Kenneth Williams, Ph.D. - US grants

Eucaryotes contain a wide variety of single-stranded (ss) nucleic acid binding proteins that are required in stoichiometric amounts for DNA replication, transcription, hetergeneous nuclear RNA (hnRNA) processing and translation. One of the best characterized ssDNA binding proteins that may play a role in DNA replication is the UP1 protein from calf thymus. Our major objectives are to better understand the function of UP1 and to examine its interaction with ssDNA at a molecular level. In addition, we would like to determine if UP1 shares certain structural features that are essential for binding single-stranded nucleic acids in common with other ssDNA and ssRNA binding proteins. UP1 has already been sequenced and its gene cloned, thus it is perhaps the only mammalian ssDNA bindign protein that is currently suitable for detailed structure/function analysis. Specifically, protein chemistry studies are planned to determine if several calf ssDNA binding proteins that are slightly larger than UP1 are, in fact, structurally related to UP1. Homologous ssDNA binding proteins from calf liver, mouse myeloma and HeLa cells will be sequenced so that highly conserved and therefore probably essential regions can be identified in UP1. Similar studies on proteins associated with hnRNA will determine if, as immunological studies suggest, these ssRNA binding proteins are structurally related to UP1. Physicochemical techniques such as fluorescence, chemical modification, photo-crosslinking, and NMR will be used to identify amino acids in UP1 that are involved in DNA binding and to determine how the presence of two 10,000 dalton globular domains in UP1 relates to its ability to bind ssDNA. High expression UP1 vectors will be constructed to help supply the large quantities of protein required by these studies and to allow future in vitro mutagenesis experiments. The function of UP1 will be examined by comparing the relative tissue distribution and ability of UP1 and UP1-like proteins to stimulate DNA polymerase alpha. Protein affinity chromatography will be used to identify those proteins that UP1 interacts with in vivo. These proposed studies are basic to our understanding of those physiological processes that require a single-stranded nucleic acid template.

We have established a protein chemistry facility that is overseen by the principal investigator and that now routinely does analyses for more than 100 different laboratories at Yale, the N.I.H. and 26 other universitiest. The Yale Facility has already demonstrated that it can successfully serve the needs of a diverse group of investigators whose problems require either peptide separation, amino acid sequence or analysis, or peptide synthesis. Amino acid sequencing of peptides isolated from enzymatic digests of sub-nanomole amounts of protein has, in particular, become one of the most essential services offered in our facility. To meet the exponentially increasing demand for this service, funds are requested to purchase an Applied Biosystems Pulsed- Liquid Protein Sequencer. The faster cycle time and higher repetitive yield of this instrument, compared to other automated sequences currently available, will allow our facility to both keep pace with the growing need for amino acid sequence data and to also significantly improve our capability to determine long stretches of sequence. Most of the protein and peptide sequencing done in our facility is directed at obtaining amino acid sequences that can serve as the basis for synthesizing oligonucleotide probes that can then be used to clone the respective gene. Once the cDNA clone is isolated it can then be sequenced. The difficulties involved in trying to automate DNA sequencing have so far discouraged our facility from offering DNA sequencing as a service. The Applied Biosystems DNA Sequencing System overcomes these limitations and offers the possibility of automatically sequencing 6,000 bases per day. This high degree of automation is clearly essential to adequately handle the increasing quantity of DNA sequencing that is required in eukaryotic systems. If funds are provided, these two instruments will immediately be brought to bear on a number of biomedically important research projects that range from inherited diseases to molecular parasitology to understanding the mechanism of synaptic transmission. Our facility has the technical expertise that is necessary to ensure that these instruments are maintained and operated continuously at peak efficiency. The potential impact of these two instruments on biomedical research at Yale, the N.I.H., as well as several other universities is indeed great.

This proposal requests instrumentation for a protein and nucleic acid chemistry facility at Yale University. The facility serves l38 investigators at Yale, the NIH, and 30 outside institutions. Thus, it serves as a regional resource center that meets the needs of a diverse group of investigators whose problems require either peptide separation, amino acid sequencing or analysis, or peptide synthesis. The facility is a clearing house for protein chemistry techniques and methodologies.

The single-stranded DNA binding proteins encoded by gene 32 of bacteriophage T4 (gp32) and ssb of E. coli (SSB) have served as prototypes for a class of proteins required in stoichiometric amounts in DNA replication, repair and recombination. In general, binding of these proteins imposes a conformation onto the ssDNA that is both resistant to nucleases and optimum to then serve as a substrate for other enzymes, such as DNA polymerases, involved in nucleic acid metabolism. Our research increasingly suggests that gp32 and SSB share many features in common with functionally unrelated eukaryotic nucleic acid binding proteins. Hence the function of the acidic COOH-terminus of these proteins appears analogous to the corresponding region in high mobility group proteins; as in the case of gp32 and SSB, hydrophobic interactions involving aromatic amino acid residues have also been implicated in the binding of heterogeneous nuclear RNA binding proteins. Finally, the zinc binding domain of gp32 appears to represent a nucleic acid binding motif shared by eukaryotic transcription factors as well as nucleic acid binding proteins encoded by murine leukemia and acquired immune deficiency viruses. To continue this line of research, we propose to more clearly define the role of the zinc ion in gp32 and to use an in vitro mutagenesis/1H-NMR/physicochemical approach to identify amino acids in gp32 and SSB that are directly involved in ssDNA binding. High priority will be given towards crystallizing gp32. Two new conditional lethal mutations in SSB will be characterized and limited proteolysis as well as photocross-linking experiments will be undertaken to determine how the tetrameric structure of SSB relates to ssDNA binding. A novel approach using a thermolabile SSB mutant and peptide competition experiments is planned to identify amino acids essential for SSB tetramer formation. This research will culminate in the synthesis and characterization of peptides corresponding to presumed functional domains in SSB and gp32 as well as to other related nucleic acid binding proteins. The structures of these synthetic peptides will then be varied in such a way as to test our ideas concerning the relationship of structure and function in nucleic acid binding proteins. These proposed studies are basic to our understanding of those physiological processes such as DNA replication, transcription and translation that require a single-stranded nucleic acid template.

This award will provide funds to a group of six scientists who direct microchemical facilities that serve large numbers of investigators at their own and other institutions. The funds will be used to obtain and distribute a synthetic peptide useful for measuring the sensitivity and accuracy of protein sequencing procedures at these and many similar facilities, and to increase cooperation and contact between such facilities. The overall aim of the project is to increase the quality of service provided by the facilities. The use of microchemical techniques for the sequencing and synthesis of protein and DNA now occupies an important role in modern biology. Because of the substantial investment in equipment and personnel required, such work is most often performed in central facilities that serve a large group of investigators. There are now nearly a hundred such facilities in the U.S. This award will help insure that the service provided by each is of a uniform high quality and is consistent with the state of the art in protein sequencing.

