david pearce - US grants

In many economic settings the efficiency of explicit and implicit contractual arrangements is limited by the presence of imperfect monitoring: some agents cannot observe perfectly the actions of others. Often the economic problem of interest involves the indefinite repetition of some strategic situation such as an oligopoly, a partnership, or any organizational hierarchy. Cooperation in such problems depends upon the participants responding aggressively to indications that not all participants are honoring the implicit agreement. It would seem then that the possibilities for cooperation are enhanced when signals related to players' behavior are observed without delay, and when players can respond quickly to new information. Under their previous grant the investigators demonstrated that the opposite is true. Cooperation is enhanced by delays in the observation of signals and slower reaction speeds for players. The delays and slower reaction speeds made it possible to use a single punishment to deter a multitude of different potential deviations from the implicit agreement. This is described as "reusable punishments." Exploiting reusability enhances organizational efficiency dramatically. In their previous work the investigators used standard repeated games with discounting. These games assume a fixed frequency with which players take actions and fixed frequency with which players receive information regarding the play of the game. The contribution of this grant comes from relaxing these assumptions, studying information flow in other problems, and generalizing the results into a new theory of organizational design. The new topics considered include implicit contract theory in competitive markets, renegotiation in supergames, strategic complexity, partial moral hazard in repeated agency, and the strategic dynamics of collusion in markets with renewable resources.

A five year program of didactic training and research is proposed to examine the role of gene regulation in renal physiologic adaptive mechanisms. During phase one of the program I will undertake formal instruction in molecular biology and genetics while studying the regulation of renin gene expression. During phase two I will use the concepts and techniques developed in the first phase to study the genetic control of renal transport processes. The phase one didactic program will be designed around the core curriculum of the graduate program in Biochemistry and Biophysics and UCSF in conjunction with the research conferences in the Metabolic Research Unit and division of Nephrology. The research during phase one will be directed at localizing and characterizing the enhancer and promoter elements which confer tissue-specific expression to the renin gene. A cell lines has been found which expresses the renin gene only in certain media. This property will be exploited to localize the tissue-specific gene regulatory regions by use of DNase I hypersensitivity, "gel shift" and "footprint" assays. Candidate regulatory sites will be tested for enhancer, promoter, or repressor activity by assessing their effect on transcription from a homologous or heterologous promoter in a recombinant construction using transfer. The trans-acting factors which act on these sites will then be identified and characterized. The Na+/N+ antiporter responds to a number of stimuli (e.g., acidosis) in a manner consistent with an increase in its biosynthesis. The specific goal in phase two of the program will be to design and implement an independent research project directed at determining whether transcritpional control is exerted in the response of the antiporter to these stimuli, and if so, to determine the underlying mechanisms. This program of didactic training and research will provide the basis for subsequent study of not only the antiporter but also potentially of the gene regulatory events implicated in the control of other transport systems.

This project addresses a neglected but crucial area of research in the theory of strategic dynamics, i.e., renegotiation in repeated games. Renegotiation and bargaining power are combined in a unified, coherent theory. Much of the project is concerned with providing clean characterizations which make the theory tractable in applied economic problems. Both the formulation of the appropriate definitions and the characterizations become progressively more challenging as more complicated supergames are studied. The project gives definitions and conjectured results for asymmetric strictly repeated games and for some dynamic and stochastic games. This is extremely important research because it corrects a major flaw in models of repeated games and repeated game models are widely used in macro theory, industrial organization, international trade and many other areas of economic theory. The validity of repeated game models depend on the credibility of the agreements reached by the players. If players think they can escape punishment for violating agreements by renegotiating the agreement after they violate it, then they no longer have an incentive to abide by the agreements. Therefore, a model of repeated games without a theory of renegotiation is flawed because many different outcomes other than the one agreed upon are possible.

DESCRIPTION (Adapted from the Applicant's Abstract): The MR is a member of the nuclear receptor superfamily that is of fundamental importance in the regulation of blood pressure, electrolyte balance and responses to stress. The long-term goal of the applicant is to understand the molecular mechanisms of MR transcriptional control of gene networks in order to gain insight into its role in the pathogenesis of hypertension. Physiological observations imply that MR possesses two fundamental regulatory properties: 1) Transcriptional specificity; 2) Interaction with other steroid receptors, in particular a close relative, the glucocorticoid receptor (GR). However, MR and GR fall within a receptor subfamily, the members of which were thought, on the basis of molecular biological evidence,neither to possess distinct transcriptional specificities, nor to interact with each other. The applicant has shown that MR and GR do indeed possess specificity, acquired through protein-protein interactions with other transcriptional regulators at specific DNA sites. Furthermore, the applicant has recently shown that the receptors also interact with each other, in vivo, through heterodimer formation at a common DNA regulatory element. Thus, the goals of the work described in this proposal are: 1) To characterize the functional and physical interactions of MR with other members of its nuclear receptor subfamily. First, the applicant plans to establish the underlying mechanistic basis for MR-GR heterodimer formation. The applicant will functionally localize an MR-GR heterodimer interface by assessing the activity of chimeric receptors in cultured cells, and then, once localized, to characterize it using biochemical methods. He will also characterize interactions between MR and other members of its subfamily,particularly the androgen receptor; 2) To assess the role of non-receptor protein factors, in particular members of the AP1 family, in controlling MR transcriptional activity and specificity. The applicant has surprisingly identified functional interactions at the transcriptional level between MR and AP1 family member, c-Jun, which will be biochemically characterized. Functional interactions will also be further characterized; 3) To identify MR response elements (MREs) in candidate target genes in the central nervous system, smooth muscle cells and kidney collecting tubule. MR demonstrates specificity in both enhancement and repression of its genetic targets in these tissues, however, the role of MR-specific response elements in the implicated genes remains unknown. Thus, the applicant plans to identify MREs in candidate genes using a yeast enhancer trap method. He will then use a combination of molecular genetic and biochemical approaches to determine if MR's specificity in both enhancement and repression is controlled entirely by protein-protein interactions or if protein-DNA interactions play a role.

