• Cardiovascular
• Endocrine
• Gastroenterology
• Genetics
• Geriatrics
• Hematology
• HIV
• Immunology
• Infectious Diseases

• Nephrology
• Oncology
• Pharmacology
• Pulmonary
• Rheumatology
• Sports Medicine

Title of Research Project:Apoptosis and autophagy in cardiomyocytes
My research involves the response of cardiomyocytes to stress, such as ischemia or aging. Using laboratory and animal studies, we are investigating why and how cardiomyocytes die in response to various stimuli, in particular we study cardiomyocytes after myocardial infarction and in aging. We explore molecular, cellular and genetic aspects of cell death, cellular protection and turnover. This links very closely with stem cell function and therapy for the failing heart. In addition, we are commencing pilot observational human studies in this area.

Title of Research Project:Post-Translational Trafficking of Cardiac Ion Channels
The basic function of the heart is to work as a pump, circulating blood through the lungs, brain, and body. For each successful heartbeat the activity of millions of individual heart cells must to be coordinated for them to contract in synchrony. The cardiac electrical system, based on ion channels and the flow of ions exists for this coordination. In ischemic heart, ion channels are both poorly expressed and improperly localized, which result in dangerous heart rhythms such as sudden cardiac death. There are 325,000 cases of sudden cardiac death in the United States each year. In addition, ischemia related congestive heart failure, which affects five million Americans, is due in part to altered ion channel regulation.

For these reasons, we are very interested in ion channel regulation in both normal and damaged heart cells. We recently developed a new paradigm describing the mechanism by which the ion channels are targeted from inside the cell to their proper location on the cell membrane. We are continuing a cell biology based approach to study the movement of ion channels from the time they are formed to their placement at specific locations on the cell membrane. The techniques of the lab involve live cell imaging and total internal reflection microscopy (TIRF) as well as standard immunocytochemistry and biochemistry. The goal of our research is to use the insights gained by these basic cell biological studies to develop therapeutic interventions that decrease the incidence and impact of sudden cardiac death and heart failure.

Title of Research Project:
Angiogenic therapy has been investigated as a potential therapeutic strategy for ischemic cardiomyopathy for two decades, but there is still no product available for clinical use. The problems include short duration of action of the angiogenic factors, inadequate transduction or expression of the genes, and immune reactions against the delivery vectors. Also, uncontrolled expression of angiogenic factors may cause complications. To overcome these difficulties we used the adeno-associated viral (AAV) vector to mediate long-term angiogenic gene expression and used cardiac-specific promoter and hypoxia response element to limit the angiogenic gene expression in ischemic myocardium. We use mouse and porcine as model for our study. Our goal is to development an effective and safe angiogenic gene therapy for the treatment of ischemic cardiomyopathy.

Irreversible myocardial-injury occurs shortly after the onset of coronary occlusion. Angiogenic therapy can reduce post-infarct remodeling and improve cardiac function, but it has little effect on repairing existing scars. Transplantation of stem cells from various sources into infarcted hearts has the potential to regenerate injured myocardium. We have tested to use bone marrow derived mesenchymal stem cells (MSCs) myocardial regeneration. We noticed that the survival rate of MSCs was very low in ischemic myocardium. Thus, we proposed a strategy of combining cell therapy with angiogenic gene therapy. We hypothesized that angiogenic factors will stimulate neovascular formation in ischemic myocardium, which in turn will support transplanted stem cell survival.

Recent studies indicated that embryonic stem cells (ESCs) may be a better source for cardiac regeneration. However, none of the current methods can mediate ESC to differentiate into 100% pure cardiomyocytes. Using a mixture of differentiated cardiomyocytes and undifferentiated ESC may potentially cause tumor. We study to isolate ESC-derived cardiomyocytes by genetically labeling the human ESCs with a cardiac-specifically expressed surface marker. AAV vector will be used to deliver the genes. The purity, protein expression profile, the tumorigenicity and the regenerative ability of the selected cells will be studied.