DESCRIPTION: Structure and function of A1 heterogeneous nuclear ribonuclear protein (hnRNP) will be studied. This is an abundant protein that binds pre-mRNA. There is disagreement in the literature over whether or not it binds sequence specifically. One goal of this project is to measure the sequence specificity of binding using an equilibrium titration method developed in this lab. The effects of posttranslational modifications, such as methylation and phosphorylation on binding, will also be studied. "A major effort....will be to solve the structure of an A1:RNA complex." Both x-ray crystallography and NMR methods will be tried. The effect on binding of the length of linker arms connecting two RNA binding domains will also be studied and compared with theory developed by Crothers. A yeast two-hybrid system will be used to identify proteins that interact with A1. The sequence specificity of RNA binding and the proteins that bind should provide insight into the function of A1, including how it modulates selection of alternative 5'-splice sites. Some conclusions may be relevant to the more than 100 other RNA binding proteins that use the same RNA binding motif as A1.

The W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University provides a full range of protein and nucleic acid analytical and synthetic services to nearly 600 principal investigators at Yale University, Cornell University, Columbia University, Harvard Medical School, Johns Hopkins Medical School, Mt. Sinai School of Medicine, Rockefeller University, Sloan-Kettering Cancer Center, Texas A&M University, and the universities of California, Illinois, Michigan, Texas and Virginia as well as at another 123 institutions. Each year this facility completes more than 10,000 services including the sequencing of ~750 proteins and peptides. The vast majority of the latter samples are HPLC-purified peptides that have either been eluted from MHC molecules or that have been isolated from tryptic digests carried out by the Keck Facility on SDS PAGE- separated proteins. Although all these peptides gave symmetrical absorbance peaks upon HPLC, amino acid sequencing demonstrated that nearly one-third of these samples were, in fact, either mixtures of peptides, artifact peaks resulting from Coomassie Blue and other reagents or, were lost prior to sequencing due to adsorption onto plastic surfaces. The ability of the requested laser desorption mass spectrometer (LDMS) to routinely determine masses on 50- 500 femtomole amounts of peptide will, in most instances, enable the prior identification of those two-thirds of the samples which contain only a single species in the expected mass range (for a 10-40 residue peptide) and which are therefore suitable for amino acid sequencing. In addition to preventing the needless sequencing of unsuitable samples, the resulting masses will confirm residues which would have been only tentatively assigned by Edman degradation and will establish the length of these peptides, which is of particular concern in characterizing MHC bound peptides. Other applications envisioned for LDMS are in characterizing posttranslational modificati ons of proteins and, possibly, as a quality control monitor of oligonucleotide synthesis. The other instrument that is requested, a multiple peptide synthesizer capable of the simultaneous synthesis and automated cleavage of 96 peptides, will need the increasing demand for large numbers of economically priced, high quality synthetic peptides. Currently, several laboratories at Yale are synthesizing as many as 100 peptides/year using a manual system of multiple peptide synthesis. Obviously, acquisition of a multiple peptide synthesizer would significantly increase research productivity in these laboratories and would bring the cost of these essential oligomers within reach of many more investigators. If funds are provided, these two instruments will immediately be brought to bear on a large number of important research projects that range from characterizing peptides bound to MHC molecules in T cell responses to increasing our understanding of the biochemical basis of autoimmunity, protein folding, posttranslational processing of a parathyroid-like hormone related protein, isotype switch recombination in activated B lymphocytes to biological applications of peptide nucleic acids. The high level of technical expertise in the Keck Facility virtually guarantees that these instruments would be expertly maintained and the extremely large number of Yale and non- Yale investigators served by this facility ensures that the positive impact of this equipment on biochemical research would be large and would extend well beyond Yale University.

9451456 Williams This project proposes to upgrade the teaching of NMR in organic chemistry, analytical chemistry & physical chemistry and facilitate undergraduate research, through the replacement of computer- simulations and photocopies with hand-on use of an FT-NMR Spectrometer by students. The organic chemistry lab will be divided into "mini-research" projects all of which will employ NMR for the characterization of products and the analysis of reaction mixtures. Students in Quantitative analysis will observe a single signal for 2 exchangeable protons and relate it to the concentration of the mixture. Instrumental Analysis students will determine the signal to noise ratio and resolution under various experimental conditions. In the Physical Chemistry lab NMR will be integral in experiments involving relaxation times, magnetic susceptibility and inorganic synthesis. Implementation of this project will considerably affect the level of education offered by the Chemistry Program at Francis Marion University.

9411519 K. Williams This Minority Research Initiation Planning Grant affords the Principal Investigator the opportunity to plan a project looking at legislative decision making within the Congressional Black Caucus and the Women's Caucus. Employing a principal-agent framework the Principal Investigator will develop a project looking at the relationship between caucuses and interest groups. The objective is to establish a way of assessing how effective caucuses are in passing legislation which has been drafted by associate interest groups.

electrospray ionization mass spectrometry; peptide chemical synthesis; biomedical equipment purchase; synthetic peptide;

Immediately following transcription heterogenous RNA is complexed with six proteins (A1,A2,B1,B2,C1,C2) that together form a 40S ribonucleoprotein particle that appears to be essential for pre-mRNA packaging, splicing and transport. These six proteins have apparent molecular weights on SDS PAGE that range from 32,000-44,000. All of these "core" hnRNP proteins share the same structural organization consisting of one (type C) or two (type A or B), 90 amino acid residue RNA binding domains at their NH2-termini followed by either a glycine-rich (type A or B) or acidic (type C) domain that contains sites for specific protein:protein interactions. The 90 residue hnRNP binding domain represents a common structural motif found in many other eukaryotic proteins including nucleolin, snRNP associated, sexual differentiation, neural development and poly A binding proteins. In each case this "mobile" RNA binding element is then linked to other domains that are specific to the function of each of the above proteins. The degree of sequence conservation at 21 positions distributed throughout this domain is sufficiently high that it provides on of the few instances where an "activity", RNA binding, can be inferred based solely on an amino acid sequence. Our laboratory and others have isolated cDNA clones and sequenced 5 of the core hnRNP proteins so that we can now address some more interesting and detailed questions. An NMR/X-ray crystallographic approach will be used to solve the three dimensional structure of the 90 residue domain at the NH2- terminus of A1. This prototypical RNA binding domain can be isolated in good yield following limited proteolysis of A1. Functionally important residues within this domain will be identified via a synthetic peptide analogue/in vitro mutagenesis approach. The nucleic acid binding properties of the A1 and type C hnRNP proteins will be examined with particular reference to the role of multiple RNA binding domains in A1 and the possible existence of high affinity chromatography in the absence and presence of synthetic peptide analogues will be used to identify peptide regions that are directly involved in hnRNP protein:protein interactions. In vitro particle reconstitution and nucleic acid binding studies will assess the role of post-translational modifications in hnRNP proteins. These studies are basic to understanding structure/function relationships in a family of eukaryotic, single-stranded RNA binding proteins that are intimately involved in RNA metabolism.

