David Stern, Ph.D. - US grants

Many oncogenes act by disrupting pathways regulated by peptide growth factors. These oncogenes transform cells by causing inappropriate production of growth factors or by encoding mutated growth factor receptors. These mechanisms for transformation will be investigated using two different experimental systems. The neu oncogene will be studied to determine the mechanism of activation of an oncogenic growth factor receptor. A model autocrine system, in which cells are transformed because they constitutively produce EGF, will be used to develop methods for reverting transformation mediated by autocrine circuits and to identify genes that cooperate with the EGF gene in cell transformation. The neu oncogene encodes a protein designated p185 that is closely related to, but distinct from, the receptor for EGF. The product of the neu proto-oncogene is itself likely to be a growth factor receptor. Because normal and transforming forms of p185 differ by only a single amino acid replacement, this system is ideal for investigating the mechanism of oncogenic activation of a growth factor receptor. The effects of the activating mutation on activity and specificity of the p185 kinase, sites of phosphorylation of p185, and oligomerization will be examined. These studies will yield insights into the mechanism of signal transduction by the tyrosine kinase family of growth factor receptors. Cells expressing a transfected EGF gene at high levels are tumorigenic but differ from ras-transformed cells in being anchorage-dependent and growing only slowly as tumors. These partially transformed cells will be used to study multi-step carcinogenesis in which one of the affected genes induces production of EGF-like factors. Genes that are known to act synergistically with EGF in stimulating cell proliferation will be tested for the ability to cooperate with EGF in tumorigenesis. Transformation by the EGF expression plasmid is mediated by an extracellular autocrine circuit that can be interrupted with an anti-EGF monoclonal antibody. EGF-transformed cells will be used as a model therapeutic system for tumors mediated by autocrine circuits. These cells will be used to test the efficacy of anti-EGF antibodies, anti-EGF receptor antibodies, and EGF antagonists in blocking tumorigenicity.

[unreadable] DESCRIPTION (provided by applicant): The gene encoding the receptor tyrosine kinase (RTK) HER2/neu/ErbB2 is an important human breast cancer oncogene, and a validated therapeutic target. We hypothesize that optimal development and use of ErbB2-targeted therapeutics will require a thorough understanding of the normal biological functions of ErbB2 and its regulation by interactions with other ErbB family receptors. Aim 1 continues our analysis of normal functions of ErbB2 and ErbBS in mammary gland development. Aim 2 investigates a new hypothesis in which excessive signaling through ErbB2 drives genomic instability in breast cancer through actuation of DNA checkpoint signaling and selection for checkpoint bypass. Aim 3 will evaluate phosphorylation markers for measuring for ErbB and pathway activation in human cancer n the best-case setting of core biopsies. The significance of these studies extends well beyond breast cancer, since the Epidermal Growth Factor Receptor (EGFR) and ErbB2 are mutated or overexpressed in many types of adenocarcinoma. Since ErbBs are forefront targets for new RTK inhibitors in cancer treatment, success of our efforts to merge ErbB biology with tumor studies will pave the way for similar approaches to cancers caused by other RTKs. [unreadable] [unreadable] [unreadable]

Regulation of endothelial cell (EC) coagulant, barrier, adhesive and proliferative functions is central to inflammation, and the host response to tumors. We have identified, cloned and characterized a novel polypeptide, designated Endothelial-Monocyte Activating Polypeptide II (EMAPII), which modulates these EC functions, and is phlogogenic and angiogenic. EMAPII is synthesized by activated murine mononuclear phagocytes (MPs) and constitutively by murine Meth A sarcoma (Meth A) cells, and is secreted as an about 18-20 kDA single chain molecule. EMAPII also activates both MPs, stimulating cell migration and tissue factor synthesis, and polymorphonuclear leukocytes (PMNs), promoting chemotaxis and superoxide generation. In vivo, subcutaneously administered EMAPII elicits inflammation; but Meth A tumor cells transfected to constitutively overexpress EMAPII exhibit increased growth. We hypothesize that the context and kinetics of EMAPII production determine its biologic properties, either as phlogogenic agent in inflammatory settings, or as inducer of neoangiogenesis in tumors. The specific aims are to understand mechanisms by which EMAPII elicits inflammation, and influences tumor-host reactions. Using cDNA probes and antibodies to EMAPII (murine and human), we will identify sites of synthesis and accumulation in a spectrum of tumors, inflammatory and vascular lesions. Functional activity of a region of mature EMAPII near the HN2-terminus, which, in synthetic peptides, stimulates PMN and MP migration, binds specifically to cells, and cross-links to an about 73 kDa cell surface polypeptide will be examined using additional peptides and site-directed mutagenesis. These reagents will also be used to characterize and isolate the EMAPII cell surface binding site. Based on strong sequence homology between the NH2-terminal region of EMAPII and von Willebrand antigen II (vWAgII, a polypeptide in platelet alpha- granules and released by stimulated ECs), we have shown that vWAgII has properties resembling EMAPII. Because of the role of secreted platelet proteins in tissue repair, we will assess the effects of vWAgII on ECs, MPs, and PMNs, and determine its contribution to inflammation and wound repair in animal models. And since elevated levels of vWAgII, consequent on infusion of the arginine vasopressin analog, DDAVP, induced MP tissue factor, we will find out whether vWAgII is capable of initiating activation of procoagulant mechanisms in vivo, and the thrombosis complicating DDAVP therapy. These studies offer new means of linking coagulation with inflammation, and define a new ligand-receptor interaction, involving EMAPII, relevant to host responses in inflammation, neoplasia and, vasculopathies, such as atherosclerosis.

Advanced glycosylation endproducts (AGEs) are nonenzymatically glycated proteins present in plasma and in tissues, and which accumulate in the vasculature at an accelerated rate in diabetes. Exposure of cultured endothelial cells to AGEs increases monolayer permeability, alters cellular proliferation and thrombogenicity. The presence of AGEs in the basal lamina can promote monocyte migration, followed by monocyte interaction with AGEs, thereby leading to cell activation with cytokine and growth factor release. The basis for these perturbations of monocyte and endothelial functions in vitro, which could set the stage for the development of vascular lesions in vivo, results from the interaction of AGEs with specific cellular binding proteins. The central hypothesis of this work is that AGEs contribute to the pathogenesis of vascular lesions via their interactions with cellular receptors central in the pathogenesis. The specific aims of our application are to assess endothelial expression of AGE receptors and deposition of AGEs in a hamster model of hyperglycemia, hypercholesterolemia, and hyperglycemia + hypercholesterolemia. These studies will utilize antibodies raised to cellular AGE receptors and antibodies to AGEs in order to localize these molecules in vascular lesions, and to determine the effect of blocking AGE- cellular interactions on lesion formation/progression. The experiments outlined in this application should provide insights into an additional ligand-receptor interaction, the binding of AGEs to their receptors, which is likely to contribute to the pathogenesis of atherosclerotic vascular lesions.

