David Miller - US grants

Research at the University of Wisconsin-Milwaukee (UWM) Field Station was begun in 1967. Since then the UWM Field Station has grown steadily in research use and productivity. The attractiveness of the Station for research can be attributed to the quality of its natural areas and facilities, the baseline information available, ongoing research, its location, and the level of assistance and on-site support provided by the staff. Located near Milwaukee, Madison and Chicago, the 850 ha research area includes one of the finest remaining examples of beech-maple woods in Wisconsin, nearly all of the predominant wetland vegetation types found in northwestern North America, old fields and agricultural fields. Long-range plans for the Field Station research program emphasize: 1) development of the Station and the Cedarburg Bog as a wetlands research center, 2) maintenance of its strong program in avian behavioral ecology, and 3) experimental plant ecology, population biology, and ecological genetics. The Station has an extensive and diverse long-term data base that has been developed over 20 years. Expansion of the data base and enhancement of its utility and accessibility are currently being stressed. This project will support replacement of the Station's outdated and increasingly unreliable weather station in order to maintain and improve the quality of the data base and to fulfill research program plans.

A convenient source of radionuclides used in biomedical applications is often a radionuclide generator. In such a system an efficient radionuclide separation is used to periodically isolate a short-lived parent. The stringent requirements imposed on such systems, especially for nuclear medicine applications, have limited the number of radionuclide generators that have ben successfully developed and made commercially available for routine use. The development of a new radionuclide generator, based on 118Te/118Sb, is the main objective of this research project. The 3.5-min 118Sb daughter decays principally by positron emission, and has a potential use in first- pass angiography. Column chromatography procedures will be developed to achieve the necessary separation of the 118Sb from the 118Te parent. Activated carbon, along with other commercially available adsorbents, and in combination with a variety of mobile phases, will be systematically evaluated for conditions of optimal 118Sb elution and minimal 118Te breakthrough. The stability and reproducibility of the experimentally determined optimal generator conditions will also be evaluated. The 118Te parent will be produced by proton bombardment of antimony targets, and radiochemically separated using existing procedures. A careful measurement of Sb(p,x) cross sections in the proton energy range of 28-46 MeV will be made using stacked foil and activation techniques. This cross section information will permit optimization of parent production in thick targets, as well as serve as a test of models used to calculate excitation functions.

DESCRIPTION (Adapted from applicant's abstract): The mammalian brain is composed of billions of neurons. Each of these highly specialized cells is estimated to make hundreds, if not thousands, of synaptic connections to other neurons. The staggering complexity of this network parallels the remarkable intellectual and creative abilities of the human mind. Only a fraction of these circuits have been defined at the level of individual cells, however. And it seems unlikely that we can even begin to understand the origins of these complex behaviors until a much more detailed wiring diagram of the vertebrate brain is known. The purpose of this proposal is to develop microscopic methods that can reveal synapses between specific neurons. We have chosen to test the reliability of these approaches in a model organism with a simple, well defined nervous system. In the nematode C. elegans the nervous system is composed of precisely 302 neurons. The morphology and synaptic connectivity of each of these neurons has been defined by serial section electron microscopy. Thus, we have a complete circuit diagram for the entire C. elegans nervous system. Our goal is to test the feasibility of observing these neurons and their specific synapses in intact animals in the light microscope. Because the methods that we will develop would obviate the need for EM reconstruction, our approaches should greatly facilitate efforts to identify specific mutations that perturb the wiring diagram of this simple nervous system and thereby lead to the cloning of the genes that control synaptic specificity. In addition, it is expected that the methods that we will develop can be utilized in the future to define the detailed neuroanatomy of other, more complex nervous systems. We will develop methods that can be utilized to observe specific subsets of these connections in the light microscope. Our strategy exploits the following tools: 1) green fluorescent protein (GFP) and epitope tags to label synapse-specific proteins and the neurons in which they are expressed; 2) cloned gene regulatory regions to drive expression of these marked proteins in specific neurons; 3) confocal and multiphoton excitation microscopy to resolve these labeled neuronal processes and their synaptic connections.

We calculate normal modes of alpha-lytic protease, both for the wild type and for the M190A mutant, in order to investigate how internal vibrations in the enzyme might contribute to a characteristic feature of its catalytic specificity. While the wild type is highly specific for small substrates in its primary specificity pocket, the M190A mutant has a much broader specificity, catalyzing both large and small substrates. We hypothesize that for the atoms lining the walls of the specificity pocket, the wild-type normal modes have a more symmetric character, with the walls vibrating in phase, and the size of the pocket remaining relatively fixed. This is in agreement with previous X-ray crystallographic results. In contrast, we expect that the mutant modes have a more antiymmetric character, with the walls vibrating out of phase, and the pocket able to expand and contract. These results would suggest that the internal vibrations of a molecule may play a role in determining both binding and catalytic specificity. The use of resources has been very important for the detailed graphical analyses of our normal-mode results. An example of the types of calculations we make is that of the changes in active-site volume and surface area that take place as the protein structure is perturbed along each of its normal modes. These computationally-intensive calculations are made using an algorithm included in the Computer Graphics Labroatory's MidasPlus package. In addition to such calculations, we have used the CGL extensively for detailed three-dimensional rendering of both our crystallographic and modeled protein structures.

An ?-secretase inhibitor differentially affects the formation of ?-amyloid peptides in cultured cell lines additional evidence for multiple processing pathways. The amyloid plaques that accumulate in the neuropil and cerebral blood vessels of Alzheimer disease victims conain an array of peptides, which are cleaved from the juxta-membrane and transmembrane domains of the ?-amyloid peptide presursor (APP). The enzyme that cleaves APP(695) at Met-597 to yield Asp-1 of A? has been denoted ?-secretase, whereas (-secretase cleaves APP(695) at either Val-636 or Ala-638 to yield the C-terminus of A? (1-40) or A? (1-42). The enzyme ?-secretrase cleaves APP(695) at Lys-612, near the middle of the A?-forming sequence. An ??secretase in CHO cells appears to be the zinc-metalloprotease TACE, a tumor necrosis factor-?converting enzyme. Both TACE and ?-secretase are stimulated by phorbol esters, and both are inhibited by certain peptide-hydroxamate derivatives such as TAPI. The PMA stimulated ?-secretase is virtually absent in fibroblasts from mice lacking the TACE gene, which confirms that TACE is essential for this activitiy. We sought to determine whether ?-secretase competes with ?-secretase in the processing of APP, that is, whether ?-secretase action can preclude A? formation. We chose to study APP processing in two non-transfected cell lines rat PC-12 pheochromocytoma cells, which exhibit some properties of neuronal cells, and human A-204 rhabdomyosarcoma cells, which have been found to form A? deposits. In PC-12 cells TAPI inhibited ?-secretase to the extent of about 50%, and the yields of A??(1-40) and A? (11-40) were elevated about 2-fold. In contrast, in A-204 cells TAPI inhibited A? formation by about 60%. Mass spectral analysis of the products showed that, in addition to the usual form of A?, A-204 produced a unique form, which resulted from ?-secretase cleavage 3 residues before Met-597. The cleavage that produces this form is strongly inhibited by TAPI. These results show that A? can be formed through different pathways, which may give unexpected responses to inhibitors, either lowering or raising the rate of A? secretion depending upon the cell-type. It appears likely that more than one ?-secretase and one ?-secretase exist in these cells, since saturating amounts of the inhibitor only lowered the rate of cleavage by about 50%. One ?-secretase and one ?-secretase are Zn-metalloproteases.