9514179 Williams Heterogeneous nuclear RNAs (hnRNA) are found in the nucleus in complexes with more than 20 hnRNP proteins. A1 hnRNP is unusual in that it appears to have several functions in addition to its structural role in hnRNP fiber formation. A1 interacts with intermediate filaments; modulates alternative 5'-splicing; can recognize a high affinity RNA target sequence that is similar to splice site consensus sequences; and facilitates annealing of complementary nucleic acids. The latter "activity" is controlled by phosphorylation of a critical serine residue in A1. A1 hnRNP also contains two conserved RNA binding domains at its NH2 terminus, and has an RGG repeat which may be a significant RNA binding domain. In these studies, the yeast two hybrid system will be used to identify other proteins that interact with A1 in vivo. Circular dichroism, fluorescence spectroscopy, and nuclear magnetic resonance will be used to probe the structure and nucleic acid binding properties of the RGG motif, and to locate sites of methylation within the domain. The methylase that carries out the posttranslational modification of the RGG will be cloned and analyzed. The results of these studies will significantly increase our understanding of a multi-functional hnRNP protein that plays a key role in pre-mRNA biogenesis. %%% Although all of the information contained within a cell is encoded in its DNA, most of the actual functions of cells and the organs and systems that they make up are carried out by proteins that are in turn composed of varying sequences of amino acids. Since proteins are made in the cytoplasm of the cell while the DNA that encodes them is in the nucleus, a molecule called messenger RNA (mRNA), structurally related to DNA, serves to transport the "message" for protein synthesis from the nucleus to the cytoplasm. In general, there is a unique mRNA for each protein. This project is directed at understanding how a particular class of nuclear proteins (hnRNP proteins) that are cri tical for the formation and transport of mRNA carry out their functions. In particular, how hnRNP proteins bind to mRNA and to other proteins will be investigated so that this critical step in gene expression will be better understood. ***

Traditional formal theoretical analyses of spatial voting applied to large elections have focused almost exclusively on elections in which voters make their choices simultaneously. In many cases this is an accurate portrayal and although voting may take place sequentially over a period of time, voters typically make their choices without the knowledge of the choices made by those who have voted earlier. However, there are other notable large elections in which groups of voters make choices with knowledge of the choices made by earlier voters in the same election. In this investigation, the researchers focus on a particularly interesting case of sequential voting: presidential primaries in the United States. In the presidential primary system voters in states that have later primaries know the outcomes of the primaries in earlier states when they make their choices. However, the nomination of the party is a function of the votes in both early and later states. Thus, voting that determines the nomination, because of the primary system, occurs sequentially. In this research the investigators focus on two ways in which sequential voting is different from simultaneous voting and the possibility that the voting process leads to different outcomes as a consequence. First, there is the possibility for information aggregation during the sequential voting process that is not possible under simultaneous voting. Second, when voting is sequential there is the unique possibility for candidates to withdraw from the election during the voting process. The current research extends an earlier investigation of information aggregation by examining the role of differences in risk preferences on the voting behavior and candidate selected. The research is then extends the theoretical model to consider the effects of candidate exit on these factors. The research consists of experimental tests of the theoretical predictions.

The circumstances that predispose to development of HIV and SIV encephalitis (SIVE) are not known; current evidence underscores the importance of the CNS macrophage. The role of CNS macrophages as a target of HIV and SIV infection, and their contribution to the development of CNS lesions is well established. However the identification of distinct monocyte/macrophage subpopulations that carry virus to the CNS and macrophage subsets within the CNS that are the target for infection is not resolved. We have used immunohistochemistry to define phenotypic differences between the resident parenchymal microglia, perivascular macrophages, and multinucleated giant cells. All of these cells express antigens of myeloid lineage including complement receptor 3 (CD11b), CD4, and CD68. Perivascular macrophages and multinucleated giant cells (MNGC) but not parenchymal microglia expressed LPS receptor (CD14) and LCA (CD45). Perivascular macrophages and parenchymal microglia upregulate or express de novo CD11b, CD40, and B7-2 (CD86) with higher level of expression of these antigens within the subcortical white matter. In several cases we observed that macrophages making up MNGC had either lost or down regulated CD4. Using this immunophenotype pattern to differentiate between brain macrophages and either double staining for viral protein or in situ hybridization for viral nucleic acid, we found that a population of perivascular brain macrophages is the major cell infected in the CNS of animals with SIVE. We further demonstrate based upon immunohistochemistry for CD14 and CD45 that the MNGC are derived from a population of perivascular macrophages and not the parenchymal microglia. These studies demonstrate the ability to differentiate between subpopulations of brain macrophages underscoring the primary role of the perivascular macrophage as an early and major target of SIV.

Over the past few years there has been tremendous advances in the sensitivity of mass spectrometers for peptide sequencing, the ability of parent ion scanning approaches to rapidly detect and identify a plethora of protein posttranslational modifications and the promise of gentle ionization techniques to extend mass spectroscopy into the realm of non-covalent protein interactions and folding. These techniques have been disseminated slowly because of the small number of laboratories with demonstrated track record in these new techniques. The current proposal is intended to bring state-of-the-art mass spectroscopy to Yale University through the purchase of a QTOF mass spectrometer. This instrument will support research in 10 institutions that do not currently have a comparable instrument. The projects include a broad spectrum of biological research areas including tRNA synthesis; pre- mRNA maturation; early neurogenesis; the control of cellular differentiation and growth; intracellular membrane transport; protein structure and dynamics; the generation of antigen receptor diversity; and the role of cell surface glycosylation in establishing cellular identity.

Experimental allergic encephalomyelitis (EAE), used as an animal model of multiple sclerosis (MS), can be induced by the adoptive transfer of CD4+ T cells that are specific for central nervous system (CNS) antigens While the mechanism(s) responsible for the pathological changes in EAE and MS are not fully defined, the traffic of lymphocytes through the CNS and antigen presentation within the CNS are required events for the development of inflammation Several studies using monoclonal antibodies (mAb) against known molecules expressed on T cells, endothelial cells, or CNS glial cells, have shown that it is possible to inhibit the induction of CNS inflammation in the EAE model In the present study we report a mAb TLD-4A2, obtained by immunizing mice with rat microglia, that recognizes a previously uncharacterized molecule The TLD-4A2 mAb stains resting and activated lymphocytes, as well as activated CNS endothelial cells and microglia The staining pattern of this antibody when screened on tissues from the CNS, lymph node, thymus, and spleen, and by immune- precipitation studies followed by one and two dimension western blots, suggest that it recognizes a novel antigen Treatment of rats with the purified 4A2 mAb resulted in either total inhibition of EAE or a significant delay in its induction and a milder clinical disease TLD-4A2 treatment also resulted in a decreased number T cells and monocyte/macrophages accumulating within perivascular cuffs and penetrating into the CNS parenchyma TLD-4A2 antibody apparently does not directly interfere with T cell function or viability as demonstrated by the ability to recover and stimulate CD4+ encephalitogenic peptide specific T cells from cervical lymph nodes of 4A2 treated animals, and from antibody containing T cell proliferation assays These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hemato genous cell traffic and the initiation of CNS inflammation