Tissue hypoxia (H), common to many cardiopulmonary/vascular disorders, affects vascular homeostasis by modulating central properties of endothelial cells (ECs) and smooth muscle cells (SMCs). In ECs: P-selectin is translocated to the cell surface; von Willebrand factor is released; cytokines, such as Interleukins (ILs)-1, -6, and -8, are produced; permeability of th EC monolayer increases in parallel with a decline in intracellular cAMP levels; thrombogenicity increases; and nitric oxide levels are reduced. In SMCs, cAMP levels fall, potentially promoting vasoconstriction. Effects of acute H on EC/SMC properties, as well as their consequences for vascular function, are observed in cardiac and pulmonary grafts after prolonged preservation, in which increased permeability, vasoconstriction, leukocyte (PMN) infiltration and thrombus formation contribute to graft failure. We have shown that by addition of cAMP and NO/cGMP agonists to cardiac preservation solutions, these vascular homeostatic properties are maintained, thereby enhancing successful function of the graft. In pilot work, we have developed a reproducible rat model of orthotopic lung transplantation, and have similarly found that maintenance of vascular homeostatic properties within the graft is essential for graft function/survival. Based on these finding, we hypothesize that H primes ECs and SMCs for subsequent vascular dysfunction, by enhancing PMN-EC interactions, inducing cytokine production, and perturbing cAMP/cGMP messenger pathways. Our specific aims are; (1) to determine mechanisms underlying hypoxic modulation of increased PMN-EC interactions, and suppression of intracellular second messenger cyclic nucleotide levels: and (2) to use insights from these in vitro studies to develop an improved strategy for lung preservation, where regimens for optimal organ storage are still emerging, and rapid vasoconstriction and oxidant stress are well documented. We propose to enhance lung preservation by utilizing cAMP and NO/cGMP agonists, and other interventions targeted to the vasculature, such as blocking antibodies to cell adhesion molecules. The long-term goal of these studies is to understand the contribution of H-induced perturbation of ECs/SMCs to the pathogenesis of a variety of disorders, ranging from vascular dysfunction accompanying organ preservation to atherosclerosis, in order to design more effective preventive and therapeutic strategies.

DESCRIPTION: (Adapted from the Investigator's Abstract) Accelerated vascular disease, involving both the micro- and macro-vasculature accompanies diabetes mellitus (both types I and II) and syndromes of insulin resistance. Nonenzymatic glycation of proteins and lipids occurs with hyperglycemia and is most marked in macromolecules with long turnover times, ultimately leading to formation of advanced glycation endproducts (AGEs). A principle means by which AGEs affect cellular properties is by interacting with binding proteins, the best characterized of which is receptor for AGEs (RAGE), a member of the immunoglobulin superfamily. Based on the applicant's pilot data, they hypothesize that engagement of RAGE by AGEs underlies changes in those cells critical to atherogenesis, especially endothelial cells and monocytes, affecting cell function in ways that predispose to the development of atherosclerosis and microvascular disease. The major goal of this project is to develop and utilize molecular tools allowing dissection of the contribution of RAGE to diabetic atherosclerosis. Their specific aims are: (1) To develop transgenic (Tg) mice overexpressing sRAGE and RAGE gene knock-out (0) mice, in order to study the role of RAGE in hyperfibrinogenemia, elevated plasminogen activator inhibitor-I (PAI-I) and vascular hyperpermeability associated with diabetes; (2) to develop a diabetic murine model of accelerated atherosclerosis, and, using this model, to determine the contribution of RAGE to lesion formation. These experiments will initially employ apoE 0 mice rendered diabetic with streptozotocin, based on the applicant's pilot data showing more extensive atherosclerosis in these animals than in euglycemic apoE 0 mice, as well as cross-breeding with murine genetic models of diabetes. Tg mice overexpressing apoB, an excellent substrate for nonenzymatic glycation and oxidation, will also be used to develop an atherosclerosis/diabetes model. (3) To assess if AGE engagement of RAGE leading to increased plasma soluble (s) VCAM-1 antigen provides a marker of ongoing endothelial perturbation in diabetes. The impact of RAGE blockade and antioxidant therapy on plasma sVCAM-1 and indices of vascular dysfunction in experimental murine diabetes will be evaluated. Then, using optimal conditions from their murine study, a clinical trial of antioxidants will be undertaken in diabetic patients with microalbuminuria to determine the effect on sVCAM-1 and plasma markers of oxidant stress. The results of these experiments are directed towards our long-term goal, understanding mechanisms important in the pathogenesis of accelerated atherosclerosis in diabetes.

Hypoxia/hypoxemia (H) which occurs in a wide spectrum of cardiopulmonary and vascular disorders has especially severe consequences in the perinatal period. Endothelial cells (ECs), most immediately subject to environmental perturbation, rapidly respond with alterations in hemostatic functions important in vascular homeostasis, and by setting in motion compensatory mechanisms facilitating adaptation to oxygen deprivation. The first aim of this proposal is to elucidate mechanisms through which H rapidly modulates the character of the EC surface to one which ultimately favors procoagulant events leading ultimately to the development of thrombosis. This proposal is based on pilot studies showing the H induces release of prothrombotic von Willebrand fat or from ECs, decreases EC surface expression of the anticoagulant cofactor thrombomodulin, and induces translocation of the neutrophil adherence molecule P-selectin to the EC surface (bound/activated leukocytes damage the endothelium and initiate clotting in the subendothelium). This hypothesis will be examined in cell culture and animal models. Because our previous work has shown that augmentation of EC cGMP suppresses H-induced binding of leukocytes, the ability of maneuvers which elevate intracellular cGMP levels to block P-selectin expression will be studied as a potentially protective therapeutic intervention. The second aim is to study H-mediated induction of Interleukin 6 (IL-6) a cytokine with anti-inflammatory and neuroprotective properties, which may promote cellular survival during oxygen deprivation. These experiments will examine molecular mechanisms underlying enhanced expression of Il-6 in H at the promoter level, based on our recent finding that the NF-IL-6 promoter has a central role in gene expression under H. We will also address the potential protective effects of il-6 in models of pulmonary and cerebral ischemia using wild-type and deletionally mutant Il-6 transgenic mice. Work in project V will be carried out closely with studies proposed in other parts of the PERC: (i) we will assist in experiments to determine mechanisms underlying the H-induced increase in corticotropin-releasing hormone expression in the placenta (Goland); and (ii) we will participate int he sheep (Dr. Daniel) and baboon models (Dr. Stark) of placental ischemia to determine if mechanisms identified in our studies are operative in these settings relevant to perinatology. Through a combination of in vitro and in vivo studies, involving Project V and its interactions with the other projects in the PERC, we seek to accomplish the long-term objective of understanding how the response to H perturbs cellular functions and brings about adaptations critical to survival during oxygen deprivation.

9808478 Stern Sporopollenin, often referred to as the most resistant biopolymer on earth, is an essential component of mature pollen grains. Sporopollenin is also found in the spore walls of non seed plants, and in the resting cells of diverse microorganisms. Despite the importance of this apparently ancient compound, the routes of synthesis, secretion, and assembly of sporopollenin remain unknown. The zygospore wall of Chlamydomonas monoica contains sporopollenin and the availability of zygospore mutants of this unicellular alga suggests development of C. monoica as a model for the study of sporopollenin biosynthesis and function. The composition of the zygospore wall will be analyzed using differential fixation and staining followed by fluorescence or transmission electron microscopy and by electrophoresis of proteins solubilized during wall morphogenesis in wildtype and mutant strains. A nuclear transformation procedure will be developed and wildtype alleles of genes responsible for zygospore defects will be isolated by their ability to complement mutant phenotypes, using an indexed cosmid library for transformation. Alternatively, insertional maturation mutants will be generated by nuclear transformation and the molecular tag will then be used to clone the defective gene. The expression of genes cloned by these approaches will be compared in wildtype and maturation mutants.