DESCRIPTION Corticosteroid actions in the brain are critical for blood pressure regulation, neural plasticity, and responses to stress. The two major classes of corticosteroids, mineralocorticoids and glucocorticoids, exert their effects primarily through intracellular receptors- the mineralocorticoid (MR) and glucocorticoid (GR) receptors- that elicit changes in cellular behavior through direct effects on the transcriptional activity of specific target genes. While in some brain sites, MR acts as a receptor for the mineralocorticoid, aldosterone, in other brain structures, including hippocampus, MR acts as a high affinity glucocorticoid receptor, mediating the stimulatory effects on neuronal excitability of low levels of corticosterone (the principal glucocorticoid in rats). High corticosterone levels, as seen in stress, depression, or Cushing's syndrome activate both MR and GR and suppress neuronal activity. While intermittent elevation of corticosterone is important for behavioral plasticity, chronic elevations can damage neurons, cause cognitive dysfunction and may be implicated in the pathogenesis of hypertension. Our long-term aims are to gain mechanistic insight into how MR and GR function as transcriptional regulators with distinct specificities in brain, initially focusing on hippocampal neurons. Towards this end we will: 1)Localize the N-terminal activation domain of rat MR and identify specific amino acids required for MR activity using a random mutagenesis screen in yeast. Once activation mutants have been identified they will be tested in transfection experiments using a hippocampal neuronal cell line. We will then go on to identify, clone and characterize hippocampal factors that interact specifically with MR (and/or GR) N-terminal regions; 2)Determine the mechanistic basis of hippocampal-specific expression and corticosteroid repression of the 5-HT1A receptor gene, a key steroid target gene in hippocampus. Using cultured hippocampal neurons and cloned 5-HT1A genomic DNA, we will identify hippocampal-specific and steroid-regulated response elements;3) Identify and characterize target genes in hippocampus. We will being by characterizing the expression pattern and steroid response of Synaptotagmin I and Tmp21, genes recently found by us to be steroid regulated in hippocampus. We will then to go on to study the transcriptional regulation of these genes using approaches similar to those described above for 5-HT1A. Finally, we will use a PCR-based cDNA subtraction method to identify genes differentially regulated by MR and GR.

A human gene probably responsible for Batten s disease has recently been cloned. This is a very serious disease causing neurodegeneration and death in many children. The nature of the defect causing the disease is not known. This human gene, CLN3, has a homologue in yeast, BTN1. A btn1 null mutant is viable, but does have a subtle phenotype in that under some conditions the mutant is somewhat resistant to D-(-)three-2-amino-1-[p-nitropheny1]-1,3-propanediol (ANP). The investigators propose to investigate Batten s disease using yeast and the BTN1 gene as a model system. The P.I. s will investigate expression of normal and mutationally altered forms of Cln3p and Btnip, and will determine critical regions of the proteins. The subcellular location of Btnip will be determined using GFP or epitope tags. Proteins interacting with Btnip will be found using the two-hybrid system, as well as by isolating possible complexes using a GST-Ttnip fusion or Btnip tagged with poly-histidine. Genetic screens will be done to find other mutants resistant to APN, to find suppressors of btn1, and to find mutations that are synthetically lethal with btn1.

DESCRIPTION: (Adapted from investigator abstract) Batten disease is a common progressive neurodegenerative disease in children, with an incidence as high as one in 12,500 live births. The first major consequence of Batten disease is the onset of blindness due to retinal degeneration, followed by deteriorating psychomotor and mental function and premature death. Although the gene responsible for this disease, CLN3, was positionally cloned in 1995, little has been determined about the function of CLN3 in mammalian cell models. The major goal of the proposed research is to develop and utilize a neuronal cell model for the study of Cln3-mediated neurodegeneration. The investigator will use intact retinue and a retinal cell culture model, derived from the Cln3 knockout mouse for this purpose. They propose to examine the survival of retinal cells, and whether Cln3 mice undergo photoreceptor-specific apoptosis, using DNA laddering, DNA fragmentation and ssDNA analysis. Furthermore they will examine by electron microscopy the ultrastructure of retinal cells, in particular for the occurrence of lysosomal curvilinear or fingerprint-like deposits of membrane structures characteristic of Batten disease. The most promising studies into the function of CLN3 have come from the yeast homolog, Btn1p. It has been established that deletion of Btn1p results in a more acidic vacuole (lysosomal), and that this altered pH is corrected by manipulating proton pumping at the plasma membrane through increased expression of HSP30, and through increased expression of BTN2, which is implicated in a novel endocytic pathway. The investigator intends to measure lysosomal pH in primary retinal culture from Cln2 and control normal mice and to determine whether pH homeostasis is disturbed in Cln3 cells. Endocytosis will be monitored by confocal microscopy through the uptake of fluorescent dyes that associate to acidified compartments, and the recycling of tagged transferrin conjugates in both Cln3 and control normal primary culture. Furthermore, for the first time this model system will allow the test of a potential therapeutic agent, chloroquine, which has been shown in the yeast model to reverse the phenotypic effects of deleting Btn1p. Overall this proposal will provide for the first time a retinal cell based model-system for the study of neurodegeneration, morphological and biochemical abnormalities characteristic of Batten disease and to test the efficacy of chloroquine as a potential therapy.