The identification of distinct monocyte/macrophage populations that carry virus to the CNS and subsets within the CNS that are the target for infection by HIV/SIV is not defined In this study we have used immunohistochemistry and combined immunohistochemistry with in situ hybridization to define which populations of brain macrophages are infected at peak viremia and in SIVE Perivascular brain macrophages and the resident brain macrophage, microglia, are both CDllb and CD68 positive The perivascular macrophages, but not microglia, are also positive for CD14, CD45, and CD86 Multi-nucleated giant cells (MNGC), when present, are CD11b and CD68 positive like parenchymal microglia and perivascular macrophages The MNGC also are CD14 and CD45 positive, but express variable to non-detectable levels of CD4 Using combined immunohistochemistry and in situ hybridization and/or double label immunohistochemistry for myeloid cell markers followed by gp120, we demonstrat e th at the majority of gp120 or viral RNA positive cells are CD14 positive perivascular macrophages These perivascular cells are the major cell infected within the CNS during peak viremia (2 weeks post infection) and in animals with SIVE Less than 1% of the infected cells are resident microglia based upon immunohistochemistry These data underscore the importance of the perivascular macrophage as the major target of SIV in the CNS

Experimental allergic encephalomyelitis (EAE), used as an animal model of multiple sclerosis (MS), can be induced by the adoptive transfer of CD4+ T cells that are specific for central nervous system (CNS) antigens While the mechanism(s) responsible for the pathological changes in EAE and MS are not fully defined, the traffic of lymphocytes through the CNS and antigen presentation within the CNS are required events for the development of inflammation Several studies using monoclonal antibodies (mAb) against known molecules expressed on T cells, endothelial cells, or CNS glial cells, have shown that it is possible to inhibit the induction of CNS inflammation in the EAE model In the present study we report a mAb TLD-4A2, obtained by immunizing mice with rat microglia, that recognizes a previously uncharacterized molecule The TLD-4A2 mAb stains resting and activated lymphocytes, as well as activated CNS endothelial cells and microglia The staining pattern of this antibody when screened on tissues from the CNS, lymph node, thymus, and spleen, and by immune- precipitation studies followed by one and two dimension western blots, suggest that it recognizes a novel antigen Treatment of rats with the purified 4A2 mAb resulted in either total inhibition of EAE or a significant delay in its induction and a milder clinical disease TLD-4A2 treatment also resulted in a decreased number T cells and monocyte/macrophages accumulating within perivascular cuffs and penetrating into the CNS parenchyma TLD-4A2 antibody apparently does not directly interfere with T cell function or viability as demonstrated by the ability to recover and stimulate CD4+ encephalitogenic peptide specific T cells from cervical lymph nodes of 4A2 treated animals, and from antibody containing T cell proliferation assays These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hemato genous cell traffic and the initiation of CNS inflammation

The driving forces for this proposal are the ever increasing number of genes that have been sequenced and whose protein products have been expressed in quantities sufficient for biophysical study of their interactions with other proteins, nucleic acids and ligands and the difficulty in accessing these technologies. We propose, therefore, to initiate a new section within the Keck Foundation Biotechnology Resource Laboratory (KFBRL) that will bring state-of-the-art biophysical technologies suitable for detecting and quantifying macromolecular interactions to hundreds of Yale and non-Yale users of the KFBRL so these investigators may transcend the primary sequences that are increasingly yielding to genome projects and more rapidly and fully answer the more interesting questions pertaining, for instance, to how the approximately 80,000 human proteins accomplish their functions. To begin this task we are requesting funds for an HPLC SEC/laser light scattering system. In support of this request are research projects from 17 investigators at 6 institutions that do not have access to the proven ability of this instrumentation to detect and quantify a wide array of protein: protein, protein: nucleic acid and other biomolecular interactions. The requested instrumentation is needed to advance our knowledge across a broad biochemical and biomedical front that includes but is not limited to developmental regulation, DNA damage recognition, blood pressure recognition., tumorigenesis, signal transduction, pre mRNA biogenesis, immune responses, and molecular mechanisms of exo and endocytosis. The strengths of this proposal include the extremely wide, diverse and productive user base that supports the KFBRL; the demonstrated ability of the KFBRL to bring biotechnology to bear on the hundreds of difficult research problems. That confront these users; the complementarity between the biophysical instrumentation requested and the instrumentation in the KFBRL; the technical and organization expertise to bring the requested instrumentation immediately "on-line" and to keep it operating optimally and continuously far into the future; and the extensive expertise and preliminary results that have been gained with an SEC/LS system that was on loan to the KFBRL for several months. These results demonstrate the potential for the requested instrumentation to make to major contribution to biomedical and biochemical research that will extend to many of the greater than 800 investigators from greater than 200 institutions that currently rely on the KFBRL for state-of-the-art biotechnological analyses and syntheses.

The identification of distinct monocyte/macrophage populations that carry virus to the CNS and subsets within the CNS that are the target for infection by HIV/SIV is not defined In this study we have used immunohistochemistry and combined immunohistochemistry with in situ hybridization to define which populations of brain macrophages are infected at peak viremia and in SIVE Perivascular brain macrophages and the resident brain macrophage, microglia, are both CDllb and CD68 positive The perivascular macrophages, but not microglia, are also positive for CD14, CD45, and CD86 Multi-nucleated giant cells (MNGC), when present, are CD11b and CD68 positive like parenchymal microglia and perivascular macrophages The MNGC also are CD14 and CD45 positive, but express variable to non-detectable levels of CD4 Using combined immunohistochemistry and in situ hybridization and/or double label immunohistochemistry for myeloid cell markers followed by gp120, we demonstrat e th at the majority of gp120 or viral RNA positive cells are CD14 positive perivascular macrophages These perivascular cells are the major cell infected within the CNS during peak viremia (2 weeks post infection) and in animals with SIVE Less than 1% of the infected cells are resident microglia based upon immunohistochemistry These data underscore the importance of the perivascular macrophage as the major target of SIV in the CNS