Sustained endothelial and mononuclear phagocyte dysfunction is critical to the pathogenesis of chronic vascular disorders. Non-enzymatic glycoxidation of proteins and lipids forming Advanced Glycation Endproducts (AGEs) in the vasculature and tissues is accelerated in atherosclerosis, diabetes and renal failure. Interaction of AGES with Receptor for AGE (RAGE) on endothelium and monocytes perturbs cellular properties critical to vascular and tissue homeostatic processes, and causes chronic cellular activation. The central hypothesis of the Program Project is that AGE-RAGE-mediated modulation of endothelial and monocyte functions compromises physiologic effector mechanisms and eventuates in aggressive atherosclerosis, delayed wound repair, and impaired resolution of local inflammation. Employing glucose intolerance as the stimulus for enhanced AGE formation, our pilot studies have shown that antagonism of AGE-RAGE interaction suppresses accelerated atherosclerosis, ameliorates wound healing and diminishes inflammatory consequences of soft tissue infection. Project 1 will exploit our recently developed murine model of accelerated atherosclerosis associated with glucose intolerance to probe the role of RAGE in rapid formation of vascular lesions. Project 2 will extend our concept to a secondary intention wound model in insulin- resistant mice in which AGE-RAGE-mediated cellular dysfunction underlies compromised tissues reparative mechanisms. Project 3 will focus on local inflammation/infection in AGE-rich soft tissues using a model of gingivitis triggered by bacterial infection. The overlapping host response mechanisms triggered by atherogenesis, wound repair and local inflammation, the intimate involvement of endothelium and monocytes, as well as the central role of AGE binding to RAGE, provide the basis for close interactions among the three Projects. By collaborative studies between each of the Projects, the contribution of RAGE will be determined using transgenic mice and mutated RAGE molecules. At the end of this Program Project, we expect to have generated new and important information related to vascular and monocyte dysfunction underlying accelerated atherosclerosis, impaired wound healing and the compromised host response to local inflammation common to disorders characterize by tissue deposition of AGEs. These data should provide insight into a novel target for the development of future therapeutic agents.

DESCRIPTION (provided by applicant): The four members of the epidermal growth factor receptor (ErbB) family of hormone receptors are forefront targets for the new generation of FDA-approved rational cancer therapeutics, including Herceptin, Cetuximab, Tykerb, and Tarceva. The first receptor (EGF receptor) is a therapeutic target in two of the greatest human cancer killers: non-small cell lung carcinoma and colon carcinoma. The second receptor, HER2/neu/ErbB2, is a validated therapeutic target in breast cancer. In contrast, the role of the fourth receptor, ErbB4, is uncertain. There are four different forms of this receptor with different structures. Preliminary evidence indicates that each form of ErbB4 has unique functions, with some forms having opposite effects on processes related to cancer. We will determine what the functions of these four ErbB4 receptor forms are, and how this relates to the well-being of cancer patients expressing these forms. The results will have important implications for human cancer treatment, including whether ErbB4 is a reasonable therapeutic target, and may identify unique biological functions for ErbB4 that may be harnessed in cancer therapies. The work will reveal whether there are subsets of patients with strong ErbB4 activity that potentially would be harmed by EGFR and ErbB2-directed therapies. This work will also be important in other human diseases involving ErbB4. Neuregulin-1, one of the hormones that activates ErbB4, may have therapeutic potential in cardiovascular disease, and has been linked to schizophrenia and bipolar disorder. Presenilin-dependent cleavage of ErbB4, may be important in Alzheimer's Disease. Aim 1 will create mouse models for determining the differential effects of the ErbB4 forms on mammary development. This will reveal which of these forms affects which normal processes. Aim 2 will first use the mouse models from Aim 1 to evaluate positive and negative effects on development of breast cancer. Then, we will determine the clinical correlations of excessive expression of these individual ErbB4 isoforms in a set of nearly 1000 human breast cancers with sixteen years patient follow up information. This will directly test the impact of individual isoforms on patient prognosis. Aim 3 will evaluate unique features of cell regulation by ErbB4 forms that act in the cell nucleus. This affords ErbB4 the ability to reprogram major cell activities, some relevant to cancer. These experiments will provide a global view of these functions. PUBLIC HEALTH RELEVANCE: The four different forms of the hormone receptor ErbB4 potentially induce or antagonize cancer, depending on which form is expressed. The focus of this application is on determining the processes regulated by these individual forms, and how this impacts on development of breast cancer and other cancers. Besides the possibility that ErbB4 functions autonomously as an oncogene or tumor suppressor, ErbB4 forms complexes with the related EGF receptor and ErbB2, which are the targets for new drugs that are used in treatment of breast, colon, and lung cancers. Our findings will reveal whether ErbB4 tempers or augments their activities in cancer, with important therapeutic implications. ErbB4 is also important in cardiovascular and nervous systems. Neuregulins, which are hormones that bind and activate ErbB4, may be useful in promoting recovery from cardiac injury. These hormones have also been linked to familial schizophrenia and bipolar disorder. Similarly, polymorphisms in ErbB4 itself have been genetically linked to schizophrenia. Finally, ErbB4 is potentially an important biological target for presenilin cleavage in Alzheimer's disease.

[unreadable] DESCRIPTION (provided by applicant): DNA damage response networks are common targets for carcinogenic mutations and may be excellent targets for response-modifying drugs. The central core of these networks consists of phosphatidyl inositol 3-kinase-related kinases (PIKKs), e.g. human Atm and Atr, which activate downstream pathways in response to DNA damage. PIKKs phosphorylate components of complexes that are recruited to damageinduced foci, including the Mrell/Rad50/Nbsl complex, 53BP1, and Brcal, and activate effector kinases Chkl and Chk2 that amplify the signal and help convey it to effector proteins. In budding yeast, the PIKK Mecl (Atm/Atr ortholog) is coupled to protein kinase Rad53 (Chk2 ortholog) through Mecl-dependent phosphorylation of the "mediator" Rad9 (unrelated to human Rad9). Mammalian BRCA1, 53BP1, and now MDC1/NFBD1 are candidate Rad9 orthologs that resemble Rad9 in having twin carboxyl terminal BRCT domains. Like Rad9, they undergo PIKK-dependent phosphorylation, form damage-induced complexes with other checkpoint proteins and are recruited to sites of DNA damage. MDC1/NFBD1 is intimately involved in DNA damage response mechanisms. The PIKK-Mediator-Chk core of DNA damage signaling will be investigated using both the simpler yeast cascade Mecl to Rad9 to Rad53, and the more complex interactions of ATM/ATR in regulation of NFBD1. [unreadable] [unreadable] Aim 1. A combination of biochemical and molecular methods will be used to determine the mechanism [unreadable] through which the mediator Rad9 and the PIKK Mecl regulate Rad53. [unreadable] [unreadable] Aim 2.. siRNA oligonucleotide knockdowns will be used to identify unique and overlapping functions of BRCA1, 53BP1, and NFBD1 in DNA checkpoint responses. [unreadable] [unreadable] Aim 3. Molecular techniques will be used for functional analysis of NFBD1/MDC1 in the activities [unreadable] identified in Aim 2. [unreadable] [unreadable]