Extracellular fluid volume in vertebrates is regulated primarily by the mineralocorticoid hormone, aldosterone, which stimulates Na+ absorption in tight epithelia, particularly the kidney collecting duct. This process plays a pivotal role in the control of blood pressure, and its dysregulation, either due to excess hormone or to target cell defects, is an important cause of hypertension. Although aldosterone is known to act through intracellular receptors to regulate gene transcription, the mechanistic basis of its effects on Na+ transport remains poorly understood. The earliest effect of aldosterone is to stimulate apical membrane ENaC insertion and/or increase ENaC open probability without altering ENaC subunit gene transcription. Recently, significant progress was made in understanding the molecular basis of aldosterone-regulated Na+ transport with the discovery that serum and glucocorticoid-regulated kinase (SGK) is an aldosterone-induced ENaC modulator. SGK mRNA and protein are rapidly increased by aldosterone in kidney collecting duct and wild type SGK stimulates ENaC-mediated Na+ current more than seven-fold in Xenopus oocytes. Interestingly, an SGK mutant that cannot bind ATP ("kinase-dead mutant") not only fails to increase Na+ current but actually decreases it. These opposite functional effects of wild type and mutant SGK are correlated with striking opposite effects on ENaC plasma membrane localization in oocytes. Moreover, antagonism of the phosphatidylinositol-3-kinase (PI3K) signaling pathway has an effect on SGK activity and ENaC-mediated Na+ transport similar to the kinase-dead mutant. These findings strongly implicate SGK as an aldosterone-induced regulator of ENaC activity. However, it remains unknown how SGK modulates ENaC activity in CD cells and what role P13K plays in this process. With these observations in mind, the major goals of the work described in this proposal are to: (1) Determine if SGK is necessary and/or sufficient to mediate the effects of mineralocorticoids on Na+ transport in cultured CD cells. We will express wild type SGK and the kinase-dead mutant in cultured CD cells under the control of a heterologous promoter so that their levels can be controlled independently of mineralocorticoids and their selective effects on Na+ transport examined. We will also determine if mineralocorticoid-stimulated Na+ transport in collecting duct cells is delayed or blocked by disruption of SGK expression or activity. (2) Examine the role of ENaC phosphorylation and apical membrane relocalization in SGK- stimulated Na+ transport. We will determine if SGK directly interacts with and/or phosphorylates ENaC subunits. We will determine if SGK stimulates ENaC localization to the apical membrane of cultured cells. We will use mutagenesis to examine SGK structure-function relationships in oocytes and cultured cells. (3) Examine the role of PI3K in activating SGK and in mediating the effects of mineralocorticoids on Na+ transport. We will examine the effect on ENaC-mediated Na+ transport of blocking PI3K activity. We will determine if constitutively active mutants of SGK, or downstream products of PI3K, overcome the effect of PI3K inhibition on Na+ transport. Together, these studies promise to lead to a greater understanding of the mechanistic basis of blood pressure regulation and potentially to new avenues of treatment for salt sensitive hypertension.

By arriving at self-enforcing agreements, agents in an ongoing strategic situation create surplus that benefits them both. Little is known about how that surplus will be divided. This project concerns the role of reputation formation in such environments. It studies a model in which players entertain the slight possibility that their opponents may be subject to a variety of behavioral biases. Happily, it seems that these informational complications make it easier to predict what will usually happen in the repeated game. There emerges a theory of bargaining in repeated games that strengthens some links between cooperative games and noncooperative game theory.

The proposal also addresses dynamic games, in which the different periods of play are con-nected by "state variables" such as capital stock or inventories. Here one can examine some of the most important problems in industrial organization, including collusion in the face of potential entry. One of the goals is to derive a generalization of the "Nash bargaining solution with threats" that can be applied in dynamic games.

DESCRIPTION: The gene responsible for juvenile neuronal ceroid lipofuscinosis, CLN3, (also denoted CLN3 or Batten's disease) has a homolog in the yeast Saccharonzyces cerevisiae, designated BTNI. Disruption of BTN1 revealed a distinct phenotype, resistance to ANP (or D-(-)-threo-2-amino-1- aboutp-nitrophenyl]-l,3-propanediol). This phenotype is complemented by plasmids containing either the yeast BTN1 gene, or the human CLN3 gene. Thus, the human CLN3 gene functions in yeast. We have established that Btn1p is a vacuolar protein and that btn1-delta strains have an elevated activity of the plasma membrane H+-ATPase in the early phases of growth due to a decreased vacuolar pH. The activity of the plasma membrane H+-ATPase and vacuolar pH return to normal in the later phases of growth, through altered gene expression. Furthermore, we have demonstrated that chloroquine can also return vacuolar pH to normal in btnl-delta strains. Our long term goals are to determine the essential features of CLN3 and Btn1p in yeast, to determine other genes and proteins associated with the BTN1 pathway, and to establish whether yeast can provide a model for phenotypic reversal of Batten disease. Altered vacuolar pH regulation in btnl-delta strains will be investigated with a number of studies, including measurement of the assembly and disassembly of vacuolar H+-ATPaso and analysis of vacuolar content to see if altered pH is due to altered transport of an as yet unidentified substrate. Expression of normal and mutationally altered forms of CLN3 and Btn1p in yeast will be examined to determine the critical regions of the proteins. We propose to identify the proteins interacting with Btnlp, and Btn2p, a novel component of endocytosis with elevated expression in btn]-A strains, which may alter vesicular delivery to the vacuole, using the two hybrid system, as well as by isolating possible complexes using a Btn1p or Btn2p tagged with poly-histidine. Genetic approaches will be utilized for isolating and characterizing genes resembling or interacting with BTN1, including suppressors of btn 1-delta and synthetic lethality/enhancement with b/ni-LI and btn2-delta. A newly uncovered gene, BTN3, and R VS161, are synthetically lethal and synthetically lethal on a non-fermentable carbon source, respectively, in btn1-delta strains and will be studied for physical and genetic interaction with Btn1p. The human homolog to RvsI6lp is amphiphysin, an auto-antigen associated to the neurological disorder, Stiff Man Syndrome. An understanding of the function of Btnlp (and Cln3p) in yeast could furnish valuable information on Batten's disease in humans.