DESCRIPTION (Abstract of the application) The thrust of this application is to utilize the power of DNA microarray technology to advance knowledge on a wide front of research directed at understanding, diagnosing, treating, and preventing diabetes, digestive and kidney diseases. To rapidly bridge the large gap between what is theoretically possible and what is now available to NIDDK-supported investigators, we propose to establish an NIDDK Biotechnology Center that will build upon the foundation that is already in place in the form of the DNA Microarray Section of the Keck Foundation Biotechnology Resource Laboratory at Yale University. We propose to purchase, process, and array onto glass slides tens of thousands of sequence-verified mouse and human cDNAs annually; to select, process and array onto a custom "NIDDK" glass slide replicate sets of sequence-verified human, mouse, and/or rat cDNAs involved in diabetes, digestive and kidney diseases; to establish a Bioinformatics and Biostatistics Core that will be necessary to interpret and archive the avalanche of resulting data, and to construct the public Web interface needed to make this data available to the scientific community. In short, we propose to provide comprehensive, state-of-the-art microarray analysis services to all 60 NIDDK supported investigators at Yale University and to as many other Yale and non-Yale investigators as possible. The strengths of this proposal are manifold and include the high quality of the research; the instrumentation and support that are already in-place, the relevant expertise that is available in the Keck Facility and in the Center for Medical Informatics; the demonstrated ability of this group of investigators to process and prepare arrayable PCR products from 8,100 mouse cDNA clones and to print slides containing 16,200 features; and the close association between the Keck Facility and the Yale Liver and Kidney Centers. In 1998 the Keck Facility completed 76,348 protein, peptide, and nucleic acid syntheses and analyses for 311 Yale Principal Investigators from 49 departments and sections and 500 non-Yale investigators from 212 institutions. Over the next three years we propose to provide these same users with the ability to economically and rapidly quantify the level of expression of nearly 50,000 sequence verified mouse and 50,000 human cDNAs and to establish an NIDDK Biotechnology Center that will continue to provide DNA microarray analyses and support far into the future.

EIA-0002217
Kim, Jung H.
North Carolina A&T State University

MII: Infrastructure for Intelligent Mobile Information Systems

This proposal involves the creation of an infrastructure for research and graduate education in Intelligent Mobile Information Systems (IMIS) at North Carolina A&T State University. The infrastructure will support the efforts by the Department of Computer Science, the Department of Electrical Engineering and t he College of Engineering to enhance the effectiveness of North Carolina A&T State University as a pipeline to graduate study by African-Americans in computer science and computer engineering. The proposal outlines an aggressive mentoring program oriented toward encouraging African-American students to continue to graduate school. This will involve identifying undergraduate students with high academic performance and recruiting them to participate in the IMIS research program.

This proposal requests funds to purchase a PE Biosystems Voyager DE- PRO MALDI mass spectrometer together with advanced robotics and software. This system is especially configured to allow high throughput identification of proteins isolated from ID and 2D polyacrylamide gels and multiplexed genotyping of single nucleotide polymorphisms (SNPs). The robotics accessories include an automatic gel spot cutter, an automated protein digester, and a robot to deposit samples of either protein digests or oligonucleotides onto a MALDI target plate for automated protein identification or SNP analysis. This instrumentation package will substantially expand and improve an existing service for protein identification already provided by the Keck Foundation Biotechnology Resource Laboratory (KFBRL). It will also be the basis for a new service, namely the identification of SNPs. With several genome projects completed or nearing completion emphasis is rapidly being directed towards proteomics including understanding protein function and its control by posttranslational modifications and large scale protein expression studies. A critical feature of proteomics is the need for rapid, cost-effective, and accurate protein identification - which we believe can be met by the requested instrumentation. Similarly there is rapidly increasing interest in the identification of single nucleotide polymorphisms particularly as to how these polymorphisms relate to diseases and again, this grant application seeks to meet this critical need. The strengths of this proposal include the extremely wide, diverse and productive user base that supports the KFBRL; the demonstrated ability of the KFBRL to bring biotechnology to bear on the hundreds of difficult research problems that confront these users; the technical and organizational expertise to bring the requested instrumentation immediately "on-line" and to keep it operating optimally and continuously far into the future; and the extensive expertise and preliminary results that have been gained with a DE-PRO system that was on loan to the KFBRL. These results demonstrate the ability of the requested instrumentation to make a major contribution to biomedical research that will extend to many of the >800 investigators from >200 institutions that currently rely on the KFBRL for state-of-the-art biotechnological analyses and syntheses.

DESCRIPTION (provided by applicant): The tenet of this proposal is that FF-ICR mass spectrometers are sufficiently advanced and the required methodologies are sufficiently well proven to place such an instrument in a core facility like the Keck Laboratory for the purpose of bringing quantitative protein expression and phosphorylation analysis within reach of 346 Yale and 633 non-Yale principal investigators from 224 institutions who used this Laboratory in 2000. With several genome projects completed or nearing completion, interest is rapidly being directed towards proteomics--including protein expression and understanding protein function and its control by post-translational modifications. A major goal of the Keck Laboratory is to begin to bridge the large gap between what is theoretically possible with FT-ICR mass spectrometry and what is actually available to most NIH-supported investigators. Rather than only reading about break through protein expression methodologies like isotope coded affinity tags, we would like many more NIH-supported investigators to be able to bring these technologies to bear on the challenging biomedical problems they seek to solve. The strengths of this proposal include the diverse and productive user base that supports the Keck Laboratory; the demonstrated ability of this Lab to bring mass spectrometric and other technologies to bear on hundreds of difficult research problems that confront these users; the technical and organizational expertise to bring the requested instrumentation "on-line" and to design, optimize, and implement well defined services; the long term stability of the Keck Lab and its proven ability to continually operate and maintain sophisticated biotechnological instrumentation; the rapid progress of the Keck Microarray Resource - which provides an excellent opportunity to compare mRNA versus protein expression analysis; and the extensive support and "infrastructure" which is in place to ensure the success of the proposed research. This support ranges from the MS and separation technology expertise needed to ensure maximum benefit is derived from the requested instrumentation to the bioinformatic, computer programming, and biostatistical expertise needed to ensure the resulting data is properly interpreted and is archived such that it can be readily retrieved, subjected to further analysis, and cross-correlated with other relevant data. We believe the requested instrument would make a major contribution to biomedical research that would extend far beyond Yale University.

Sequential voting among collegial courts during deliberations is a phenomenon that is fairly consistent among different types of court systems. However, the method of sequential voting varies widely from court to court. The U.S. Supreme Court uses a seniority sequential voting system during the certiorari vote and the original votes of merits, in which the most senior justice votes first, and so on to the least senior member. U. S. state courts use a multitude of sequential voting mechanisms which range from the seniority system described above, to a reverse seniority system (in which the least tenured justice votes first and so on to the most tenured justice voting last), to a rotation or random seniority system (in which the order of voting is determined randomly). Sequential voting processes affect the level of uncertainty judges confront when they cast their vote on a case. The amount of uncertainty that judges confront when they cast their vote is relevant because this influences a judge's ability to vote strategically. Specifically, it may influence a judge's decision to join the majority coalition. Being in the majority has two benefits for judges: an individual-level benefit and a collective benefit. Individually, in certain judicial systems, a judge who is in the majority has he opportunity to draft the initial opinion where bargaining occurs. Collectively, the formation of supermajorities is attractive since the legitimacy of the court's judgment is heightened when there is consensus about a legal issue. To test the effect of voting sequence, the researchers propose a series of laboratory experiments. Laboratory experiments also allow control for relevant parameters, such as the type of voting system, risk aversion of the judges, size of the court, the level of uncertainty, and the saliency of the issue under consideration. This research will provide evidence about which judge is more likely to vote strategically given his or her position in the voting queue, and the rate of strategic voting that should occur in each of the court systems. The experiments should also provide some useful results about the social welfare properties of each system.