DESCRIPTION (from applicant's abstract): Increasing evidence points to a causative role for amyloid-beta peptide (Abeta) in the pathogenesis of Alzheimer's disease (AD). Thus, mechanisms through which Abeta affects cellular properties have come under intensive study. Higher concentrations of Abeta(1-40/42), present later in the course of AD, form aggregates and fibrils which nonspecifically damage membranes of virtually any cell. Earlier in AD, when lower levels of Abeta are present, specific interactions of Abeta with particular cells, as well as sensitive subcellular loci, could also mediate cellular toxicity. We have identified a novel endoplasmic reticulum-associated Abeta binding protein termed ERAB which has properties of a b-hydroxyacyl-Coenzyme A dehydrogenase/short-chain alcohol dehydrogenase. Recombinant human ERAB binds Abeta(1-40/42) specifically, though it does not interact with beta-amyloid precursor protein (betaAPP) or forms of Abeta containing C-terminal amino acids from the precursor. ERAB, constitutively produced by neuroblastoma cells, was co-immunoprecipitated with Abeta; following exposure of cultures to exogenous Abeta, ERAB was rapidly redistributed to the inner aspect of the plasma membrane. Toxicity of Abeta to neuroblastoma cells was prevented by liposome-mediated introduction of blocking anti-ERAB F(ab')2 into the cells, and was enhanced by overexpression of ERAB in COS cells. We propose that ERAB is a critical intracellular target potentiating Abeta-mediated cellular perturbation and, ultimately, cytotoxicity. We hypothesized that Abeta influences ERAB by effecting ERAB translocation to the plasma/nuclear membrane, where induction of cell stress, marked by generation of toxic aldehydes and events triggering apoptosis, occurs. Our first aim is to determine the contribution of ERAB enzymatic activity and ERAB binding to Abeta on cytotoxicity by analyzing ERAB-Abeta-induced generation of toxic aldehydes and apoptosis. Our second aim is to analyze the contribution of ERAB to Abeta-induced cell stress using transgenic mice; we hypothesize that mice with targeted overexpression of ERAB will display exaggerated cytotoxicity in an Abeta-rich environment, and that this will be further exacerbated by ischemic stress. The long-term goal of this wok is to determine if ERAB is an important cellular cofactor for Abeta cytotoxicity, in order to assess whether inhibition of ERAB might be a novel neuroprotective strategy for future drug development.

9975765
Grossman

The personnel involved in this project are Arthur Grossman (PI, Carnegie Institution of Washington), Paul Lefebvre and Carloyn Silflow (Co-Pls, University of Minnesota), John Davies (Co-PI, Iowa State University), Elizabeth Harris (Co-PI, Duke University), David Stem (Co-PI, Boyce Thompson Institute, Cornell University), and Ronald Davis (Co-PI, Stanford University).
Chlamydomonas reinhardtii, a unicellular haploid green alga, has been and will continue to be a very important model system for elucidating basic biological processes in plants. Experimentation with Chlamydomonas is particularly important for the dissection of photosynthetic processes since this alga can be grown rapidly on an exogenous source of fixed carbon and is the only genetically tractable eukaryote for which mutations that affect all aspects of photosynthesis and carbon assimilation are conditional rather than lethal. The application of sophisticated genome-wide methodologies to studies of Chlamydomonas will augment its utility as a model for the analysis of photosynthetic function and regulation. Specifically, as a first goal, normalized Chlamydomonas CDNA libraries will be generated using RNA isolated from cells grown under a variety of environmental conditions. The 3' and 5' sequences of the cDNAs will help generate unique sets of ESTs that will be arrayed onto polylysine coated slides. These arrays will be used to examine global gene expression under conditions that markedly alter the activity and biosynthesis of the photosynthetic apparatus. The microarrays will also be used to analyze gene expression in those photosynthetic mutants that appear to be regulatory in nature (to identify potential gene targets for regulatory elements). Furthermore, full-length sequences will be generated for most of the cDNAs, which would represent almost the complete coding capacity of the Chlamydomonas nuclear genome. A second goal is to establish a partial physical map of the nuclear genome that is aligned with the genetic map with the aim of using the information to perform rapid and efficient map-based cloning of genes. The mapping studies will focus on specific regions of the nuclear genome that are centered around mutations that define genes having critical functions in photosynthesis. A third goal is to complete the sequence of the chloroplast genome, generate and analyze strains that are deleted for each of the chloroplast open reading frames, and use microarrays containing each of the chloroplast open reading frames to characterize global expression of chloroplast genes under different environmental conditions. The microarray technology will also be used to evaluate the abundance of all chloroplast mRNAs in nuclear mutants that exhibit aberrant transcriptional and post-transcriptional control of plastid gene expression. Much of the information and many of the tools that will be developed during the course of this work will allow studies of photosynthesis to attain a global dimension, which is critical for elucidating the dynamic nature of chloroplast function and the mechanisms involved in regulating chloroplast gene expression.

Tissue and vascular deposition of Advanced Glycation Endproducts (AGEs), irreversible products of glycoxidation of proteins and lipids, occur during normal aging and are accelerated by glucose intolerance, atherosclerosis and renal dysfunction. Thus, host response mechanisms triggered by local inflammation and/or infection operate in an AGE-rich environment in these settings. Cells critical to resolution of such inflamed foci, especially endothelial cells (ECs) and mononuclear phagocytes (MPs), express Receptor for AGEs (RAGE), a principal cell surface binding site for AGEs. Consequent to engagement by AGEs, RAGE brings about changes in which local inflammation is initiated by porphyromonas gingivalis (Pg) in AGE-rich tissues using glucose intolerant animals similarly challenged with Pg. Gingiva from glucose-intolerant mice showed extensive deposition of AGEs and increased expression of RAGE in MPs and ECs. Interruption of AGE interaction with cellular RAGE, by administration, by administration of a truncated form of RAGE (sRAGE) spanning the extracellular domain, suppressed gingival inflammation and alveolar bone loss. We hypothesize that AGEs, via their interaction with endothelial and monocyte RAGE, prime gingival tissue for an exaggerated inflammatory response eventuating in enhanced alveolar bone loss. Thus, gingivitis in glucose-intolerant mice provides an opportunity to extend our concept of AGE-RAGE-mediated cellular activation as a basis for non- resolving and destructive inflammation. Our aims are: (1) to compare production of inflammatory cytokines, collagen synthesis and degradation, influx of inflammatory cells into affected gingival tissue and extent of alveolar bone loss in glucose-intolerant and normal mice with/without infection with Pg; (2) to determine how blockade of AGE-RAGE interaction attenuates periodontal inflammation and bone loss; and, (3) to use murine transgenic (Tg) models in which wild-type or dominant negative RAGE is selective over-expressed in ECs or MPs to test the concept that RAGE contributes to PD in AGE-rich gingiva. Project will work closely with Projects 1&2 and will obtain technical assistance from the Cores. Collaborative interactions include: development and characterization of Tg RAGE mice (Projects 1-2 and Core C), transcriptional analysis of RAGE expression and dissection of the RAGE ligand binding domain (Project 1-2), cytokine analysis (Project 2), and a pathologic study of tissues (Core B).