[unreadable] DESCRIPTION (provided by applicant): The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are hormone activated transcription factors that impact on the function of virtually every mammalian tissue. This proposal focuses on the mechanistic basis of MR and GR regulation of gene expression, particularly in systems with importance to blood pressure regulation and stress responses. The following hypotheses motivate our future goals: 1) Non-receptor cellular factors are important determinants of the robust and transient induction of a subset of MR- and GR-regulated genes, in particular, SGK1; 2) Synergy control is an important determinant of MR and GR activities, particularly in regulating phenylethanolamine N-methyltransferase (PNMT), the glucocorticoid-regulated enzyme that catalyzes the conversion of norepinephrine to epinephrine. Synergy control is determined by interactions between the DBD and N-terminal domains and requires receptor modification by non-receptor factors, which include the recently discovered ubiquitin-like protein, SUMO1 Based on these considerations, we will: 1) Determine the mechanism of rapid induction of SGK1 gene transcription by MR and GR. We will: (a) identify and characterize hormone response elements involved in the induction of SGK1 gene transcription by MR and GR; (b) determine the role of chromatin modification and transcriptional synergy in the induction of SGK1 gene transcription. 2) Determine the mechanism of down-regulation ("deinduction") of SGK1 gene expression. We will: (a) Characterize the time course of SGK1 mRNA regulation in rat kidney; (b) determine if new protein synthesis is required for the deinduction of SGK1 gene expression in CCD cells; (C) assess the role of chromatin and non-chromatin factors in SGK1 deinduction; (d) examine the role of mRNA stability in SGK1 deinduction. 3) Determine the mechanistic basis of synergy control and its role in MR and GR activity and specificity. We will: (a) functionally characterize the 5' regulatory region of PNMT; (b) identify cellular factors required for controlling MR and GR synergistic activity; (c) examine the determinants and consequences of SUMO1 modification of GR and MR. Together, these studies will extend our understanding of the gene regulatory mechanisms that underlie blood pressure regulation and responses to stress and potentially lead to new avenues for the prevention and treatment of disease.

Batten disease is the most common progressive neurodegenerative disorder in children, with an incidence as high as one in 12,500 live births. Batten disease results from mutation of CLN3, and is characterized pathologically by the accumulation of autofluorescent hydrophobic material in the lysosome of neurons and other cell types. However, the mechanism driving these cellular alterations and the manner in which they relate to the neurodegeneration in Batten disease is unknown. Individuals with Batten disease, and a cln3-knockout mouse model for Batten disease have a circulating autoantibody to glutamic acid decarboxylase (GAD65) that is inhibitory to this enzyme's ability to convert glutamic acid to 7-aminobutyric acid (GABA). The GAD65 autoantibody associates with the brain in cln3-knockout mice, inhibits the activity of GAD, and results in a subsequent elevation of glutamate and altered expression of genes involved in the regulation and utilization of glutamate. The major goal of this proposal is to establish and characterize the contribution of the GAD65 autoantibody to the pathogenesis of Batten disease. As the model for this study is a genetically defined I knockout mouse, we will establish whether the presence of a GAD65 autoantibody is simply an epl- phenomenon, and whether it contributes directly to Batten disease. We shall: (i) construct cln3-knockout mice that are unable to mount an immune response to GAD65 by crossing to MuMT and C-alpha knockout mice, which lack the ability to generate B-cells, and CD4+ and CD8+ T-cells, respectively. This will enable us to determine whether the autoantibody contributes to the pathology of Batten disease. (ii) test by passive transfer of GAD65 autoantibodies to normal mice the degree of pathogenicity on the CNS mediated by these autoantibodies. Also, by transfer of GAD65 autoantibodies to immune deficient mice we will determine if the cln3-defect allows for preferential deposition of IgG in the brain (iii) define the inflammatory response to the presence of autoantibodies and the participation of the innate immune system in the disease process in cln3- knockout mice by examining cytokines such as IL-1 and TNF, complement proteins and microglial activation, respectively. (iv) characterize cell type specific molecular effects that elevated glutamate and exposure to the autoantibody have on primary neuronal culture from cln3-knockout mice.

Economists have developed a wide variety of models to predict how economic agents will interact strategically over time. However, many of these models suffer from a lack of predictive power because multiple equilibria are predicted by the standard equilibrium definitions used in game theory. Recent research shows that in some special classes of games, adding a richer set of possible behavior to the game allows reputations to form that dictate a single outcome. This project will apply these ideas to a large and important class of games, those that model bargaining behavior.

The project will place particular emphasis on understanding what happens when bargainers are not fully informed about some of the opponent's goals or payoffs. The goal is to develop stronger and more robust predictions in this central area of economic theory.