LeRoy Peterson and Kenneth Williams, Francis Marion University, and Hans-Conrad zur Loye, University of South Carolina, are jointly supported for research in organic-inorganic hybrid materials. They will prepare and characterize new ligands derived from bipyridines, incorporate them into new organic-inorganic hybrid materials, investigate new organically templated polyhalobismuthates, and synthesize new II/VI and III/V supertetrahedral clusters.

These hybrid materials often have porous structures that can support catalysts or selectively separate gases. They also show interesting electronic and optical behaviors. This project also builds a collaborative project between a primarily undergraduate institution and a Ph.D. granting institution in South Carolina. Undergraduates from Francis Marion University will conduct cutting-edge materials chemistry research and interact with faculty and graduate students at the University of South Carolina. This project will also involve female and African-American students, contributing to a diverse and well-trained future workforce.

[unreadable] DESCRIPTION (provided by applicant): The tenet of this proposal is that progress in biomedical research is increasingly dependent upon high throughput biotechnological advances (e.g., automated DNA sequencing, DNA microarray analysis of mRNA expression, mass spectrometric-based proteome profiling, and several emerging technologies for SNP genotyping) that are increasingly limited by the inability of the "personal computers" and small departmental clusters available to Yale investigators to adequately and timely analyze the enormous volume of the resulting data. While recent biotechnological breakthroughs have brought us to the exciting threshold of systems level biomedical research, to take advantage of these technologies Yale investigators will need far more powerful computers than are now within their reach and a commensurate level of technical programming and systems administration support. The proposed Center for High Performance Computation in Biomedicine at Yale would utilize the requested instrumentation and would serve as a focal point for the staff and physical infrastructure needed to bring computer science to bear on challenging, yet amenable problems that stand in the way of biomedical research. The strengths of this proposal include the very diverse and productive investigator user base that would support the proposed Center, the demonstrated ability over the last 24 years of the PI and the Keck Laboratory he oversees to continually operate and maintain sophisticated biotechnological instrumentation and to bring it within reach of hundreds of Yale and non-Yale researchers, the extensive infrastructure and expertise that is available to bring the requested instrumentation on-line and to oversee and support its continuous use, and the careful planning and very firm support of both Yale University and its School of Medicine to ensure that this proposal represents a coordinated and well conceived institutional response to the challenge of providing the high performance computing needed to drive biomedical research forward. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to maintain and to continuously improve the NIDA Neuroproteomics Center at the Yale University School of Medicine. Faculty members from Yale and other institutions (Univ. Chicago, Univ. Connecticut, Rockefeller Univ., Stanford Univ., Univ. Texas) with established records of research into the molecular actions of psychostimulants and psychotropic drugs, as well as of other basic aspects of neurobiology, will continue to work together with members of the W.M. Keck Foundation Laboratory to create the NIDA Neuroproteomics Center. The main goal of the Center is to apply high-throughput, state-of-the-art proteomic methods to analyze adaptive changes in neuronal protein expression and regulation that occur in response to drugs of abuse. In addition, the Center will provide training in proteomics technologies for members of the Center, and will apply new proteomics technologies to answer biological questions related to the actions of drugs of abuse. Under the direction of Dr. Kenneth Williams (Center Director) and Dr. Angus Nairn (Co-Investigator, Psychiatry) in the Administration Core, the Center will include a Protein Profiling and Identification Core, a Protein Post-translational Identification and Profiling Core, and a Targeted Proteomics Core. Lipid and Biophysics analyses will be included in the Protein Profiling Core. Finally, a Protein Database, Biostatistics, Bioinformatics and a High Performance Computing Core will provide essential support that will positively leverage the value of each of the proteomic technology cores. The behavioral adaptations that accompany drug addiction are believed to result from both short and long-term adaptive changes in brain reward centers. Thus, exposure to drugs of abuse regulates intracellular signaling processes and in turn results in alteration of gene expression, protein translation and post-translational modifications of proteins. Repeated exposure to drugs of abuse leads to long-term, stable alterations in these signaling systems that are critical for the changes in brain chemistry and structure of the addicted brain. The Center, through its multidisciplinary and collaborative organization, brings together Yale faculty and their collaborators to gain a deeper insight into the mechanisms of neuronal signaling processes and into how drugs of abuse alter these signaling mechanisms. Specific goals of the research supported by the Center will include analysis of the actions of the psychostimulants, cocaine and amphetamine, nicotine and opiates. Newly developed methods will be applied to the study of protein phosphorylation and other post-translational modifications of neuronal proteins, as well as to the targeted analysis of neuronal proteins implicated in the actions of drugs of abuse. PUBLIC HEALTH RELEVANCE: Drugs of abuse usurp and modify communication between neurons, resulting in stable changes in the expression and activities of proteins (the "neuroproteome") required for normal neuronal function. Improving our fundamental understanding of how drugs of abuse alter the neuroproteome will provide deeper insight into the process of drug addiction and thus help in the design of new therapeutic strategies.

Specific Aim. Many tasks in molecular biology and genetics involve repetitive processes or assays. The performance of many of these is best accomplished with specialized instrumentation and dedicated staff in a core facility. In the context of the Yale SCOR in Hypertension, there are a number of such bench tasks and analysis tools that benefit from such core facilities. The Specific Aim of the Molecular Core is to provide infrastructure support for the following activities. 1. Oligonucleotide synthesis 2. DNA sequencing 3. Microsatellite genotyping 4. SNP identification and genotyping 5. Preparation and archiving of genomic DNA 6. Analysis of linkage and association 7. Bioinformatics

Carolina Cyber Defender Scholarship program is jointly offered by the University of North Carolina at Charlotte, NC A&T State University, and Johnson C. Smith University. This program prepares students to become intellectual leaders in cybersecurity. Students can pursue baccalaureate, masters as well as doctoral studies. Scholarship students engage in rigorous academic programs that emphasize both theory and practice of cybersecurity and incorporate research and experiential learning.