The career development of physician-scientists requires a broad approach in preparation for a career in academic medicine with research, teaching, and clinical responsibilities. This grant proposes a four-year career development plan consisting primarily of targeted hypothesis-driven research, but also incorporating teaching and mentoring activities, participation in institutional and national conferences and meetings, and continued clinical training. The University of Wisconsin-Madison provides the ideal environment to develop a career as a physician-scientist, with a reputation for premiere medical care and cutting-edge biomedical research, state-of-the-art facilities, extensive technical and intellectual resources, and a strong tradition of open collaboration among researchers and clinicians. The proposed research will investigate the dynamic and complex host-pathogen interactions involved in viral replication and pathogenesis. The simple genomic structure of positive-strand RNA viruses and experimental evidence suggest that host factors are indispensable for their replication, although the identity of these host factors and their functional impact on viral replication remain largely unknown. The recent development of novel viral replication systems in the well-studied yeast Saccharomyces cerevisiae permits the functional investigation of the cellular host factors involved in positive-strand RNA virus replication. Flock house virus (FHV), a bipartite positive-strand RNA virus and member of the Nodaviridae family, is the only animal virus shown to replicate in S. cerevisiae, and consequently represents a unique model to investigate the mechanisms of viral replication. The S. cerevisiae experimental system will be used to isolate and characterize yeast mutants unable to support FHV replication. A concurrent immunochemical approach will also be used to isolate and characterize cellular host proteins associated with the FHV RNA polymerase. The identification and characterization of host-pathogen interactions, with a particular emphasis on the impact of cellular host factors, are crucial both to understand the basic mechanisms of viral replication and pathogenesis and to design rational antiviral therapies.

Neuronal Nicotinic Acetylcholine Receptors (nAChRs) are an abundantly expressed and molecularly diverse family of cation channel proteins that mediated key physiological functions in the brain. Learning, memory, and nociception have been linked to cholinergic activity. Nicotine addiction depends on nAChR function and important human diseases (epilepsy, schizophrenia, Alzheimer's) have been linked to defects in cholinergic signaling. Neuronal nAChR subunits associate in a variety of combinations to produce pentameric receptors that facilitate cation transport upon binding to the neurotransmitter acetylcholine. Although at least 15 different types of vertebrate nAChR subunits have yet to be identified. This prediction is underscored by the recent discovery of at least 40 distinct nAChR subunit genes in the genome of a much smaller and simpler organism, the nematode, C. elegans. The goal of this project is to define the physiological and behavioral roles of a subset of these nAChR proteins in C. elegans motor neurons. At least five different nAChR subunit genes ("MnAChRs") are expressed in C. elegans motor neurons. Although nAChR expression has also been detected in the vertebrate spinal cord, the roles of nAChRs in mammalian motor neuron function are largely unknown. The well-defined neuroanatomy and relative simplicity of the C. elegans motor neuron circuit will facilitate our effort to detect motor neuron-specific expression and to define the intracellular localization of these nAChR subunits. In addition, we will exploit the powerful genetics of C. elegans to detect functional interactions between the nAChR genes and to perform genetic screens that can reveal new components of these signaling pathways. Ultimately, our approach should reveal functionally important and evolutionarily conserved proteins that also mediate cholinergic signaling in the human brain.

Our long-range objective is to develop a clearer fundamental understanding of the molecular basis of fertilization so that the process can be controlled to either promote fertilization or block it. Much progress has been made to identify ZP3, the protein in the mammalian egg coat that binds sperm and stimulates the acrosome reaction, the release of the acrosomal vesicle that must occur for sperm to penetrate the egg coat. Despite this progress, the identity of the ZP3 receptor on sperm has been elusive. One strong candidate is beta1,4-galactosyltransferase, an enzyme first discovered in the Golgi apparatus of somatic cells but later found on the surface of sperm. Beta1,4-Galactosyltransferase binds ZP3 but not other egg coat proteins. In addition to its role in gamete adhesion, beta1,4- galactosyltransferase acts to trigger the acrosome reaction in sperm. The acrosome reaction can be stimulated with antibodies to beta1,4-galactosyltransferase that act as mimics of ZP3 to bind and crosslink beta1,4-galactosyltransferase. There is evidence that binding beta1,4-galactosyltransferase stimulates at least some of the same signaling systems inside sperm that ZP3 stimulates, leading to the proposal that beta1,4- galactosyltransferase may be the major ZP3 receptor signaling the acrosome reaction. Mice with a targeted deletion of the beta1,4-galactosyltransferase gene, although fertile, produce sperm with severely compromised ability to acrosome react and penetrate the egg coat. In this proposal, the signaling systems that are activated by beta1,4-galactosyltransferase will be studied. The goals are to determine if binding beta1,4-galactosyltransferase specifically can completely reproduce all of the identified signaling systems activated by ZP3 binding, to study beta1,4-galactosyltransferase mutants to identify structural features necessary for signaling, and to identify proteins that interact with the portion of beta1,4- galactosyltransferase found in the cytosol. These studies will clarify the role of a sperm receptor for the egg coat in the acrosome reaction, a necessary step in the process of fertilization.

There has been a rapid growth in the use of digital imaging at Vanderbilt over the last three years. Some of this due to newly faculty, but much of it has arisen from success of the Cell Imaging Shared Resource in teaching researchers to use the confocal microscopy cannot be met by the one instrument that is currently available to Vanderbilt faculty. In an effort to remedy this deficit, several well established users of the existing confocal microscope began discussion a plan for sharing the use and maintenance costs of a new instrument. The Major Users included in this proposal compromise a core group share related research goals and employ similar techniques. For example, both Denison and Solnica-Krezel are performing time-lapse imaging experiments on live cells and embryos and at least two other investigators (Hogan, Wright) anticipate using this approach. The PI, Miller, in collaboration with Dave Piston, the Director of the Cell Imaging Resource, have developed methods for multicolor-GFP imaging in the LSM510. Denison, Solnica-Kerezel and Miller intend to exploit GFP multiplexing in their projects. Severak of the Major Users will employ multicolor labeling in fixed tissues with immunochemical or cytological dyes (Denison, Hogan, Wright, Solnica-Kerezel). The computer controlled, multiple laser lines of the LSM510 (458, 488, 514, 633) provide the full range of excitation wavelengths and powers needed by these investigators. Although the microscope will be largely utilized by the Major Users, microscope time will be allocated through the Cell Imaging Shared Resource to insure that allotments are fair and that unscheduled time can be used by other investigators at Vanderbilt.