Broader implications include the ability to conduct much stronger empirical tests of economic models. Once tested, the models will allow economists to give much better advice to policy-makers and others who use bargaining to reach agreements between nations, interest groups, firms, and individuals.

[unreadable] DESCRIPTION (provided by applicant): Hormone-regulated Na transport in tight epithelia is essential for maintenance of circulatory volume and regulation of the ionic milieu in mammals. SGK1 is an aldosterone-regulated PI3-kinase-dependent serine-threonine kinase that plays an important role in mediating hormonal regulation of Na transport through its effects on trafficking of the epithelial Na channel (ENaC), the rate limiting step in Na+ reabsorption in tight epithelia. SGK1 phosphorylate and inhibits Nedd4-2, a ubiquitin ligase that inhibits ENaC primarily by altering its trafficking and degradation, thus leading to ENaC accumulation in the plasma membrane. We recently found that SGK1-mediated phosphorylation of Nedd4-2 triggers recruitment of a chaperone protein of the 14-3-3 family. These observations and other recent studies suggest the following hypotheses: 1) Interactions with phosphoinositide lipids activate SGK1 and help direct it to the appropriate cellular location to inhibit Nedd4-2 and stimulate ENaC; 2) SGK1 inhibits Nedd4-2 by stimulating its interaction with the chaperone inhibitor 14-3-3sigma, or another 14-3-3 protein; 3) The opposing effects of Nedd4-2 and SGK1 are manifested in changes in ENaC endocytosis and endosomal localization. We propose the following aims: 1) Determine the functional roles and physical basis of SGK1 interactions with phosphoinositides. We will determine which phosphoinositides interact with SGK1 using phosphoinositide lipids fixed to nitrocellulose membranes, and incorporated into liposomes. We will determine the relationship of phosphoinositide binding and function of this novel domain by examining the effect of SGK1 mutants on ENaC-mediated Na+ currents and on in vitro kinase activity. We will determine the role of the SGK1 phosphoinositide interaction domain in targeting SGK1 to specific cellular compartments in response to PI3K activation. 2) Determine the role of 14-3-3 proteins in inhibiting Nedd4-2. We will test the ability of SGK1 to stimulate ENaC-mediated Na+ currents in the presence of Nedd4-2 mutants that have their 14-3-3 interaction motif disrupted. We will determine which 14-3-3 isoforms are expressed in kidney CD and in cultured CCD cells, and interact with Nedd4-2 in cultured CCD cells using isoform specific antibodies in coimmunoprecipitation assays. We will determine the effect on aldosterone-stimulated Na+ transport of eliminating expression of specific 14-3-3 isoforms using siRNA. 3) Determine the effect of SGK1 and Nedd4-2 on ENaC trafficking, and characterize the mechanistic basis of this effect. We will use live cell staining of epitope-tagged transfected ENaC to examine the effects of SGK1 and Nedd4-2 on plasma membrane localization of ENaC. We will determine the role(s) of exocytosis and endocytosis in regulated ENaC trafficking by examining the effects of inhibitors and dominant negative dynamin on cell surface ENaC, and examine the correlation of ENaC currents and apical trafficking by biotinylation of endogenous ENaC in a model CCD cell (mpkCCD). We will establish systems for examining ENaC post-endocytosis. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Identification of biomarkers for neurodegeneration and for progression of neurodegeneration would be important for diagnosis, for following clinical progression of disease and, most importantly, for identification of, and monitoring efficiency of treatments. We propose to systematically characterize blood serum markers in a well defined genetically inherited neurodegenerative disease. The juvenile neuronal ceroid lipofuscinoses (JNCL or Batten disease) is a lysosomal storage disorder that represents one of the most common groups of neurodegenerative diseases of childhood. Clinical manifestations that include progressive blindness, decline in cognitive function, seizures, and motor dysfunction culminating in premature death. The objective of this proposal is to identify candidate protein biomarkers in serum of NCL patients, and more specifically in the juvenile subtype (JNCL or Batten disease), and develop a panel of disease biomarkers that can aid in assessing disease progression and possible diagnosis of this disorder. We have serum samples from 60 patients with genetic confirmation of JNCL at various ages, and state of disease progression. In addition, we have serum samples from a cohort of patients drawn over a period of 5 years that will allow for comparative analysis. A sub-set of these samples include serum taken at a pre-symptomatic time point. In addition, through the Batten Disease Diagnostic and Clinical Research Center we have clinical profiles in the form of the Unified Batten Disease Rating Scale on patients whose serum will be used for this study. We propose two aims: (i) An exploratory characterization of the protein species present in blood serum from JNCL patients and unaffected subjects will be performed using two-dimensional gel electrophoresis (2DGE) to identify differentially expressed proteins. (ii) Profiling of the leukocyte proteome by 2DGE may reveal the activation and expression of specific inflammatory response related proteins that can be used as disease biomarkers and potential indicators of disease progression. As our understanding of the neurodegenerative pathways in the NCLs increases, it is beginning to become apparent that the NCL disorders may also share some common pathological features with other, more common, neurodegenerative diseases such as Alzheimer's and Parkinson's. Thus our studies have the potential to broadly impact other neurodegenerative diseases and has wide ranging significance to CNS disease. Completion of the proposed studies would likely prove valuable in identification of markers of more complex neurodegenerative diseases [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The Neuronal Ceroid Lipofuscinoses (NCLs) are the most common group of neurodegenerative diseases of childhood with a frequency of 7-10 per 100,000 live births. They are progressive and fatal, reducing patients' lives to very few meaningful years of early normal development followed by progressive deterioration of motor, visual, and mental skills, confinement to bed and complete helplessness further complicated by frequent epileptic seizures and ultimately premature death. Over the past decade, major discoveries elucidating this enigmatic group of lysosomal disorders have been accomplished. Currently, eight different genetic loci have been postulated. Two early clinical types, CLN1 and CLN2, are caused by deficiencies of two lysosomal enzymes, respectively, palmitoyl protein thioesterase 1 (PPT1) and tripeptidyl peptidase 1 (TPP1). Four NCL types, CLN3, CLN5, CLN6 and CLN8, respectively, are caused by mutations in genes encoding four new transmembrane proteins, the physiological functions of which are still unknown. In addition, deficiency of cathepsin D is known to cause a congenital form of NCL in sheep was reported to result in congenital NCL in humans. The International Congress has alternated between the USA and Europe for the past 20 years at an interval of 2-3 years, and the last Conference was held in Helsinki, Finland in June of 2005. It represents the main forum for the exchange of data and ideas about NCL research and this Conference is the only one of its kind. The Congress will be held at the Hyatt Hotel, Rochester, NY, USA from July 14th to 17th, 2007. For the first time this Congress will be held in conjunction with the yearly family conference for families with Batten Disease, namely the National Batten Disease Support and Research Association (BDSRA) meeting, July 12th 15th, at the same location. Thus, there will be one overlapping session for both meetings on therapeutic strategies, the topic of most interest to parents with children with Batten Disease. The aims of this congress are: 1. Provide a forum for presentation of the latest results by all the major international groups of researchers in Batten disease and to receive input from leading scientists whose work is relevant to understanding Batten disease. 2. Encourage students, junior investigators and minorities to present their work at the conference. 3. Encourage new researchers in ancillary fields to present their work on Batten gene products and to collaborate with other research groups. 4. To translate advances at the basic research level into therapeutic strategies and improved patient care. 5. To promote interactions among NCL investigators and affected children their families. The Congress will aid interactions of researchers that will hopefully expedite a greater understanding for these diseases. The Neuronal Ceroid Lipofuscinoses (NCLs) are the most common group of neurodegenerative diseases of childhood with a frequency of 7-10 per 100,000 live births. The Congress will aid interactions of researchers that will hopefully expedite a greater understanding for these diseases. [unreadable] [unreadable] [unreadable]

