21) phase of this project we will examine human skin fibroblasts to determine if the increasing age of the donor is accompanied by organizational changes in VIF networks. Studies will also be carried out to determine how experimentally induced glycation alters VIF network organization. Also during the R21 phase we will prepare antibodies specific for the regions that are glycated in vimentin and cDNAs expressing vimentin with amino acid substitutions for the relevant lysine residues. These reagents will be used for the R33 phase which will focus on determining the effects of glycation on the dynamics of vimentin IF, on their subcellular organization and on their specific roles in altering the micromechanical properties of cells. This will involve using both state of the art live cell assays; and in vitro analyses of the effects of glycation on vimentin assembly, structure, and mechanical properties. Some of these studies will be carried out with a group of collaborators that we have enlisted for this project. Finally, attempts will be made to correlate the changes in vimentin glycation with age related parameters of tissues obtained from young and old mice. The studies proposed will provide important new insights into how age-dependent protein glycation alters the organization and expression of the vimentin cytoskeleton and how these alterations impact the micromechanical properties of the cell.

PROJECT SUMMARY-- CORE A The Administrative Core will provide structure to facilitate the activities and meet the needs of the individual research Projects and Core B of the Program Project Grant. The principal missions of the Core are: to reduce administrative barriers within and between Projects so researchers can focus on advancing their research; foster team science by facilitating communication and regular meetings and interactions between the Projects and Core B within the PPG; evaluate progress in order to speed the pace and quality of research and to resolve any problems such as project overlap. The Core will also maintain a shared Dropbox that will be a central repository for protocols and generated data that can be accessed by all participants and facilitate preparation of reports for the NIH. !