0208335
Steele

A complete description of the evolutionary history of multicellular animals will require detailed information on the content, organization, and regulatory mechanisms of the genomes of animals from phyla throughout the animal phylogenetic tree. Of particular importance for such studies are species that diverged early from the rest of the animals and unicellular organisms which are close relatives of animals. Features of the genomes of such organisms which are shared with more recently diverged animals would then be ones which were present in the ancestor of all modern animals. The goal of this project is to prepare and archive bacterial artificial chromosome libraries from Nematostella vectensis (a sea anemome which is a member of the early diverging animal phylum Cnidaria) and from the choanoflagellate Monosiga brevicollis, a unicellular organism closely related to multicellular animals. These libraries will provide important resources for addressing a variety of questions related to the origins, evolution, and development of multicellular animals. Such questions include: How did the Hox family of developmental regulatory genes evolve? When did various other developmental regulatory gene families appear during animal evolution? How closely related are choanoflagellates and multicellular animals, and what genes were necessary to make the transition from unicellularity to multicellularity? What happened at the genome level when nerves were added to multicellular animals? What cell adhesion and intercellular signaling molecules were present in the common ancestor of modern animals? Are cnidarian genes organized into operons, as suggested by the presence of spliced leader addition in this phylum? In addition to providing material for analysis of animal evolution, the libraries will facilitate the isolation of gene promoters, which will be essential for the development of functional assays in these organisms and for dissecting gene regulatory networks. The libraries generated by this project will be freely available to the research community. Information on the libraries and their availability will be provided through various means, including appropriate scientific newsgroups and web sites.

Networks and computer systems are becoming increasingly attractive targets to large-scale programmed
attacks such as worms and Distributed Denial of Service attacks (DDoS), which can compromise a vast
number of vulnerable targets in a few minutes. Critical end-user applications vulnerable to such attacks
include e-commerce, e-medicine, command-and-control applications, video surveillance and tracking, and
many other applications. While there is a growing body of research techniques, prototypes, and commercial
products that purport to protect these applications and the network infrastructure on which they rely, there
is little existing scientific methodology by which to objectively evaluate the merits of such claims. Moreover,
thorough testing of a defense system for worms or for attacks on the infrastructure cannot be evaluated
safely on a live network without affecting its operation.

To make rapid advancements in defending against these and future attacks, the state of the art in the
evaluation of network security mechanisms must be improved. This will require the emergence of large-scale
security testbeds coupled with new standards for testing and benchmarking that can make these testbeds
truly useful. Current shortcomings and impediments to evaluating network security mechanisms include lack
of scientific rigor;lack of relevant and representative network data;inadequate models of defense mechanisms;
and inadequate models of both the network and the transmitted data (benign and attack traffic). The latter
is challenging because of the complexity of interactions among traffic, topology and protocols.

The researchers propose to develop thorough, realistic,and scientifically rigorous testing frameworks and methodologies for particular classes of network attacks and defense mechanisms. These testing frameworks will be adapted for different kinds of testbeds, including simulators such as NS, emulation facilities such as Emulab, and both small and large hardware testbeds. They will include attack scenarios; attack simulators;
generators for topology and background traffic; data sets derived from live traffic; and tools to monitor and
summarize test results. These frameworks will allow researchers to experiment with a variety of parameters representing the network environment, attack behaviors, and the configuration of the mechanisms under test.

In addition to developing testing frameworks, the researchers propose to validate them by conducting tests on representative network defense mechanisms. Defense mechanisms of interest include network-based Intrusion Detection Systems (IDS); automated attack traceback mechanisms;t raffic rate-limiting to control DDoS attacks; and mechanisms to detect large-scale worm attacks. Conducting these tests will require incorporating real defense mechanisms into a testbed, and applying and evaluating frameworks and methodologies. Conducting these tests will also help us to ensure that the testbed framework allows other researchers to easily integrate and test network defense echanisms of their own.

The research team includes experts in security, networking, data analysis, software engineering, and operating systems who are committed to developing these challenging integrated testing frameworks.

Intellectual Merit: The development of testing methodologies for network defense mechanisms requires
significant advances in our understanding of network attacks and the interactions between attacks and their
environment including:deployed defense technology, traffic, topology, protocols, and applications. It will
also require advances in our understanding of metrics for evaluating defenses.

Education: The research into testing methodologies for network defense mechanisms will involve
graduate students and provide new curriculum material for universities.

Broader Impact: By providing new testing frameworks, the work will accelerate improvements in
network defense mechanisms and facilitate their evaluation and deployment. The researchers will hold yearly workshops to disseminate results and obtain community feedback.

Viral pathogenesis is intimately linked with dynamic and complex host-pathogen interactions. The mechanisms underlying these essential interactions, in particular those shared by viruses with similar genomic structures, represent attractive potential targets for antiviral drugs. Viruses that contain a positive-sense single-stranded RNA genome, such as hepatitis C virus, West Nile virus, and the SARS coronavirus, represent a diverse group of pathogens responsible for significant human diseases for which few effective therapies exist. Despite the varied clinical syndromes caused by these viruses, all characterized positive-strand RNA viruses use intracellular membranes for viral RNA replication complex formation and function. However, the mechanisms whereby viral RNA replication complexes assemble on specific intracellular membranes are not well understood. The long-term objectives of this project are to elucidate the host-pathogen interactions that facilitate positive-strand RNA virus replication complex assembly and function. A detailed understanding of these interactions will provide insight into the mechanisms of viral pathogenesis, and potentially identify novel targets for broadly effective antiviral drugs. The specific focus of this proposal is to define the early events in viral RNA replication complex assembly. The targeting, transport, and initial interactions of virus-encoded RNA replication complex proteins with intracellular membranes are essential steps in replication complex assembly, and therefore are important determinants of viral pathogenesis. The general strategy of the proposed research is to use Flock house virus, an established and versatile model used to study positive-strand RNA virus structure and replication, to investigate the mechanisms of viral RNA replication complex assembly. Biochemical, molecular, and genetic approaches will be used to investigate the targeting and transport of the Flock house virus RNA-dependent RNA polymerase to intracellular membranes. The specific aims of this proposal are designed to accomplish two goals: 1) understand the role of cellular chaperone proteins in Flock house virus RNA replication complex assembly; and 2) define the membrane receptor responsible for Flock house virus RNA replication complex intracellular localization.