This research addresses one of the most ubiquitous problems in social science: how do two parties divide the gains that can be achieved through cooperation? The two parties may be neighbors discussing drainage, fencing or greenery, a corporation and its supplier considering investments that would enhance their business relationship, a union and a firm bargaining over wages and benefits, or two countries negotiating a trade agreement.

Nobel Laureate John Nash showed almost fifty-five years ago that, for a simple class of bargaining problems, two branches of game theory ("cooperative" and "noncooperative") agreed on how the surplus from cooperation should be divided. Since that time, numerous developments in economics have lent support to the results Nash obtained. A major limitation of the analysis is that it is restricted to "static" situations, where the two parties' actions, and the world they inhabit, are completely unchanging. In other words, there is no scope for thinking about investments, learning by doing, economic growth, technological change or global warming.

This Proposal moves the investigation from static strategic settings to dynamic situations called stochastic games. These models are sufficiently flexible to capture any of the phenomena mentioned above. The first step is to rephrase the classic results of Nash in an intertemporal (albeit unchanging) setting. Next, one needs to allow for all the complexities of a stochastic game, and express the strategic situation as a family of interlocking Nash bargaining problems. If that can be done, a full solution should emerge in a manner analogous to what happens in the original static analysis of Nash.

Nash shed light not only on how the benefits of cooperation would be shared, but also on what each party will do to gain maximum advantage in negotiations. This research project will similarly generate predictions regarding behavior before a contract is signed. Applications include the speed at which patent races are fought, destructive behavior by negotiating parties, and the development of aggressive weaponry

The broader impact of this research is the development of a new game theory tool that will be of great use to social scientists and policymakers who seek to understand how individuals and nations compete over time.

The University of Rochester is offering a REU Site program in Cellular and Molecular Biology to promising undergraduates. The goal is to encourage students to pursue graduate education and careers in the Biological Sciences. Participants will experience the excitement of hands-on, hypothesis-driven research under the mentorship of a faculty. The Site will recruit underrepresented minority students and students from smaller, non-research-intensive, undergraduate colleges. Students will be selected from a large national pool of applicants, and will participate in a 10-week independent research project under the supervision of a faculty mentor. Students will attend research seminars as well as seminars on ethics, graduate school and career issues, and will also be offered the opportunity to participate in a GRE prep course and in discussion groups. Participants will present their research in poster form at a mini-symposium at the end of the summer. Finally, continuation of research work during the academic year will be strongly supported and used as a mechanism to support linkages between UR and other colleges. More information is available by contacting Dr. David A Pearce at David_Pearce@urmc.rochester.edu, or by visiting the program website at http://www.urmc.rochester.edu/GEBS/summer.htm.