Each scholarship student is required to contribute to one of many cutting edge research projects led by experienced professors and develop capabilities in critical thinking, problem formulation and problem solving. The program has a rich set of extracurricular activities including frequent seminar speakers from industry and academia as well as participation in national and international hands-on cybersecurity competitions. Professional development, including effective communication as well as leadership, is emphasized. The curriculum covers a broad spectrum of cybersecurity topics including network security, configuration security, penetration testing, software assurance, data privacy, mobile and wireless security, usable security, access and identity management, computer forensics, policy and governance, security risk management, and cryptography.

The scholarship program is aimed at creating a cadre of diverse cyber security professionals to address the nation's critical need for talent in key national security as well as critical infrastructure protection positions.

Growing evidence indicates that incorporating hands-on exercises, such as cyber games and interactive simulations, increases student interest in Information Assurance (IA) studies and enhances their learning experience. Cyber games are highly interactive hands-on exercises in which students are asked to build IT network infrastructures and services while simultaneously managing and defending against realistic cyber attacks.

As part of an ongoing collaboration, UNC Charlotte and NC A&T State University (an historically Black institution), are developing faculty development workshops where participants will learn how to incorporate these games and interactive simulations into their classes. The workshop materials, based on successful experiences as well as incorporating best practices reported in literature, are being designed to satisfy a six key objectives: (i) targeting faculty members with traditional Computer Science backgrounds with limited system administration experience, (ii) building their instructional capability to provide students with cyber games and interactive simulations, (iii) keeping institutional resource commitment at a manageable level through the use of virtualization techniques, (iv) engaging and motivating students through challenging exercises based on realistic business scenarios, (v) increasing student interests in pursing IA studies through building their confidence in hands-on problem solving skills, and (vi) promoting students to work in teams.

Workshop participants include current IA faculty or those from institutions with a strong interest in, and with appropriate institutional commitment to introducing hands-on experiences during the academic year following each workshop. Special effort is being made to include faculty from institutions serving underrepresented populations.

Advanced Instrumentation; All Sites; Articulation; Arts; Bears; Biological; Biotechnology; Budgets; Businesses; Collaborations; Communication; Complement; Complement Proteins; Computer Instrumentation; Computers and Advanced Instrumentation; Data; Data Storage and Retrieval; Discipline; Ensure; Evaluation; Fostering; Foundations; Funding; Future; Generations; Goals; Grant; Guidelines; Human Resources; Individual; Infrastructure; Instrumentation, Other; Internet; Investigators; Joints; Laboratories; Manpower; Method LOINC Axis 6; Methodology; Methods and Techniques; Methods, Other; NIDA; National Institute of Drug Abuse; Nerve Cells; Nerve Unit; Neural Cell; Neurobiology; Neurocyte; Neurons; Neurosciences Research; On-Line Systems; Online Systems; Participant; Pilot Projects; Play; Productivity; Programs (PT); Programs [Publication Type]; Progress Reports; Proteomics; Qualifying; Range; Recruitment Activity; Regulation; Reports, Progress; Research; Research Infrastructure; Research Personnel; Researchers; Role; Sampling; Scientist; Services; Shapes; Solid; Techniques; Technology; Thinking; Thinking, function; Training; Training Activity; Training of Investigators; Ursidae; Ursidae Family; WWW; Work; Writing; abused drugs; base; cost; data retrieval; data storage; drug of abuse; drugs abused; drugs of abuse; improved; instrumentation; interdisciplinary approach; investigator training; member; neurobiological; neuronal; new technology; online computer; outreach; personnel; pilot study; programs; protein expression; recruit; response; skills; social role; web; web based; world wide web

The University of North Carolina at Charlotte (UNCC) and North Carolina Agricultural and Technical State University (NCAT) propose to continue and expand the Carolina Cyber Defender Scholarship Program supported by the CyberCorps(R) Scholarship for Service (SFS) program. The project will prepare highly-qualified cybersecurity professionals for entry into the government workforce, and will have an immediate impact on the information assurance and forensics capabilities of the federal workforce by developing well-trained cybersecurity professionals. The project will support community college students at the Forsyth Tech Community College (FTCC) through early mentoring and advising activities before their transfer to either UNCC or NCAT through established 2+2 pathways. Further, the proposed collaboration will help NCAT obtain CAE-R designation in research and contribute towards graduating under-represented cybersecurity researchers at the PhD level.

This project involves students in cybersecurity research and hands-on activities guided by a large team of experienced faculty, with sixteen current research initiatives and active funding from government agencies and private corporations. The core expertise is focused on Secure Software Development; Secure Network Administration; Cyber intelligence and Analytics; and Cloud Security. The NSF supported Industry/University Collaborative Research Center on Configuration Analytics and Automation provides a rich environment where students work on cutting-edge cybersecurity challenges and integrate with classroom education, which has generated many innovations, including a U.S. patent awarded to a team of SFS students at UNC Charlotte. The proposed program is designed to provide students with a solid foundation of cybersecurity, exposure to real-world cybersecurity problems, and hands-on experiences through competitions so that upon graduation they are well-prepared to meet the challenges of defending the nation's critical infrastructure. NCAT is implementing a new Process Oriented Guided Inquiry Learning (POGIL) approach for teaching cybersecurity. POGIL is based on the process of guided inquiry - a learning cycle of exploration, concept invention and application based on carefully designed materials that students use to guide them to construct cybersecurity knowledge.

Three universities, North Carolina A & T State University, the University of North Carolina at Charlotte, and the University of Tennessee at Chattanooga, are collaboratively developing faculty expertise in information assurance (IA) and computer security through two faculty development workshops. Covering two years, the workshops involve thirty-six faculty members from two-year, four-year, and minority colleges and universities. The workshops are helping participants to integrate IA labs and case studies into the curricula at the participants' home institutions. The workshops are designed to increase the faculty members' expertise and their home institution's capacity to produce a diverse workforce in the area of information assurance. Personalized instruction plans and follow-up assistance are provided to faculty participants. Besides building faculty expertise, the project is improving partnerships in information assurance education.

This project is improving overall information security and critical infrastructure protection in the U.S. by producing a larger and more diverse IA workforce with better IA skills. The curriculum materials disseminated through the workshops can be adopted across multiple disciplines such as computer science, software engineering, information technology and business, and are being made available to a broader audience through conferences, publication, and a dedicated website. The project is building a community of security education experts across multiple disciplines. Broad coverage of IA topics and the introduction of innovative teaching techniques are attracting faculty participants with diverse backgrounds.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Symptomatic abnormalities of peripheral nerve function are among the most common complications of HIV-1 infection as HIV-sensory neuropathy (HIV-SN). There are presently no effective therapies for HIV-SN, and there are few animal models of HIV-SN in which candidate therapeutic agents can be tested. The CD8+ T lymphocyte depleted SIV-infected rhesus model, which is highly reproducible and results in rapid disease with consistent pathology will be used. Given that the CD8+ T lymphocyte depletion SIV infection model has close similarities to HIV-induced CNS disease and a high incidence of SIV encephalitis (SIVE) (~85%), we hypothesize that PNS lesions also develop at a high frequency, thus providing a model for the study of the mechanisms of HIV-SN disease. The main goal is to use the CD8+ T lymphocyte depletion SIV infection macaque model to determine the role of macrophage activation, monocyte traffic and virus driving peripheral nervous system (PNS) disease. The specific aim for this project is to define the role of resident macrophage activation, monocyte infiltration and virus in the development of HIV-induced PNS injury using CD8+ T lymphocyte depleted SIV infected rhesus macaques.