[unreadable] DESCRIPTION (provided by applicant): Idiosyncratic surgical practice patterns may contribute to unwarranted variation in the use of laparoscopy and partial nephrectomy (PN) among patients with early-stage renal cell carcinoma (RCC). In order to test this hypothesis, we will explore the following specific aims: 1) To evaluate geographic variation in the use of laparoscopic surgery among patients with RCC; 2) To characterize geographic variation in the use of PN among patients with RCC; and 3) To assess for an interaction between the use of laparoscopy and the use of PN. Linked data from SEER and Medicare will be used to identify a national sample of incident kidney cancer cases diagnosed between 1998 and 2002. Temporal trends in annual incidence rates for RCC- specific laparoscopy and PN will be calculated and compared for individual hospital referral regions, adjusted for relevant covariates. Interactions between the use laparoscopy and PN will be evaluated a priori. This data will provide more precise characterization of unwarranted variation in the use of laparoscopy and PN among patients with early-stage kidney cancer and may, in turn, motivate patient-, provider- and policy-level interventions aimed at making the surgical treatment of RCC both more effective and more patient-centered. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In the nervous system, the pattern of synaptic inputs and outputs defines the direction of information flow. A neuron detects stimuli via elongated processes called dendrites and releases signals from a morphologically distinct class of cytoplasmic extensions called axons. These asymmetric features of neurons are developmentally controlled and fundamentally important to brain function. In the nematode, C. elegans, the DD class of GABA motor neurons undergoes a developmentally regulated reversal of synaptic polarity in which a dendrite switches to become an axon and an axon adopts the properties of a dendrite. Remarkably, DD neuronal processes are not withdrawn to accommodate this change but are remodeled in situ to interconvert pre- and post-synaptic specializations. The implementation of this program is regulated by UNC- 55, a conserved member of the COUP family of nuclear hormone receptor transcription factors. Therefore, we hypothesize that polarity reversal and synaptic remodeling of the DD motor neurons are transcriptionally controlled. We will use new, powerful, cell-specific microarray technology to (1) profile DD motor neurons during the developmental period in which metamorphosis occurs and (2) exploit unc-55 mutants to identify UNC-55 regulated transcripts. A comparison of these data sets should reveal strong candidates for genes that orchestrate polarity reversal and the consequent synaptic remodeling of DD motor neurons. The evident plasticity of vertebrate neuronal asymmetry and the evolutionary conservation of molecules that specify neuronal polarity suggest that our discoveries in a nematode model system will reveal genes with fundamental roles in regulating these events. [unreadable] [unreadable]

[unreadable] Description (provided by applicant): Innate cellular antiviral responses are essential first lines of defense against viral infections, and also play crucial roles in the initiation of adaptive immunity and hence eventual virus control or clearance. However, the inappropriate activation of innate immune responses can also contribute to viral pathogenesis. Intracellular pathways that suppress or potentiate steps in innate antiviral responses therefore represent crucial components of the host-pathogen interactions that ultimately determine the outcome of viral infections. Furthermore, a detailed understanding of the cellular and molecular mechanisms underlying these pathways is important for the rationale design of novel antiviral or immunomodulatory drugs. Although our knowledge regarding innate cellular antiviral responses has progressed tremendously over the past decade, significant gaps still exist. For example, innate antiviral immune responses consist of a complex network of interactions that exhibit both cell type- and pathogen-specific differences, but the functional significance and impact of these differences on viral pathogenesis, particularly within the central nervous system, have not been well studied. We hypothesize that intrinsic maturation-dependent innate antiviral responses are key determinants in the pathogenesis of neurotropic virus infections. We propose to explore these responses in human neuronal cells using western equine encephalitis virus (WEEV), a mosquito-borne positive-strand RNA virus belonging to the Togaviridae family (genus: Alphavirus). WEEV and the closely related eastern and Venezuelan equine encephalitis viruses are category B bioterrorism agents, and cause central nervous system infections that are associated with high clinical morbidity and mortality for which there are no effective therapies. Arboviral diseases, such as those caused by neurotropic alphaviruses, have also seen both an emergence and resurgence as significant public health threats over the past several decades. The long-term objectives of this project are to elucidate the molecular mechanisms underlying the cell- autonomous recognition, activation, and effector pathways involved in the innate antiviral responses of human central nervous system neurons to arbovirus infections. The focus of this proposal is to employ targeted and global genetic and chemical genomic approaches with cultured human neuronal cells to examine the activation pathways responsible for WEEV recognition and initiation of innate antiviral responses in mature neurons. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The sensation of pain triggers self-protective responses to noxious insults. Pain is perceived by specialized sensory neurons called nociceptors that utilize elaborate webs of dendritic processes to detect harmful stimuli. These features of nociceptive neurons are universally observed in animals and are thus likely to depend on conserved genetic programs that control nociceptor morphogenesis throughout phylogeny. The mechanisms that govern the creation of these complex structures are poorly understood but recent studies in Drosophila have established that sensory neuron morphology is defined by surprisingly diverse arrays of transcription factors. The identities of downstream genes and their orchestration by these transcriptional networks are largely unknown, however. Here, we propose to solve this problem by exploiting a powerful gene expression profiling method to identify targets of transcription factors that control morphogenesis of a model sensory neuron. In the nematode, C. elegans, a single pair of bilaterally symmetric neurons, PVDL and PVDR envelop the body with a regular array of highly branched dendrites that can be readily visualized with a fluorescent (GFP) marker in living animals. The PVDs respond to strong mechanical force ("harsh touch") to trigger a withdrawal reflex, a behavior indicative of nociceptive function. We have used a cell specific microarray profiling strategy to identify transcripts that are highly expressed in PVD neurons. Specifically, we will: (1) Use available mutants and RNAi clones to test transcription factor genes in this data set for roles in PVD dendritic morphogenesis;(2) Use the mRNA tagging method to reveal genes downstream of the conserved LIM- homeodomain protein, MEC-3, an established determinant of PVD dendritic branching and;(3) Extend this mRNA tagging strategy to at least five additional key transcription factor genes identified in Aim 1. These data sets of transcription factor target genes will provide a foundation for future studies that link transcriptional control to the cell biology of dendritic arborization. We believe that our approach represents a useful alternative to direct studies of mammalian tissues and moreover, that it offers a uniquely powerful strategy for identifying molecules with fundamental, conserved roles in nociceptor morphogenesis. PUBLIC HEALTH RELEVANCE: Specialized nerve cells that mediate the sensation of pain utilize an elaborate array of spaghetti-like sensory processes directly beneath the skin to detect noxious stimuli. To facilitate the identification of genes that govern the creation of these sensory networks, we propose a genetic approach in the nematode C. elegans, a simple model organism with a single pair of PVD sensory neurons that display a stereotypical and readily visible array of nerve processes. The results of this work should reveal similar human genes with crucial roles in the creation of sensory neuron arrays and therefore potentially lead to treatments for injuries or diseases that disrupt human sensory neuron development.