Cerebral amyloid angiopathy (CAA) is most commonly associated with vascular deposition of amyloid-beta peptide (Abeta), as well as the presence of advanced glycation endproducts (AGEs), the irreversible products of non-enzymatic glycoxidation. One of the principal means by which Abeta and AGEs impact on vascular function is by interacting with cellular elements, especially endothelial (EC) and smooth muscle (SMC) cells, resulting in changes in cellular properties, and, in certain instances, cell death. Whereas Abeta and AGEs are structurally distinct and derive from different biochemical pathways, they share in common accumulation in CAA and recognition by Receptor for Advanced Glycation Endproducts or RAGE. Engagement of RAGE perturbs cellular functions resulting in sustained oxidant stress, activation of the transport factor NF-kappaB, and, in certain instances, apoptosis. We hypothesize that CAA occurs, in part according to a "two-hit" model; the first hit is comprised of sustained Abeta/AGE binding to and activation of cellular RAGE; and the second hit includes superimposed environmental challenges such as intermittent ischemia, as in stroke. The focus of Project 3 is to dissect the contribution of RAGE in SMCs to vascular dysfunction in CAA since RAGE is expressed at highest levels in vascular SMC, which is in intimate contact with accumulated Abeta and AGE-modified matrix, and SMCs are known to undergo degeneration in cerebrovascular amyloidosis. The first aim of Project 3 is to determine if transgenic mice with targeted over expression of wild-type RAGE in SMCs display enhanced vascular perturbation, eventuation in disruption of the vessel wall in Abeta- and/or AGE-rich vascular micro environment. The second aim is to determine if transgenic mice with targeted over expression of wild-type RAGE in SMCs display enhanced vascular perturbation, eventuating in disruption of the vessel wall in Abeta-and/or AGE-rich vascular micro environment. The second aim is to determine if transgenic mice with targeted over-expression of dominant negative 9DN) RAGE are protected from vascular perturbation by Abeta and AGEs. The long-term goal of Project 3 is to determine the role of RAGE in CAA in order to evaluate whether antagonism of Abeta-AGE interaction with this receptor is a therapeutic target for patients with chronic cerebrovascular dysfunction.

The chloroplast is an essential organelle whose biogenesis is a complex, orchestrated process central to plant growth and development. Many hundreds of nuclear genes are involved in the import of nucleus-encoded proteins, the intra-organellar sorting of nucleus- and chloroplast-encoded proteins, the expression of chloroplast genes, the assembly of chloroplast enzymes, and the regulation of these processes. However, only a small proportion of such genes have been characterized in any detail. The goal of this project is to develop, use, and disseminate a set of complementary and powerful resources for the genetic and biochemical dissection of this complex process.

Maize offers an ideal set of attributes for this project. In addition to its superb genetic tools and the capacity to generate mutations at high frequency with Mu transposons, this project exploits the fact that non-photosynthetic mutant tissue can readily be obtained for biochemical analysis. The core resource that will be developed is a saturated collection of transposon-tagged chloroplast-defective maize mutants. Mutants will be selected from Mu-active maize lines based upon their chlorophyll-deficient leaves and/or increased chlorophyll fluorescence, an indication of a block in photosynthetic electron transport. Previous studies support the notion that one of these easily identified phenotypes will result from a disruption of most aspects of chloroplast biogenesis (import of proteins into the organelle, lipid, pigment, and prosthetic group synthesis, chloroplast gene expression, intra-chloroplast protein sorting, assembly of the photosynthetic apparatus).

The mutant collection will be used in two ways: (i) To determine the role of genes of known sequence but unknown function; and (ii) To discover new genes that play critical roles in chloroplast biogenesis and function.

(i) Genome sequencing projects have unmasked thousands of predicted chloroplast-localized proteins, the majority of which have no known function. To determine the roles of such proteins, the mutant collection will be used to develop a reverse genetics resource, called Photosynthesis Mutant Search (PMS). PMS, already functioning on a small scale, consists of DNA pools from plants with chloroplast defects caused by Mu insertions. 1200 independently-arising mutants are currently in the collection; this number will be increased to ~2000, at which point it is predicted that the collection will be saturated. The small number of DNA pools can be screened in a cost-effective manner to find mutant alleles of genes of known sequence with suspected roles in chloroplast biogenesis. Mutants will be identified and provided as a service. Users will analyze the mutant phenotypes to elucidate the function of the disrupted gene.

(ii) To discover new genes that play critical roles in chloroplast biogenesis and function, the same mutant lines will undergo "snapshot" characterization of visual phenotype and chloroplast protein and RNA defects. The description of each mutant will be incorporated into a web site and the mutants will be made available to other researchers. From these snapshots, users will order specific lines for further study and cloning of the disrupted gene.

In addition, the team of collaborators will form a synergistic partnership to use these tools to focus on one aspect of chloroplast biogenesis: chloroplast gene expression and its control. The study of many other aspects of chloroplast biology by the community-at-large will be facilitated by the unique genetic resources that will be produced. In addition to the agronomic relevance of genes that function in chloroplast processes, it is anticipated that this project will impact fields ranging from evolutionary biology to basic cell and molecular biology.

Hypoxemia/tissue hypoxia (H) has long been associated with procoagulant events, especially venous thrombosis, a major cause of morbidity and mortality. Experimental limb immobilization leads to a rapid fall in blood oxygen tension in parallel with fibrin deposition in venous valve pockets, thereby providing a nidus for thrombus formation. In a model of normobaric hypoxia, we have provided the first outline of a pathway through which oxygen deprivation triggers coagulation: hypoxia upregulates tissue factor and plasminogen activator inhibitor (PAI)-1 in mononuclear phagocytes (MPs), ultimately causing vascular fibrin deposition. We hypothesize that there are two pivotal and unexpected events in this H-triggered pathway, both of which are independent of hypoxia-inducible factor (HIF)-1: activation of protein kinase C isoform betaII (PKCbetaII) and activation of the transcription factor Egr-1. Tissue factor induction in hypoxic MPs is due to increased transcription at Egr-1 sites in the promoter, and Egr-1 null mice subject to oxygen deprivation display absence of tissue factor induction and vascular fibrin deposition. Our in vitro studies have traced the H-associated pathway culminating in Egr-1 transcription back to PKCbetaII: hypoxia activates PKCbetaII, the latter triggers a raf- and MEK-dependent pathway activating MAP kinases and Elk-1; and, activated Elk-1, in concert with Serum Response Factor, induces transcription of Egr-1. Our underlying concept is that events occurring at the earliest stage of oxygen deprivation, PKCbetaII and Egr-1 activation (each seen within minutes of H), are critical to the formation of thrombogenic foci within hypoxemic vasculature. Our first aim is to evaluate the role of PKCbeta in H-associated vascular fibrin formation using PKCbeta O mice, and to extend our concept of H-induced vascular perturbation to a model of lung ischemia by analyzing the response of PKCbeta O and Egr-10 mice. Our second aim addresses the hypothesis that activation of PKCbetaII in MPs is a key event driving the procoagulant mechanism, as well as other pathways contributing to ischemic tissue injury, by making and analyzing transgenic mice with targeted suppression or activation of PKCbetaII in Mps. Our overall goal is to determine if PKCbetaII and Egr-1 are potential therapeutic targets for preserving vascular homeostasis in response to hypoxemic and ischemic stress.