Title of Research Project:
The blood clotting system is centrally important as a means to protect from blood loss. To do so, the system must be sensitive to disruptions in blood vessels. We know from naturally occurring human genetic mutations and experiments in animals that a deficiency of function or amount of clotting related proteins leads to bleeding. Yet the system must also be specific. There is an equal body of evidence that unregulated or increased propensity to form blood clots leads to deleterious clot formation such as occurs in heart attacks, strokes, and blood clots in large veins. The clotting system therefore must maintain exquisite balance between tendency toward clotting and tendency toward bleeding. Minor changes in concentration or function of a host of known and countless unknown proteins can tip the balance in either direction. Primarily, we use the mouse as a model system to define genetic regulation of blood clotting in an attempt to define genetic changes that might predispose to tipping the balance in either direction. We hope to learn more about the molecules and pathways that lead to clot formation. We hope to define novel molecules or pathways that regulate clotting or interact with known clotting pathways. We are particularly interested in how male or female sex affects clotting in animals. We know that women are 1) less likely to form clots in clotting tests and 2) are protected as compared to men in diseases associated with increased clotting like heart attacks. This tells us that women may have evolved a system with a more favorable balance between clotting and bleeding. We hope to learn how and why that may be. Ultimately, we hope to identify new risk factors for bleeding disorders as well as the clotting associated diseases such as heart attack and stroke. Furthermore, we hope that by understanding the biological mechanisms underlying such risks, we might eventually identify novel drug targets aimed at treating or preventing bleeding, stroke, heart attack or blood clots.

Alpha-1-adrenergic receptors in the human heart and knockout (KO) mice.

Heart failure is a major clinical problem and new drugs are needed. Recently, we used KO mice to show that heart alpha-1-adrenergic receptors for norepinephrine and epinephrine are required for normal cardiac development and adaptation to stress and injury (J Clin Invest 2003 and 2006). The KO mouse and much other data suggest that activating alpha-1 receptors could be a novel treatment for heart failure, to stimulate heart muscle cells to recover when injured or grow stronger when weakened. However, there are 3 distinct alpha-1 subtypes, raising two major questions: (1) which subtypes are present in the human heart?; and (2) which of the subtypes are most important for cardiac protection in KO mice? A resident can join either project, depending on interests and background. Project 1 will involve collecting human heart samples in the OR, isolating myocytes, and doing receptor mRNA and protein assays. Project 2 will involve mouse genetics, and physiology, pharmacology, and pathology in KO mice.

Angiogenesis: We are studying differential responses of adult cardiac and skeletal muscle to angiogenic gene therapy, focusing on effects of VEGF and pleiotrophin on the vasculature and on the localized protein profile in the tissue. This continues a decade of our research aimed at understanding the response of adult tissue to exogenous VEGF gene delivery, potential deleterious effects, and potential therapeutic applications. Using virally-transduced myoblasts as a gene delivery vehicle, this research has demonstrated that VEGF expression can induce vascular growth in both ischemic and non-ischemic mouse skeletal muscle and myocardium. The newly-formed capillaries are in contact with the circulation, and new arterioles form upstream of the capillaries. Interestingly, the vascular response is extremely localized within micrometers of the VEGF source through a heparin-independent mechanism. Notably, constitutive expression of excessive amounts of VEGF causes vascular overgrowth, leading to vascular malformations and hemangiomas in both skeletal and cardiac muscle. In skeletal muscle, this effect occurs even if excessive levels are produced in only small regions of the tissue, and Dr. Springer's former colleagues have shown that implantation of homogenous populations of these myoblasts in which every cell expresses moderate levels of VEGF leads to growth of non-pathological, stable vessels that increase perfusion to ischemic regions. We are continuing this line of research in the heart to determine if such exquisite dose response and microenvironmental control determine vessel growth pattern and morphology in cardiac muscle, to evaluate the impact of these properties on cardiac function after myocardial infarction, and to determine differences in molecular responses of skeletal and cardiac muscle to VEGF gene delivery.

Cell therapy for myocardial infarction In collaboration with Dr. Yeghiazarians, we are studying the therapeutic effects of implanting bone marrow-derived cells (BMCs) into mouse hearts after myocardial infarction (MI), using a high-resolution echocardiography approach that our collaboration has developed to guide injections into the myocardial wall without surgery. Our study includes evaluation of the clinical relevance of common rodent models used for such experiments, and of approaches to overcome the limitations of these models to represent realistic cardiovascular disease and treatment. The echo-guided approach allows us to introduce BMCs to mice several days after MI, a time relevant to current clinical trials that is not feasible when using traditional open-chest injection approaches. We have shown that injection of BMCs 3 days post-MI can preserve or partially restore left ventricular function. We have also demonstrated that injection of a cell-free extract of lysed BMCs has a similar therapeutic effect, suggesting not only that BMC therapy may be beneficial by a paracrine mechanism, but also that the cells may simply die and thus deliver a bolus of therapeutic growth factors.