The objective of Project 2 is to identify epigenetic biomarkers for predicting recurrence of early stage (stages 1 and II) endometrioid endometrial carcinomas (EECs). While this type of cancer usually has a good prognosis, ~10% of patients present with local recurrence or distant metastasis within 3 years of diagnosis. The currently used clinicopathojogical parameters, however, are inadequate for predicting which patients are Iikely to recur and lwould most benefit from upfront therapies. In this regard, profiling of molecular markers"~ has recently been shown to be important for improved prognosis and for better stratification of cancer subtypes. Our preliminary studies have found that CpG island hypermethylation, a type of epigenetic alteration in cancer, represents an untapped resource of biomarkers for EECs. To identify these methylation-regulated loci, we will follow a phased-based approach established by the NCI's Early Detection Research Network. In the discovery step (Phase I), an epigenetic microarray approach will be used to comprehensively screen altered methylation of 27,000 human CpG islands in primary tumors of 54 early stage patients who recurred and 54 non-recurrent patients (sample size justified). Global profiling will identify candidate loci that are frequently hypermethylated in the recurrent group, but not in the non-recurrent group. In the validation phase (Phase II), a PCR-based assay, COBRA, will be used to confirm methylation findings in a second cohort of 54 early stage patients who recurred and 54 non-recurrent patients. A final set of 30 loci will then be used for further analysis in 2315 EECs (including all clinical stages and tumor grades) in the Phase III retrospective study. A robust assay, called MassARRAY, can efficiently quantify methylated CpG sites in 384 clinical samples in a single run. This extensive study will determine whether the identified loci are also poor prognostic indicators for some EEC patients. Clinical sensitivity and specificity of promising biomarkers will be calculated and used to assess patients'recurrence, disease-free survival, and other clinicopathological parameters. Such a study lays the foundation for a future prospective clinical trial (Phase IV) designed to test the utility of epigenetic biomarkers for accurate prediction of recurrent EECs.

This scholarship program is recruiting freshmen and sophomore science, technology, engineering, and mathematics (STEM) majors to become credentialed secondary STEM teachers through a collaborative effort by the WV University College of Human Resources and Education, Eberly College of Arts and Sciences, the WV Center for Professional Development, and the WV Regional Education Service Agencies (RESA) III, V, VII, and VIII. The project provides scholarships for 20 students to complete the Benedum Collaborative Five-year teacher education program. Scholarship recipients earn a STEM bachelor's degree, as well as a master's degree in education upon completing the program. The program objectives are to: 1) Increase retention of students who traditionally leave STEM majors; 2) Attract freshmen and sophomore students to elementary and secondary STEM teacher certification; 3) Provide a continuum of mentoring services through collaborative advising from STEM content mentors and STEM education mentors; 4) Increase the number of graduates with 5-12 STEM education certification, and 5) provide a supportive environment for the new teachers. The pedagogical strategies, specifically teaching science content in the context of societal issues through Science, Technology and Society (STS) approaches, appeal to underrepresented groups, thus expanding the pool of those who could potentially become interested in STEM fields. A focus on the need for 5-12 STEM teachers, along with assignments to encourage STEM students to "think like teachers", are utilized to encourage these students' interest in teaching as a career.

DESCRIPTION (provided by applicant): Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses are transmitted by mosquitoes, cause serious and potentially fatal central nervous system infections in humans, and are considered NIAID Category B Priority Pathogens due to their potential misuse as bioterrorism agents. Although vaccine development is in progress for several alphaviruses, there is an urgent and pressing need for broadly active antiviral agents against these virulent pathogens. Studies with experimental alphavirus encephalitis in mice have shown that while neurons are damaged directly by virus, uninfected neurons are also damaged via bystander mechanisms that involve altered homeostatic and neuroprotective functions of microglial cells or astrocytes. Thus, we hypothesize that combination therapy that directly targets virus replication and enhances neuroprotective responses will provide synergistic benefit in viral encephalitis. To identify and develop new antivirals to test this hypothesis, we recently screened a chemically defined small molecule library and identified a thieno[3,2-b]pyrrole compound that has potent activity against neurotropic alphaviruses in culture. Furthermore, a limited structure-activity relationship analysis with twenty structurally related analogs identified six additional compounds with enhanced in vitro activity. In addition, to explore the innovative use of natural products as a source for novel antivirals, we analyzed a series of extracts derived from marine actinomycetes and identified several that contained potent activity against alphaviruses. We are currently positioned to rapidly and efficiently move candidate antivirals through preclinical development, and we have assembled a team of experienced investigators with diverse expertise in the fields of virology, neurology, pathology, physiology, and medicinal chemistry to accomplish this task. The long-term goals of this highly collaborative project are to develop effective and broadly active therapies for encephalitis caused by neurotropic alphaviruses and related arboviruses. The specific aims of this proposal are: (1) targeted chemical modification of lead antiviral compounds, based on the initial structure-activity relationship analysis, to enhance potency, reduce toxicity, improve solubility and metabolic stability, and optimize membrane permeability;(2) identify molecular target(s) responsible for their antiviral activity;(3) analyze the in vivo pharmacokinetics and efficacy of candidate antivirals, including combination treatment with neuroprotective agents;and (4) isolate and characterize novel compounds with antiviral activity derived from marine microbes.