During aging, the neuronal response to endogenous and exogenous environmental stress undergoes continuous modulation. The accumulation of protein fragments, such as amyloid beta-peptide (Abeta), within brain parenchyma and vasculature provides a modified context in which homeostatic and reparative processes must be operative. The central theme of our Program Project is that specific, cell-associated cofactors, RAGE (Receptor for AGEs) and ABAD (Abeta binding alcohol dehydrogenase) are critical for concentrating the effects of low levels of Abeta, relevant to early stages of pathogenicity in Alzheimer's disease (AD) and cerebrovascular amyloid angiopathy (CAA), on vulnerable cells. Yet, in distinct settings, these same cofactors appear to have a pivotal role in triggering neuroprotective pathways and in orchestrating reparative processes through a complex integration of host response mechanisms. Project 1 evaluates the role of RAGE in the central nervous system, (CNS) in the potentiation of Abeta-induced cell stress by cross-breeding transgenic (Tg) mice with targeted over-expression of RAGE in cortical neurons and/or microglia with mice over-expressing mutant betaAPP, the latter resulting in an Abeta-rich environment. Project 2 examines a contrasting a protective role of RAGE in the peripheral nervous system (PNS); transient expression of RAGE in injured peripheral nerve and infiltrating macrophages interacts with two other RAGE ligands, amphoterin and EN-RAGEs (Extracellular, Newly- identified RAGE binding proteins), both of which are also present at sites of injury. in the PNS, to enhance identified RAGE binding proteins), both of which are also present at sites of injury in the PNS, to enhance reparative mechanisms. Projects 3-4 extrapolate our concept of chronic cellular perturbation in AD to the enzyme ABAD, an intracellular target of Abeta mediating generation of reactive oxygen intermediates and aldehydes in an Abeta-rich environment using Tg (#3) and in vitro models (#4), as well as structural studies go to probe ABAD-Abeta complex (#4). Analysis of overlapping cellular effector mechanisms triggered by activation of RAGE and ABAD results in critical shared molecular tools and animals models, and provides the basis for synergy among the four projects. At the end of this PROGRAM PROJECT, we expect to have generated new and important information related to the involvement of RAGE and ABAD in neuronal perturbation and repair as a first step in evaluating their efficacy as future therapeutic targets in neurodegenerative disorders.

DESCRIPTION (provided by applicant): The goal of this project is to identify the nucleotide changes that have led to phenotypic differences between closely related Drosophila species. The larger goal is to determine whether these evolved changes reflect a special set of all possible mutations. There are three specific aims for this proposal. First, we will survey three previously identified enhancer regions of the shavenbaby/ovo gene for all transcription factor binding sites that can be detected in vitro. This analysis will leverage the fact that all three enhancers have evolved new functions in D. sechellia, presumably through the loss or gain of transcription factor binding sites. This survey will provide the foundation for detailed study of binding sites that have evolved new functions. In particular, we will test whether these sites are required for gene function in D. melanogaster and we will further test whether altering them to a D. sechellia sequence is sufficient to alter their function in the manner in which svb function has evolved. We will then perform a population genetic analysis of the conserved and evolved transcription factor binding sites to test whether the evolving sites have evolved by natural selection. Second, we will continue development of a novel method for ultra high-resolution mapping of evolved differences between D. simulans, D. sechellia and D. mauritiana. This new method extends traditional meiotic mapping approaches by employing genetic markers that flank an evolved region to allow automated identification of individuals with informative recombination events. This approach promises to provide a transformative technology for studying the evolution of a large and diverse set of phenotypic differences, especially so-called quantitative traits that are controlled by multiple genes. Relevance: Differences between the response of individuals to disease and drug treatment are caused in part by genetic variation. In addition, the traits that make us uniquely human evolved in large part through changes in gene regulation, rather than through the evolution of new genes. This project involves detailed study of the evolution of a new pattern of gene regulation in a species of Drosophila. This detailed analysis of gene regulation promises to provide new insights into how gene regulation functions and evolves in natural species to cause possibly adaptive changes in morphology.

This award provides continuing support for the development and application of genomic resources for the study of fundamental biological processes in Chlamydomonas, an important model organism. This project will enhance the use of Chlamydomonas for elucidating the regulation of the photosynthetic apparatus with respect to changing environmental conditions. The investigators have the following five goals:
(1) Implement a Generic Model Organism Database (GMOD) for Chlamydomonas that will display the genome and its features, including coding regions, ESTs, cDNAs, splice sites, BAC positions, molecular markers and sites of insertion elements in mutant strains.
(2) Create a Phase II microarray representing 8,500-10,000 unique genes and supplement the array with an oligonucleotide-based array that will include genes that are defined by whole genome sequencing and that are not included on the Phase II array. Arrays will be used to catalog mechanisms of acclimation to environmental stress, using both wild-type and mutant strains whose biological phenotypes have already been determined; an emphasis will be placed on photosynthetic function.
(3) Build an insertional mutant collection saturated for nuclear genes whose products are linked to photosynthetic function. Insertion strains will be challenged with a battery of screens, and the insertion sites of 5,000 strains affected in photosynthetic function will be sequenced and made available to the community.
(4) Create high-throughput mapping tools to facilitate gene isolation from mutants that are not tagged by insertions. These will primarily be suppressors of other mutations, or mutations in genes for which a null lesion would be lethal. The mapping will be based on simple sequence repeats, automated and made more rapid by the use of bulked segregant analysis. A mapping service will also be offered to the community.
(5) Develop vectors to facilitate RNA interference-mediated reverse genetics.

Chlamydomonas reinhardtii, a unicellular haploid green alga, is an important model system for elucidating basic biological processes, especially in plants. The further development of sophisticated genome-wide methodologies for studies of Chlamydomonas will greatly augment its usefulness to the scientific community. This project will also produce extensive data on photosynthesis and acclimation, adding to our knowledge of how photosynthetic organisms sense, respond to, and survive in a rapidly changing environment. It will also enhance Chlamydomonas's usefulness as a 'training' organism in laboratories throughout the world, and provide a new and exciting entry point into research careers for young scientists.

[unreadable] DESCRIPTION (provided by applicant): To encourage underrepresented minority students and students from health disparities populations to pursue biomedical and behavioral research careers, the Michigan Minority Health and Health Disparities International Research Training Program offers opportunities for active participation in research on global disparities in child health. This training program engages students in research on problems that disproportionately affect children in developing countries and poor/minority children in the U.S. Student research experiences occur in conjunction with ongoing studies of University of Michigan faculty members in Chile, China, Ghana, Jamaica, and South Africa. These projects address both physical health and psychoeducational issues. Approximately 16 students will be selected each year (12 undergraduates and 4 medical/graduate students). The program includes: 1) pre-departure preparation; 2) close mentoring in the U.S. and at international sites; 3) research involving design of a study or subproject (if appropriate), collection, analysis, and interpretation of original data, and communication of the project in oral and written form; 4) training overseas for 12 weeks; and 5) post-trip follow-up, including: career guidance; facilitation of further research placements; opportunities for independent study programs; supervision of honors theses and doctoral dissertations; and assistance in preparing manuscripts and presentations. Minority and/or junior faculty investigators engaged in research on child health inequalities in the U.S. are encouraged to develop related projects in conjunction with the ongoing international studies. [unreadable] [unreadable]

Summary of Proposed Research
Different species have sometimes evolved similar appearances and behaviors as a result of exposure to similar ecological conditions, a phenomenon called "convergence". For example, marsupials evolved into a variety of forms resembling placental animals (for example, flying squirrels, anteaters, moles, mice, and wolves). Recent work in evolutionary developmental biology, or evo-devo, indicates that sometimes similar genes may underlie convergence. In one example that has been studied in detail, one species of drosophilid fly, D. sechellia, has evolved nearly-naked larvae, whereas most drosophild flies have hairy larvae. A second group of drosophilids, the D. virilis group, which diverged from D. sechellia approximately 60 million years ago, also contains some species with nearly-naked larvae. Evidence was presented previously that the same gene, called shavenbaby, caused these similar nearly-naked larvae in both cases. This hypothesis will be rigorously tested by performing a detailed analysis of shavenbaby gene evolution in the D. virilis group. This work will involve developing new methods that will allow tests of gene function within D. virilis. These critical functional tests will provide a compelling analysis of convergence and will indicate how the same gene can evolve in different species to cause the same result. This information will inform how often the same genes evolve to cause convergence.
Dr. Stern's laboratory has a strong record of promoting women in science and training undergraduates in research. Since starting the lab at Princeton approximately half of the graduate students and postdocs have been women. The laboratory trains approximately 2-4 undergraduates each year, all of whom perform independent research and write a thesis based on this work. About half of these projects have resulted in publications and several students have continued on to graduate school in evolutionary biology.