DESCRIPTION (provided by applicant): Batten disease, a childhood neurodegenerative disorder results from mutations in CLN3. Patients with Batten disease and a cln3-knockout mouse have autoantibodies to several proteins including GAD65. Moreover, these autoantibodies have been shown to associate to the brain in post-mortem Batten disease tissue and in the brain of cln3-knockout mice. In the previous cycle of this grant we had proposed to establish that the autoimmune component contributed to the pathogenesis of Batten disease. In the progress report we detail publications and preliminary data that confirm a neuroimmune component to the pathogenesis of Batten disease. In addition, completion of the main aim of the original proposal, namely construction of cln3 knockout mice that are also homozygous for deletion of MuMT (the immunoglobulin mu chain) or C-alpha-mice (the alpha chain of the T-cell receptor) so they therefore lack the ability to generate B-cells, and CD4+ and CD8+ T- cells, respectively, confirms an amelioration of deteriorating motor coordination resulting from mutation of CLN3. The goal of this competitive renewal is to more specifically identify the components of the autoimmune response that contribute to the pathogenesis of Batten disease. Having established that genetic suppression of the immune system slows disease progression, we will establish whether drug mediated immune suppression using a drug that is tolerable to children can slow progression of disease. Finally, we present published and preliminary data that the novel anti-GAD65 response forms a part of a wider autoimmune response. We will therefore identify the multiple autoantigens in Batten disease to gain a greater understanding of how the autoimmune response imparts its effect on neuronal function and to develop further targets for treatment strategies. We shall (i) define the inflammatory response to the presence of autoantibodies and the participation of the innate immune system in the disease process in cln3-knockout mice by examining cytokines such as IL-1 and TNF-a, complement proteins and microglial activation, respectively. (ii) We will treat cln3-knockout and control mice with immunosuppressant Mycophenolate Mofetil (CellCept) that is suitable for children, and could provide the foundation for a treatment for Batten disease. Finally we will identify additional autoantigens in batten disease by proteomic approaches. This work may lead to treatment strategies for the fatal disorder, Batten disease. Moreover, the described approach may be applied to other rare genetically inherited disorders. PUBLIC HEALTH RELEVANCE: The proposed research is relevant and significant as it has the potential to increase our understanding of Batten's Disease and ultimately impact therapeutic strategies for this disease.

Most of the models economists use to predict outcomes of bilateral bargaining assume symmetric information: the two bargainers know everything they need to know about one another. But bargaining in the real world is usually conducted under two-sided asymmetric information: both parties wish they knew more about the other side's goals, priorities, strengths and weaknesses. These situations have eluded economists' grasp for many decades.

This award funds work on a new approach to modeling bilateral bargaining with two-sided asymmetric information. It borrows heavily from the reputation formation literature initiated twenty-five years ago and first used to study entry into previously monopolized markets. The idea is that recognizing bargainer's incentives to form reputations (for being stubborn, or retaliatory, and so on) not only enriches the analysis, but also gives clearer results. The hope is to better understand how bargainers in these rich informational environments behave.

The kinds of bargaining problems to be studied arise between countries, between regulators and firms, between suppliers and producers, and between firms and unions. A better understanding of bargaining will provide valuable insights for the formulation of labor law, international law, and regulatory policy, among many other applications.

DESCRIPTION (provided by applicant): Aldosterone-regulated sodium transport in the kidney distal nephron is essential for fluid and electrolyte homeostasis and blood pressure regulation in mammals. Aldosterone acts through the mineralocorticoid receptor (MR) to control transcription rates of a group of target genes, which have direct and indirect effects on key ion transporters, such as the epithelial sodium channel (ENaC). We recently identified the scaffolding protein, GILZ1 (glucocorticoid-induced leucine zipper protein, isoform 1), as an aldosterone-regulated gene product, which acts within distal nephron cells to stimulate ENaC cell surface expression. Our published and unpublished data demonstrate that GILZ1 interacts with ENaC (alpha and beta subunits), and a group of proteins already known to be involved in ENaC regulation. These ENaC-regulating GILZ-interacting proteins (GILZIPs) include SGK1 (a phosphatidyl inositol-3-kinase-dependent kinase, which stimulates ENaC), Nedd4-2 (a ubiquitin ligase, which inhibits ENaC), and Raf-1 (the master regulator of the Raf-MEK-ERK signaling module, which has effects on a wide array of cellular processes, including inhibiting ENaC). Based on these and other data, we suggest the novel hypothesis that these proteins are assembled into an ENaC regulatory complex (ERC), the composition of which is modulated by aldosterone, in a GILZ1-dependent fashion. In order to explore this general hypothesis, we propose the following specific aims: 1) Determine the molecular identity of the components of the ENaC regulatory complex (ERC), and the role of GILZ in controlling its composition. GILZ1 interacts with ENaC as well as with several ENaC regulatory proteins (which we have termed "GILZIPs"). We hypothesize that a multi-protein ENaC regulatory complex (ERC) is formed, and that GILZ1 is a key component of this complex, and recruits activating proteins into the complex. We will use biochemical and yeast two-hybrid approaches to examine this hypothesis, and determine the role of GILZ in ERC composition over time in response to aldosterone. 2) Determine the functional effects of GILZ1 and GILZIPs on endogenous ENaC-mediated Na+ currents in a cortical collecting duct (CCD) cell line, using gene specific knockdown and over-expression. 3) Determine the effect of GILZ1 and GILZIPs on ENaC surface expression and processing in cultured cells. We will examine the effects of GILZ1 and GILZIPs (particularly SGK1, Nedd4- 2 and Raf-1) on ENaC surface expression using biotinylation in cultured kidney epithelial cells;examine the effects of GILZ1 and GILZIPs on ENaC proteolytic processing;examine the effect of GILZ1 on processing of endogenous ENaC in mpkCCD cells. This work will elucidate the mechanistic basis of ENaC regulation by aldosterone, a process of fundamental importance to the regulation of BP and ECF volume. Moreover, it will shed new light on how specific regulation of ion transport can be achieved without unleashing undesirable effects on other processes, which is a central question in cell biology. PUBLIC HEALTH RELVANCE: The blood pressure-regulating hormone aldosterone is involved in controlling salt retention by the kidney. Abnormalities of this hormone are a major cause of hypertension, and are implicated in fluid overload states such as congestive heart failure. This proposal examines the molecules involved in mediating aldosterone signaling, and how it causes salt retention in the kidney. This work will shed new light on the basic mechanisms, and provide avenues for better treatment of high blood pressure and edema.