Mobile platforms have become extremely popular among users and an important platform for developers. New privacy and security issues arise with the prevalence of mobile computing since mobile devices often store tremendous amount of personal, financial and commercial data, and therefore attract both targeted and mass-scale attacks. To meet the growing demand for mobile computing and security professionals, it is vitally important to provide education in mobile computing and security to students in computer science and other related disciplines. The overall goal of the project at North Carolina A&T University is to address this workforce need by developing course modules in mobile computing and security. The modules will be integrated into existing Computer Science courses such as computer programming, software development, operating systems, and information assurance courses. Each course module includes a tutorial, presentation slides, hands-on labs and/or case studies, and test questions. To actively disseminate these new course materials, two faculty summer development workshops will be offered, as well as a mobile programming contest for undergraduate students.

This project seeks to strengthen the Computer Science program at North Carolina A&T University by updating the Computer Science curriculum to address state-of-art technology. Students will be more competitive in the workforce and well-prepared for graduate studies in the areas of mobile computing and security. Therefore, this project will increase the education and career opportunities of African American students. This project will also develop and strengthen the University's Center for Cyber Defense, a Center of Academic Excellence in Information Assurance Education designated by the National Security Agency and the Department of Homeland Security, by expanding its education and research programs. Societal benefits of this project include increased educational materials and improved opportunities for training students to address mobile computing and security challenges.

We propose to continuously improve the Yale/NIDA Neuroproteomics Center that brings exceptionally strong Yale programs in proteomics and signal transduction in the brain together with neuroscientists from 8 other institutions across the U.S. to create a national resource that will collaborate to identify adaptive changes in protein signaling that occur in response to substances of abuse. Twenty-three faculty with established records of highly innovative research into the molecular actions of psychoactive addictive drugs, as well as of other basic aspects of neurobiology, will work together in a unique synergy with the Keck Foundation Biotechnology Laboratory to create the Center, whose theme is ?Proteomics of Altered Signaling in Addiction?. The Center will use cutting edge proteomic technologies to analyze neuronal signal transduction mechanisms and the adaptive changes in these processes that occur in response to drugs of abuse. With Co-Directors Angus Nairn (Psychiatry) and Kenneth Williams (Mol. Biophys. & Biochem.) in the Administrative Core, the Center includes Discovery Proteomics (DPC) and Targeted Proteomics (TPC) Cores. Biophysical technologies from the DPC will extend protein profiling analyses into the functional domain while lipid analyses from the DPC will leverage proteome level analyses to provide an increasingly biological systems level approach. A Biostatistics and Bioinformatics Core (BBC) that includes high performance computing and the Bioinformatics Support Program in the Yale Medical Library will provide essential support that will leverage the value of each of the proteomic technology cores. A Pilot Research Project Core is a cornerstone in our efforts to encourage strong mentoring relationships that will help attract and train future outstanding scientists. Behavioral adaptations that accompany drug addiction are believed to result from both short and long-term adaptive changes in brain reward centers. Thus, exposure to drugs of abuse regulates intracellular signaling processes that alter gene expression, protein translation, and protein post-translational modifications. Repeated exposure to drugs of abuse leads to stable alterations in these signaling systems that are critical for the changes in brain chemistry and structure of the addicted brain. The Center's research goals include analysis of the actions of cannabis, cocaine, nicotine, and opioids on these intracellular signaling pathways in brain reward areas and development of methods that enable proteomic analysis of the single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. Targeted and data- independent MS analyses of signaling proteins implicated in the actions of drugs of abuse will be used to analyze the impact of substance abuse on the neuroproteome with motif-based, ?Middle-down? MS/MS, and other novel approaches being used to study protein post-translational modifications and to uncover the interactomes of key proteins. A major initiative of the BBC will be to develop novel methods for deep integration of genomic, transcriptomic, and proteomic data with brain region, cell type and allele-specificity.

The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to quantitate and validate potential protein biomarkers initially identified from the protein profiling analyses carried out by the Discovery Proteomics Core (DPC), genomic studies, including the use of neuronal cell type-specific transcriptional and translational profiling, or the literature. These analyses will include relative and absolute quantitation of targeted protein levels or alterations in their post-translational modifications that have been implicated in model and other organisms as adaptive changes that occur in response to drugs of abuse. To address the huge level of cellular and sub-cellular heterogeneity in the central nervous system, another objective is to develop the targeted workflows needed to analyze subcellular organelles and sub-proteomes from the single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. In Aim 1 we will integrate high resolution Data-Independent Acquisition (DIA) into our large scale targeted proteome assays using fractionated Data-Dependent Acquisition (DDA) peptide libraries generated by the DPC from mouse, rat, and human brain regions. DIA analysis has the potential to change the proteomic landscape since each sample only needs to be run once, and then all peptide fragments can retrospectively be identified and quantified. In Aim 2 we will develop targeted, highly sensitive DIA assays to examine the proteomes of specific neuronal sub-types and their organelles (e.g., nuclei, synaptic vesicles), sub-cellular fractions or partially enriched samples (e.g., PSD) and then use Parallel Reaction Monitoring (PRM) to validate the most significantly differentially expressed proteins. In support of this Aim, we will develop improved methodologies for protein profiling of the limiting amounts of protein that can be obtained from the LCM and FACS-based approaches needed to isolate neuronal sub-types. Since eukaryotic proteins almost always are assembled into multiprotein complexes in vivo, Aim 3 will leverage the data from our targeted assays by implementing improved proximity biotinylation technologies for identifying protein interactomes for those proteins that are found to be most differentially expressed by our targeted assays. To further enhance the interpretation of the data from our targeted proteome analyses, in Aim 4 we will implement a Puromycin-associated Nascent CHain Proteomics (PUNCH-P) assay for translatome profiling. Since under steady state conditions protein levels are largely determined by transcript concentrations, it will be of considerable interest to compare the approximately steady state protein levels determined by our DIA analyses with the nascent protein levels determined by our PUNCH-P analyses before and after exposure to drugs of abuse. In Aim 5, we will continue to train Neuroproteomics Center members in targeted mass spectrometric techniques including experimental design, sample preparation and handling, digestion protocols, interpretation of PRM and DIA spectra so they can optimally utilize the advanced technologies available in the Center.