DESCRIPTION (provided by applicant): Mosquito-borne viruses, or arboviruses, are among the most important emerging pathogens worldwide with a significant potential impact on human health, and are also prominent components of the NIAID Category A, B, and C priority pathogens lists. The inclusion of many arboviruses as high priority pathogens is due in part to their virulence, the potential for vector-mediated dissemination, and the public concern regarding insect-borne viral infections. In addition, despite the worldwide impact of arboviruses, few effective treatments are currently available. The objective of this proposal is to use western equine encephalitis virus (WEEV), a category B arbovirus, to identify and develop novel antiviral compounds isolated from natural product extracts produced by marine sediment-derived microbes. Compounds derived from terrestrial and marine microorganisms have historically provided a rich source for novel therapeutics against a range of microorganisms, but have thus far been underutilized in the development of antiviral agents. We have already developed, validated, and employed a WEEV replicon-based assay to complete high-throughput screens against a library of >16,000 pre- fractionated extracts derived from a diverse array of marine microbial species. Furthermore, we have completed both analytical and preparative preliminary chromatographic fractionation procedures for two distinct extracts with validated antiviral activity. The specific aims of this proposal are designed to complete the isolation and functional characterization of active compounds from these two candidate extracts, including structural examination and initial viral target identification, and to expand the identification of microbe-derived natural products with anti-arboviral activity. The long term goals of this research project are to develop a wide range of antiviral compounds derived from marine microbes, characterize their structures and antiviral activities, and complete their preclinical testing in animal models of arbovirus-mediated disease, with the ultimate objective being clinical implementation of these candidate novel drugs. The experiments outlined in this exploratory project proposal will be used to establish the groundwork to accomplish these goals. PUBLIC HEALTH RELEVANCE: This study is designed to identify new drugs to treat central nervous system infections caused by potentially deadly viruses that are transmitted by insects. This research is important to public health because there are currently few effective medications to treat mosquito-borne viral infections, and preventative strategies such as vaccination are still in the developmental phases. In addition, routine vaccine distribution and use may be difficult to implement on a population-wide basis due to safety concerns with mass vaccination in the absence of an outbreak scenario. Therefore, the availability of effective medications to either treat established disease or prevent infection is a highly valuable component in the overall strategy to combat the potentially devastating diseases caused by these viruses.

Effective robotic assistance of infants with or at risk of developing Cerebral Palsy (CP) has the potential to reduce the significant functional limitations as well as the potential deficits in cognitive development. This project focuses on the development and testing of a sequence of robotic assistants that promote early crawling, creeping, and walking, along with a model of infant-robot interaction that encourages the continued practice of movement patterns that will ultimately lead to unassisted locomotion. Typically developing infants initially learn to crawl through the generation of spontaneous limb and trunk movements. Early in the process, these spontaneous movements transport the infant across the floor. The rewarding locomotory experience drives the infant to refine the movements to intentional and exploratory skills. Ultimately, the infant intentionally engages these skills to solve larger problems, such as obtaining an interesting toy or exploring the environment. Infants with conditions such as CP lack the muscle strength, postural control, and motor coordination necessary for these early exploratory limb and trunk movements to result in locomotion. Without this positive feedback, the development of the neural pathways for productive limb use is diminished, which results in delayed or lack of development of crawling and walking. These limitations in mobility negatively affect other domains of development such as perception and cognition, with effects being visible even into adulthood.

The robotic assistants to be developed in this project will aid the infant in developing locomotory skills by selectively supporting a portion of his/her weight and providing artificial, rewarding locomotory experiences. The PI's approach to infant-robot interaction is to first instrument the infant with a set of sensors, allowing for reconstruction of the trunk and limb positions in real time. A semi-supervised clustering process will then identify a menu of canonical spatio-temporal limb and trunk movement patterns given observations of behavior that is exhibited by children who are either typically developing or at risk of developing CP. The robot will respond to the recognition of a canonical movement by assisting in the corresponding postural support and transport of the child. The PI's hypothesis is that this positive feedback will encourage the continued practice of the canonical movements, as well as their use in solving larger problems. The infant-robot interaction model will selectively reward specific canonical movements as different levels of capabilities are exhibited. As the child becomes proficient at using a simple movement to trigger robotic assistance, the robot will reduce (and ultimately eliminate) its response to that particular canonical movement. Other canonical movements that encode related, but more complex and/or coordinated limb movements, will continue to be available. As the limb movements are mastered the vertical support will be reduced to encourage the infant to bear more of his/her own weight. The hypothesis is that this early intervention approach will help to guide the child along a progressive developmental trajectory that will end with locomotory skills and muscle strength that require little or no assistance. EEG-based neuroimaging will be used to monitor the progression of the infant's development. The hypothesis is that the degree of proficiency of certain skills will be identifiable using the EEG index related to motor output. This information will be used to guide the semi-supervised clustering process, as well as the decision process for selectively rewarding certain canonical movements.

Broader Impacts: Equipping children with CP at an early age with locomotory skills will not only bring them more in line with typically developing children, but will also reduce their reliance on long-term care while increasing their success in self-help, in education, and in the workplace. The techniques will be applicable to a range of other childhood disorders (including Down Syndrome), to retraining patients following stroke, and to the creation of tunable gestural interfaces for intelligent prostheses.

DESCRIPTION (provided by applicant): Developing neural circuits are actively remodeled as synapses are created in new locations and dismantled in others. These dynamic events are regulated by neuronal activity to produce mature circuits with specific physiological functions. This phenomenon has been observed throughout animal phylogeny which suggests that the underlying pathways are conserved. However, the molecular mechanisms that drive synaptic remodeling are largely unknown. Here we propose a strategy that exploits the simple model organism, C. elegans, to define a development program that remodels the synaptic architecture of a GABAergic circuit. Ventral synapses for DD class GABA neurons are relocated to new sites on the dorsal side during larval development. This synaptic remodeling program is blocked by the UNC-55/COUP-TF transcription factor in VD motor neurons which normally synapse with ventral muscles. We exploited this UNC- 55 function in a powerful cell-specific profiling strategy that identified 19 conserved genes with roles in synaptic remodeling. We have now shown that one of these UNC-55 targets, the DEG/ENaC cation channel, UNC-8, promotes synaptic remodeling in a mechanism that is activated by GABAergic signaling. This finding is important because DEG/ENaC proteins have been implicated in learning and memory but the mechanism that links DEG/ENaC function to synaptic plasticity is poorly understood. Specific Aim 1 tests the key prediction that UNC-8 is closely associated with GABAergic synapses that are remodeled by UNC-8 activity. Specific Aim 2 is designed to test the novel hypothesis that a Ca2+-dependent mechanism links neural activity to UNC-8 cation transport in a feedback loop that dismantles the presynaptic machinery. Specific Aim 3 defines the cellular origin and molecular components of the proposed activity-dependent pathway that regulates UNC-8 and promotes GABAergic synaptic remodeling. Together, these approaches offer a powerful opportunity to delineate an intricate molecular pathway that controls synaptic plasticity. Moreover, the conservation of these remodeling components in mammals argues that the results of this work are likely to reveal fundamental mechanisms that regulate synaptic plasticity in the human brain.