DESCRIPTION (provided by applicant): The Neuronal Ceroid Lipofuscinoses (NCLs) are the most common group of neurodegenerative diseases of childhood with a frequency of 7-10 per 100,000 live births. They are progressive and fatal, reducing patients'lives to very few meaningful years of early normal development followed by progressive deterioration of motor, visual, and mental skills, confinement to bed and complete helplessness further complicated by frequent epileptic seizures and ultimately premature death. Over the past decade, major discoveries elucidating this enigmatic group of lysosomal disorders have been accomplished. Currently, eight different genetic loci have been postulated. Two early clinical types, CLN1 and CLN2, are caused by deficiencies of two lysosomal enzymes, respectively, palmitoyl protein thioesterase 1 (PPT1) and tripeptidyl peptidase 1 (TPP1). Four NCL types, CLN3, CLN5, CLN6 and CLN8, respectively, are caused by mutations in genes encoding four new transmembrane proteins, the physiological functions of which are still unknown. In addition, deficiency of cathepsin D is known to cause a congenital form of NCL in sheep was reported to result in congenital NCl in humans. The International Congress has alternated between the USA and Europe for the past 20 years at an interval of 2-3 years, and the last Conference was held in Rochester, NY, USA in July, 2007. For the first time this Congress was held in conjunction with the yearly family conference for families with Batten Disease, namely the National Batten Disease Support and Research Association (BDSRA) meeting. The proposed next Congress will be held in June 2009 in Hamburg, Germany. The aims of this congress are: 1. Provide a forum for presentation of the latest results by all the major international groups of researchers in Batten disease and to receive input from leading scientists whose work is relevant to understanding Batten disease. 2. Encourage students, junior investigators and minorities to present their work at the conference. 3. Encourage new researchers in ancillary fields to present their work on Batten gene products and to collaborate with other research groups. 4. To translate advances at the basic research level into therapeutic strategies and improved patient care. 5. To promote interactions among NCL investigators and affected children their families. PUBLIC HEALTH RELEVANCE: The Congress will aid interactions of researchers that will hopefully expedite a greater understanding for these diseases.

A Research Experience for Undergraduates (REU) Sites award has been made to Sanford Research/USD in collaboration with Augustana College to provide research training for 10 students, for 10 weeks during the summers of 2013-2015. The Augustana College REU Site in Cell and Molecular Biology focuses on cell and molecular biology, with hypothesis-driven projects that include investigation of cell signaling events via characterization of macromolecular interactions and proteomic, genomic, and metabolomic changes across a variety of model organisms. REU students will directly contribute to the understanding of these areas of research. Sanford Research and Augustana College have 19 research mentors with exciting, hands-on projects that range from neuroscience to yeast biology. Each project allows students to learn modern molecular methods. Students will also gain valuable experience by attending career development workshops that cover professional integrity in research, the graduate school application process, careers in science, GRE-preparation, the importance of increasing diversity in research, and how to balance family and career. Students will give an oral presentation of their project at the beginning of the summer, a poster presentation at the end of the summer, and are encouraged to present at and attend national meetings. REU students will be exposed to the cutting-edge facilities and equipment that are housed amongst the various departments and cores at Sanford Research and Augustana College. Recruitment is carried out directly via faculty mentor visits to the students' institutions and attendance at undergraduate conferences, along with email and social media, with an aim to reach out to students who would not usually be exposed to such an opportunity. Students are selected based upon academic record, work ethic, scientific potential, and evidence of enthusiasm for participating in cell and molecular biology research. Upon completion of the program, alumni are surveyed to measure the influence that participation in the program had on their academic performance, scientific interests, and long-term career path. The REU common assessment tool at www.bioreu.org will be used to evaluate program impacts. More information is available by visiting http://www.sanfordresearch.org/education/undergraduates/spur/, or by contacting the PI (Dr. David Pearce at David.Pearce@sanfordhealth.org) or co-PI (Dr. Seasson Vitiello at Seasson.Vitiello@augie.edu).

Bargaining between two agents is one of the most central economic problems, and it often occurs under asymmetric information: one or more of the agents lack some relevant information about the opponent (how she weighs current versus future consumption, how much she values some commodity to be exchanged, what other options she might have, and so on). Despite the importance of this problem, it resists traditional economic analysis, and economists do not view the models currently available as offering adequate treatments of the subject.

This proposal studies a number of bargaining situations when players have the possibility of building reputations for being "tough". We hope to show that in the case of one-sided asymmetric information about a player's patience, very modest assumptions suffice to produce strong predictions about behavior. Turning to one-sided asymmetric information about valuations, the goal is to allow for a rich variety of behavioral perturbations and still obtain strong conclusions. We hope that the results of this research will ultimately help in the study of two-sided asymmetric information: neither player is sure about how much the other values an object to be sold, for example. The literature offers virtually no guidance about what to expect in such situations and this proposal moves research toward areas considered impenetrable by generations of researchers.

The broader impact of the proposal rests on the potential for the results to be applied to real-world bargaining situations. The economic and political landscapes are littered with important examples, from collective bargaining between union and firm to negotiations between a country and its neighbor regarding timber, pipelines, tariffs, or defense accords. Understanding the basic underlying principles would improve our ability to predict outcomes, and to design policies and institutions to promote more favorable results.