Plant and animal species exhibit an incredible diversity of form. Much of this morphological diversity is generated by genetic variation; however, the specific genetic modifications required to produce morphological variation are unknown in most cases. A comprehensive understanding of how diversity is produced requires determination of how differences in gene sequence alter trait form and function, as well as how natural selection acts upon the resulting variation. The fruit flies Drosophila mauritiana and Drosophila simulans have eyes that differ dramatically in size and shape. Through a combination of laboratory and field studies of these closely related species, the researchers propose (1) to identify genetic changes responsible for interspecific variation in eye size and shape, (2) to characterize the functional importance of variation in eye size and shape, and (3) to study the selective forces acting upon eye size and shape.

This study will improve understanding of how complex morphological traits evolve and why such traits differ between species. In addition, recent findings suggest that broad generalizations can be made from conclusions drawn from studies on model organisms such as Drosophila. Thus, findings from this study have the potential to be relevant for understanding the evolution of complex traits in other animal species and humans. This project will also allow the establishment of collaborations with two research labs in the UK; this association will allow the development of new research techniques such as the adaptation of software for analysis of complex traits in animals. Finally, an undergraduate student will be trained in research development and lab techniques and will have the opportunity to conduct independent research.

WestEd and the University of California-Berkeley are improving student learning and workforce preparation in science as well as information and communication technology by embedding the Global Learning and Observations to Benefit the Environment (GLOBE) program into curricula of selected high school-level green energy career academies in California. Embedding GLOBE within California's green energy academies will pave the way for broader GLOBE implementation in California and other states.

The GLOBE California Academy Program (CAP) is engaging students, particularly at-risk students, in research related to climate science and renewable energy technologies, providing professional development and support for academy teachers, connecting scientists and industry to academy classrooms, and linking students to GLOBE's international student community. GLOBE's data collection and analysis activities will become an integral and sustainable part of a rigorous inquiry-based approach to science education that prepares students for postsecondary education and science careers.

Integrating GLOBE into CAP will produce a replicable model program that improves high school students' preparation for postsecondary education and careers in science and technology fields. Outcomes for students include improved science/technology knowledge and skills, higher grades, increased interest in pursuing postsecondary education and science/technology careers, and development of 21st century workforce skills. Outcomes for teachers include increased facility with GLOBE and scientific practice.

EFRI-PSBR: Microalgae Lab-on-Chip Photobioreactor Platform for Genetic Screening and Metabolic Analysis Leading to Scalable Biofuel Production
INTELLECTUAL MERIT
The multi-disciplinary research team will develop microfluidic lab-on-chip devices with capabilities to precisely assay and manipulate parallel samples at single-cell resolution. The devices will enable the integration of multiple experimental parameters on a single user-friendly platform. These devices will then be used to analyze and optimize the growth and hydrocarbon production potential of an engineered recombinant photosynthetic microalgae. The specific test case will be a recombinant, fast growing Chlamydomonas reinhardtii algal strain that will be engineered to express a high-yielding hydrocarbon biosynthetic gene system derived from the slow-growing microalaga, Botryococcus braunii. These specific hydrocarbons produced by these organisms are of particular interest because they can be readily converted into petroleum-equivalent fuels.

BROADER IMPACTS
The proposed research will have broad scientific impact because the developed microfluidic platforms will directly contribute to and dramatically accelerate research and development in the general area of microbe-mediated biofuel and biometabolite production. Educational outcomes will include training of the next generation of microbial bioenergy engineers and scientists through research, involving undergraduate and minority students, local high school students, and high-school teachers in conjunction with existing programs at the participating institutions to provide them with early exposure to science and engineering. This project will allow students from multiple disciplines (engineering, microbiology, biochemistry) to interact and gain first hand experience in interdisciplinary research.

The repeated loss of pigmentation, eyes and vision in cave-dwelling animals presents biologists with an excellent opportunity to understand the genetic changes that have resulted in the convergent evolution of similar morphological changes in unrelated organisms. Freshwater crayfish have evolved to live in caves on at least seven occasions throughout their history, repeatedly losing vision. This project will use a well-supported evolutionary framework of freshwater crayfish to understand the role of gene regulation in producing iconic cave morphologies. This research will promote training of undergraduate and high school students in field, laboratory, and computational work. Data and analysis pipelines will be made available to the public and will contribute to invertebrate genomics research efforts. Ultimately, this research will advance our knowledge of how organisms adapt to novel habitats and contribute to the growing body of research in vision genomics.

While the genetics of vision loss has been studied in single species systems such as the Mexican blind cave fish (Astyanax mexicanus), comparative studies across multiple species aimed at revealing statistically significant and biologically meaningful correlations have been lacking. The multiple transitions to cave habitats by freshwater crayfish and the subsequent loss of vision in each case make the group an ideal model to test hypotheses concerning the loss of complex traits in response to a common selective pressure. These evolutionarily independent replicates provide statistical power as well as the potential to uncover biologically meaningful correlations. Researchers will use comparative transcriptomics to develop a set of candidate genes related to vision loss and cave adaptation in this system. Gene expression levels will be measured across a set of these genes from multiple individuals of 14 species, both cave and surface, and modeled as an evolving trait on the phylogeny. These data and methods will be used to test if regulatory changes associated with vision loss show a signature of adaptive evolution and if the transcriptome evolves in a predictable way in vision loss events. This project will help to elucidate the role of gene regulation in producing these iconic cave phenotypes, using a unique framework, new technologies, and newly developed evolutionary models.

? DESCRIPTION (provided by applicant): Recent advances in understanding cancer, combined with unprecedented access to patient DNA sequence data, and new rational therapeutic approaches create the need for a revolution in Ph.D. training of scientists. We have developed a new Cancer Biology Training Program that will take advantage of the high caliber of biologists at Yale, the commitment to multidisciplinary training in the life sciences, and the close collaboration of Yale cancer biologists and clinicians fostered by the Yale Cancer Center (YCC) to provide a unique cancer-focused training experience intended to spawn the next generation of cancer scientific leaders. Training will cover the genetic and biological underpinnings of cancer, the pathway to development of new therapies based upon this knowledge, and the practical challenges in applying these new therapies in cancer clinics. Predoctoral trainees will be trained in foundational biological areas through course work in fundamental areas of biology and physiology and join the program in their second year. Postdoctoral fellows will join the program early in postdoctoral training. All trainees will be spend 90% of their time in laboratory research projects mentored by highly qualified faculty who are leaders in disciplines of cancer research including tumor virology, cancer immunobiology, cancer genetics and epigenetics, stem cell research, pharmacology, and signal transduction. The program will begin by training two new predoctoral and four new postdoctoral trainees each year, with funding for one of the predocs and three of the postdocs to be provided through this T32. For predoctoral trainees, the training experience will include three research rotations and didactic and seminar courses, and continue with qualifying examinations and written dissertation in a projected period of five years overall. Postdoctoral training includes participation in research seminars and talks. Cancer-specific training provided for all trainees by the program will include three formal courses: 1. a general survey class covering basic principles of cancer biology and genetics; 2. a seminar course in which selected topics will be analyzed and discussed in depth, and a clinically-oriented workshop that covers clinical trials, 3. patient treatment patterns and clinical questions for major diseases; and personalized cancer medicine based on tumor resequencing. Every trainee will have a clinical co-mentor to foster exposure to clinical concepts through tumor boards and clinics.