DESCRIPTION (provided by applicant): Sensory neurons utilize complex, topical networks of dendritic processes to detect external stimuli. Models that seek to explain the creation of these elaborate structures must include mechanisms that control the key events of branch initiation, elongation and termination. These features are universally observed in both vertebrate and invertebrate systems and are therefore likely governed by evolutionarily conserved components. The simple model organism, C. elegans, displays a single pair of PVD nociceptive neurons that envelop the animal with a net-like array of dendritic branches. We used time-lapse imaging to show that the discrete topical region occupied by each branch is defined by a contact-dependent mechanism in which sister dendrites (i.e., dendrites from the same neuron) repel each other to stop outgrowth. Self-avoidance is also observed in mammals and insects but the molecular underpinning of this fundamental patterning event is poorly understood. Our work has revealed a novel mechanism in which the diffusible cue UNC-6/Netrin is captured at the tips of PVD dendrites to mediate self-avoidance in a pathway involving the receptors UNC-40/DCC and UNC-5. This discovery is significant because it describes the first example of a role for these highly conserved proteins in dendrite self-avoidance. Now, we have extended these findings to identify multiple downstream components of the UNC-6/Netrin self-avoidance pathway. On the basis of these new results, we propose that UNC-6/Netrin triggers actin filament growth at the tips of contacting sister dendrites to engage a non-muscle myosin motor that drives retraction. Experiments described in Specific Aim 1 exploit the novel application of TIRF microscopy to a living organism to test this model. These studies are significant because little is known of how signals at the cell membrane trigger dendrite withdrawal during self-avoidance. Our work has uncovered a key role for a conserved membrane protein, tomoregulin, in self-avoidance. Specific Aim 2 will define the mechanism of this effect and determine if tomoregulin is necessary for other known short-range UNC- 6/Netrin signaling events. Aim 2 is significant because it addresses the fundamental question of how the UNC-6/Netrin pathway has been uniquely adapted for contact-dependent self-avoidance. To address the mechanism of dendritic outgrowth, we exploited powerful cell-specific profiling methods to identify targets of a conserved LIM-homeodomain transcription factor, MEC-3, that is required for PVD branching. Specific Aim 3 will test a model, based on these results, that dendritic branches are stabilized by interaction with claudin-like proteins and other specific cell-surface components in the adjacent epidermis. These experiments are important because sensory neuron outgrowth is typically executed in close contact with epidermal tissue but the intercellular mechanisms that pattern dendritic architecture in this location are poorly defined. This work in C. elegans is expected to identify key determinants that also specify dendritic architecture in the human nervous system.

This project from George Mason University (Mason) creates an NSF I-Corps Site that will have a close affiliation with the Washington DC I-Corps Node.

NSF Innovation Corps (I-Corps) Sites are NSF-funded entities established at universities whose purpose is to nurture and support multiple, local teams to transition their technology concepts into the marketplace. Sites provide infrastructure, advice, resources, networking opportunities, training and modest funding to enable groups to transition their work into the marketplace or into becoming I-Corps Team applicants.
I-Corps Sites also strengthen innovation locally and regionally and contribute to the National Innovation Network of mentors, researchers, entrepreneurs and investors.

An NSF I-Corps Site at Mason will aid in the creation of a commercialization facilitation infrastructure that will enhance the impact of existing and future entrepreneurial training and support activities and will demonstrate the value of entrepreneurship education to the Mason community. The I-Corps Site will impact the preparedness of university talent for roles in technology-based entrepreneurial enterprises and as such will be a critical ingredient of the economic success of Virginia and the United States as a whole. With an I-Corps Site, Mason's commercialization facilitation activities will support a shift from an economy dependent on traditional employers to an entrepreneurially-driven economy. As more startups begin to flourish, new jobs will be created and the career prospects of future graduates will be strengthened. On the national level, there is valid concern that the US is losing its standing as the global leader in technology. Mason's activities will counter this trend by teaching university researchers to think beyond traditional academic boundaries, thus enabling this group of highly-talented scientists to become drivers of innovation within technology-based entrepreneurial companies that they help to create.

The Supporting and Sustaining Scholarly Mathematics Teaching (SSSMT) project at the University of Hartford seeks to improve teaching and learning in undergraduate mathematics courses. Recent projections suggest that the United States needs to dramatically increase the annual award rate of STEM degrees by 34% in the next decade to remain competitive internationally. A critical step towards improving the recruitment, retention and success of STEM majors is the adoption of research-based teaching practices in first-year and second-year college mathematics courses. These introductory courses often act as gatekeepers that filter students out of STEM majors. There is mounting evidence that suggests active learning can be much more effective than direct instruction in engaging students. Despite this evidence, there continues to be an overwhelming reliance on lecture as the primary method of delivering content in college mathematics courses. The goals of the Supporting and Sustaining Scholarly Mathematics Teaching (SSSMT) project are: 1) to form a collaborative community of faculty who are committed to implementing active learning and flipping pedagogy in a variety of first-year and second-year mathematics courses with diverse student populations; 2) to studying the effectiveness of this pedagogy; and 3) to sharing the results with the larger mathematics community. Collaboratively and collectively, SSSMT faculty participants will develop a shared understanding of how to best leverage these strategies and processes to support a more widespread adoption of engaged teaching and learning strategies locally, regionally, and nationally.

Drawing on existing research on active learning, flipping pedagogy, and faculty development, the SSSMT project seeks to create a multi-institutional network of faculty to implement its goals in these areas. The network is comprised of college mathematics faculty at different stages of their careers, from a variety of institutions, who are interested in implementing active learning and becoming scholarly teachers who conduct and publish research on their own teaching and their students' learning. In the short term, this project promises to identify and disseminate best practices regarding the use of engaged learning strategies in a variety of mathematics courses populated by diverse student populations. The project will produce comprehensive sets of resources for faculty interested in using active learning and flipping pedagogy in courses ranging from college algebra to introduction to proofs and will develop a more robust understanding of the challenges, opportunities and necessary supports for mathematics faculty who have different levels of experience with the scholarship of teaching and learning. Project outcomes will be investigated using a mixed-methods approach involving qualitative and quantitative data collected from participants through surveys, reflective journals, and teaching practice inventories. The longer-term goal of this project is to expand the cross-institutional network, inviting in and involving the departmental colleagues of project participants, as well as faculty at other institutions, thereby broadening and enriching the virtual community of faculty engaged in this work of enhancing the teaching and learning of undergraduate mathematics.

Project Summary Fertility depends on successful fertilization and early development, processes that occur in the oviduct. Common therapies for human infertility, such as in vitro fertilization and intracytoplasmic sperm injection, are expensive and increase risks of a variety of problems. More knowledge of how the oviduct interacts with sperm, the cumulus-oocyte complex (COC) and the developing embryo may improve fertility and reduce the need for therapies. The oviduct serves as a reservoir for sperm, after semen deposition and before fertilization. Binding to the oviduct maintains sperm viability and suppresses motility. Sperm are released to move to the upper oviduct (ampulla) to fertilize oocytes. There are many gaps in this model of sperm-oviduct interaction. Our studies have begun to fill some of these gaps. We have used a glycomic approach to screen 377 glycans and found that all glycans with affinity for porcine sperm have either of two motifs, sulfated Lewis X trisaccharide or branched 6-sialylated complex glycans. We have identified two candidate receptors for both glycans on the sperm membrane, PKDREJ and ADAM5. Notably, mouse sperm deficient in PKDREJ and other ADAMs do not accumulate beyond the utero-tubal junction, but it is not known if this is due to a problem in binding and retention in the oviduct. Remarkably, if these glycans are immobilized on beads, they can extend sperm lifespan, much like binding to oviduct cells prolongs the lifespan of sperm. Finally, we found that cumulus-oocyte complexes (COCs) secrete molecules that signal sperm release from the lower oviduct so they can move toward the site of fertilization. The Specific Aims will provide a mechanistic understanding of how sperm bind the oviduct, how binding prolongs sperm lifespan and how sperm are released from the oviduct by the COC. Aim 1: To determine the function of PKDREJ and ADAM5 in sperm by blocking each protein and mutating each gene in swine. Sperm from pigs that are deficient in each of these proteins will be examined to determine if their ability to bind oviduct cells and their fertility is affected. Aim 2. To determine if sperm binding to immobilized oviduct glycans suppresses ROS production, conserves ATP, and stabilizes the plasma membrane, which extends sperm lifespan and fertility. Sperm bound to immobilized glycans will be examined to ascertain what changes adhesion induces. The second group of experiments will determine which changes are sufficient to prolong lifespan by adding agents that scavenge ROS and stabilize the plasma membrane. Aim 3. To determine how the cumulus-oocyte complex signals release of sperm from oviduct cells and glycans. We will examine whether progesterone, prostaglandins, or proteins that may be secreted from COCs might induce porcine sperm release from oviduct cells and immobilized glycans. The completion of these Specific Aims will elucidate how sperm bind to the oviduct, are stored in the oviduct and are released in response to the cumulus-oocyte complex. This fundamental information could be used to develop simpler, safer and less costly assisted reproductive technologies.

Developing neural circuits are actively remodeled as synapses are created in new locations and dismantled in others. These dynamic changes are driven by the combined effects of genetic programs and neural activity that together shape the architecture and function of mature circuits. Synaptic plasticity has been observed throughout animal phylogeny which suggests that the underlying pathways are conserved and thus can be investigated in simple model organisms that are amenable to experimental analysis. Here we propose to use the nematode, C. elegans, to define a development program that remodels the synaptic architecture of a GABAergic circuit. During early larval development, DD-class GABAergic neurons undergo a dramatic remodeling program in which the presynaptic apparatus exchanges locations with postsynaptic components within the DD neuronal process. To reveal the mechanism of this effect, we are investigating the functional roles of ~20 conserved genes that we have determined are transcriptionally regulated to drive GABA neuron remodeling. Our work has shown that two of these targets, the DEG/ENaC cation channel protein, UNC-8, and ARX-5/p21, a conserved component of the Arp2/3 complex, function together in an activity-dependent mechanism that dismantles the presynaptic domain. Aim 1 tests the hypothesis that UNC-8 cation transport elevates intracellular calcium to drive presynaptic disassembly and that this effect is regulated by calcium- dependent phosphorylation. This goal is important because members of the DEG/ENaC protein family have been implicated in learning and memory but the mechanism that links DEG/ENaC function to synaptic plasticity is poorly understood. Aim 2 tests the hypothesis that the UNC-8 function triggers an actin-dependent endocytic mechanism that recycles presynaptic components for reassembly at new locations. These experiments derive from our surprising discovery that a key functional protein of the Arp2/3 actin-branching complex is transcriptionally regulated to effect synapse removal and that newly identified components of an endocytic recycling pathway are involved. Together, these approaches offer a powerful opportunity to delineate intricate molecular pathways that link neural activity to genetic programming in the execution of a synaptic remodeling mechanism. Moreover, the conservation of C. elegans remodeling components in mammals argues that this work is likely to reveal fundamental mechanisms that regulate synaptic plasticity in the human brain.

SUMMARY Gap junctions or ?electrical synapses? mediate the flow of ions between neurons and are thus essential to normal brain function. Circuit activity is defined by the selective placement of electrical synapses between specific neurons and in particular cellular compartments. Although much has been learned about the mechanisms that direct assembly of chemical synapses between specific neurons, little is known of the pathways that drive the creation of neuron-specific electrical synapses. With its stereotypical placement of gap junctions and powerful tools for genetic analysis and imaging, the C. elegans motor circuit offers a unique opportunity to investigate gap junction specificity. VA and VB motor neurons are connected via gap junctions to command interneurons (AVA or AVB) that drive backward (VAàAVA) or forward (VBàAVB) locomotion. Notably, VAàAVA gap junctions are placed on the VA axon whereas VBàAVB gap junctions are positioned on VB cell soma. The UNC-4 transcription factor functions in VAs to preserve VAàAVA electrical synapses; unc-4 mutants adopt VAàAVB gap junctions on VA cell bodies and are thus unable to move backward. Thus, UNC- 4 regulates a transcriptional program that defines both the cellular compartment and neuron- specificity of gap junction placement. We used VA-specific RNA-Seq data to reveal that UNC- 4 blocks expression of a phosphodiesterase, PDE-1, that degrades cAMP, and a neuropeptide receptor, FRPR-17, that functions in a GaO pathway that antagonizes cAMP synthesis. Aim 1 tests the hypothesis that UNC-4 represses specific downstream targets to maintain cAMP which in turn sustains VAàAVA gap junctions. Our RNA-Seq data revealed that another UNC- 4 target, the atypical kinesin VAB-8, is ectopically expressed in unc-4 mutant VAs where it antagonizes normal trafficking of gap junction components into the VA axon. Aim 2 tests the hypothesis that VAB-8 binds to microtubules to block the anterograde function of kinesins that drive gap junction transport, thus, facilitating the formation of VAàAVB gap junctions on VA cell soma. Aim 3 uses single molecule imaging techniques to test a ?blockade? model in which VAB-8 lacks ATPase/motor activity but binds to microtubules to impair gap junction export from the cell soma. Although studies in cultured mammalian cells have implicated cAMP signaling and trafficking in gap junction assembly, these pathways have not been tested for functional roles in neuron-specific placement of electrical synapses in an intact nervous system. Thus, our work with a model organism could provide important clues to fundamental processes governing the formation electrical synapses in the human brain.