DEVELOPMENTAL RESEARCH PROGRAM: PROJECT SUMMARY The primary goal of the Yale SPORE in Skin Cancer Developmental Research Program (DRP) is to provide limited support (maximum of $50,000/year, typically for no more than two years) for a broad spectrum of innovative skin cancer pilot projects (involving research, resources, and technology development applicable to human skin cancer risk, prevention, diagnosis, prognosis, or treatment). These pilot projects must have promising translational research potential, with anticipation that they can eventually develop into, or be incorporated into, full projects with an unequivocal clinical translational component. Such projects could either be developed into independent external funding at a scope and scale equivalent to a NIH R01 grant, or alternatively augment or replace a Project in future cycles of the Yale SPORE in Skin Cancer. A total of at least $230,000/year will be used for Developmental Research Projects ($56,262 in direct costs and at least $173,738 in institutional matching funds guaranteed by the Yale Cancer Center and the Department of Dermatology). These funds will make it possible to support up to five DRP projects annually, ranging from $30,000 to $50,000 each. A second goal of this DRP is broaden the collective of Yale investigators who are actively engaged in research related to human cutaneous oncology. During its second five-year funding period, the YSPORE DRP has funded 22 different projects (20 related to melanoma, one to cutaneous T cell lymphoma, one to keratoacanthoma) involving 28 different investigators from 15 different departments and sections at Yale, and two outside institutions. Six of the projects involved multiple PIs and encompassed multidisciplinary activities.

CAREER ENHANCEMENT PROGRAM: PROJECT SUMMARY The Career Enhancement Program (CEP) of the Yale SPORE in Skin Cancer will develop a new cadre of investigative cutaneous oncologists and scientists committed to multidisciplinary studies investigating the relevance of biological discoveries in human skin cancer risk, prevention, diagnosis, prognosis or treatment, and enhance the careers of individuals who are already productively investigating this field. $56,262 per year is requested to support CEP, which will be matched with $56,108 from the Yale Cancer Center, to be used for the support of two faculty-level CEP Awards per year. Funding is for one year, but may be renewed for a second year. Potential candidates for these awards include promising junior faculty who are either appointed at Yale or are outside candidates or new recruits for Yale appointment. Alternatively, candidates may be established investigators, either currently at Yale or in the process of being recruited to Yale, with previous research focus in other arenas, but who will re-channel a significant portion of their focus to translational research in cutaneous oncology. All junior faculty awardees are paired with an established investigator in translational cutaneous oncology with a documented record of successful mentoring. During the second five-year funding cycle, YSPORE CEP funds were used to support the career development of seven different investigators including four women. Individuals supported to date have had diverse backgrounds and previous experience; 4 PhDs, 1 MBBS, and 2 MD/PhDs, with experience including an entry level lung carcinoma geneticist refocusing to melanoma research; established clinician scientists enabled to devote significant time to B cell anti- melanoma responses, and a junior clinician-scientist who discovered driver mutations in cutaneous T cell lymphoma.

PROJECT SUMMARY/ABSTRACT: Triple negative breast cancer (TNBC) is an aggressive and difficult to manage disease that disproportionately afflicts underrepresented minority populations. Tumor cellular heterogeneity is a major source of treatment resistance and resilience, but is poorly understood. Thus, better understanding of tumor heterogeneity will lead to development of therapeutic strategies to treat underrepresented minority TNBC patients. The objective of this proposal is to identify and characterize biomarkers of cell subsets that drive tumor resilience in African- American (AA) patients. The central hypothesis is that differential cell population heterogeneity contributes to disparate TNBC prevalence and treatment outcomes in AA and other underrepresented patient groups. The rationale for the proposed research is that better understanding of tumor cellular heterogeneity will result in new and innovative approaches to TNBC treatment for underrepresented minority TNBC patients. Therefore, the hypothesis will be tested by pursuing the following specific aims: Aim 1) Identification of phenotypic states in AA TNBC with single cell RNA-seq; Aim 2) Associations of candidate biomarker sets with AA TNBC, clinical variables, immune phenotypes, genomic information, and outcomes in a TNBC neoadjuvant trial. The proposed research is innovative, because of the novel approach to use single cell profiling to define and understand novel tumor cell lineages and microenvironment for AA TNBC tumors. The proposed research is significant, because it is expected to advance and expand our understanding of tumor heterogeneity and drug resistance in AA TNBC patients. Such knowledge is a critical foundation for the development of cancer therapies targeting vulnerabilities of tumor heterogeneity in AA TNBC patients.

CCM3 is one of mutated genes responsible for the human CCM disease, a pathological condition that affects the vasculature of the central nervous system and results in stroke, seizure and cerebral hemorrhage with a high prevalence. CCM consists of dilated and multiple capillary channels formed by a single layer of endothelium, lacking all other normal vessel wall elements. Patients with inherited autosomal dominant CCM carry loss of function mutations in one of three genes: CCM1, CCM2 and CCM3. Deletion in one of the three Ccm genes in vascular EC induces CCM lesions in mice. However, it is unknown why both humans and mice with CCM3 loss exhibit more severe phenotype than those with loss of CCM1 or CCM2. Our unexpected discovery of the involvement of CCM3 in EC exocytosis, prompt us to hypothesize that alteration in CCM3-regulated EC exocytosis contributes to the pathogenesis of the CCM disease. We propose the following two specific aims: 1) To determine the role of CCM3-regulated brain EC exocytosis in CCM disease phenotypes. We will establish mouse CCM models even closer to human CCM disease by creating brain EC-specific Ccm3 deletion, and determine therapeutic effects of Angpt2 neutralization antibodies in the new CCM3 mouse models. 2) To explore crosstalk of CCM3-mediated EC exocytosis with other pathways implicated in CCM formation. We will test if inhibition of exocytosis in ECs blocks RhoA-dependent EC stress fiber formation, TGF-?/Smad/BMP-mediated EndMT signaling, and MEKK3-ERK5-KLF4-mediated matrix remodeling. Conversely, test if gain- or loss-of-function of RhoA, TGF-? and MEKK3-ERK5-KLF4 signaling regulate EC exocytosis. In summary, the complementary approaches using genetic, cell biological and imaging analyses will facilitate our understanding of the molecular mechanisms and pathogenesis involved in acquisition of cerebral cavernous malformations, and help in defining new and more effective therapies. Our findings should benefit the general understanding of the regulatory mechanisms of exocytosis, which also occurs in other cardiovascular cells and ECs of other cardiovascular organs such as heart, lung and aortae. Therefore, our present study is of broad significance in cardiovascular research.