Role of NO synthase in human endothelial progenitor cell function A related project is aimed at understanding the molecular basis of age- and disease-related impairment of endothelial progenitor cells (EPCs), a heterogeneous population of cells that are thought to be involved in several aspects of angiogenesis and endothelial maintenance. We are studying endothelial nitric oxide synthase (eNOS)-dependent and eNOS-independent mechanisms of EPC migration toward angiogenic stimuli by VEGF and pleiotrophin, and are investigating the molecular mechanisms through which NO controls EPC migration and differentiation. We are also investigating the correlation between eNOS activity and EPC function both ex vivo and in vivo, including the genetic manipulation of these cells to enhance or impair their functional profile.

For more information, please see http://cardiolab.ucsf.edu/molcardiolab/

1. Discovery of new receptors and ligands that are good targets for cancer therapy.

   We have produced a unique library of over 3000 proteins including essentially all secreted proteins (ligands) and most extracellular domains of transmembrane proteins (receptors). Using a high through put automated screening system we can quickly find receptors for ligands whose receptors are unknown. This is particularly important for ligands that are linked to cancer tissue and their receptors may be drug targets. Likewise we can find ligands for "orphan" receptors. Most of the scientists at FivePrime are working directly on protein drugs and we have not had a scientist to work full time on this receptor-ligand matching project. For this reason there is an exceptional opportunity for a resident to make multiple discoveries using our system and findings.

2. New therapies for diabetes.

   We have discovered a protein that increases glucose uptake in muscle but not fat. When given to mice with diabetes our protein (FPT038) reduces hyperglycemia, reduces insulin levels but does not cause hypoglycemia. All of these effects appear to be favorable in the treatment of diabetes. In addition there appears to be a persistent beneficial effect many hours after the protein is cleared from the circulation. FivePrime is developing this protein as a drug. Although much is understood about FPT038 there are still unanswered scientific questions about the mechanism of its persistent effect and its apparent insulin sensitizing activity that need answering.

3. Protein therapeutics for acute myocardial infarction

   We have discovered 4 proteins that appear to protect cardiac myocytes from ischemic damage in vitro. We are interested in studies of these proteins in animal models of myocardial infarction. The goal of this work is to develop therapeutic proteins that can be used clinically to rescue myocardium during the early stages of infarction.

Attention has thus turned to new methods of treating heart attack and heart failure patients in both the acute and chronic settings after their event. Heart transplantation remains the ultimate approach to treating end-stage heart failure patients but this therapy is invasive, costly, some patients are not candidates for transplantation given their other co-morbidities, and most importantly, there are not enough organs for transplanting the increasing number of patients who need this therapy. As such, newer therapies are needed to treat the millions of patients with debilitating heart conditions. Recently, it has been discovered that stem cells, which are early progenitor cells with the ability to direct the production of all different types of human cells, may hold the therapeutic potential for these patients. Experimental studies in both animals and humans have revealed encouraging results when stem cells are injected into the heart in the areas of myocardial infarction. These therapies appear to result in improvement in the contractile function of the heart.

Despite these promising early trials, many questions remain unanswered concerning the use of stem cells as therapy for patients with heart attack and heart failure. To answer these questions and to ultimately offer this therapy routinely to patients, the UCSF Cardiology Division has launched a Cardiac Stem Cell Translational Development Program to address these issues. We have numerous on-going projects in the small and large animal heart attack models; in-vitro experiments studying both adult and embryonic stem cell are underway; numerous observational human clinical trials are also currently being performed.

How are the thrombi that cause most heart attacks and strokes formed? How are normal hemostatic and inflammatory responses to tissue injury triggered? The coagulation cascade generates thrombin and related serine proteases upon disruption of vascular integrity, and thrombin is a potent activator of platelets, endothelial and other cells. How does a protease like thrombin behave like a hormone to regulate the cellular behaviors? We've characterized a family of protease-activated G protein-coupled receptors (PARs) that provide an answer. Thrombin cleaves PAR1's N-terminal exodomain to unmask a new amino terminus that then serves as a tethered peptide ligand, binding intramolecularly to the heptahelical segment of the receptor to cause transmembrane signaling. PAR1 is the prototype for a family of four receptors that appear to account for most cellular responses to coagulation and other trypsin-like proteases. Our laboratory currently focuses on understanding the roles of protease and PAR signaling and, more broadly, G protein-coupled receptors in cardiovascular biology:

PARs in physiology and disease. Using mice with individual and combined PAR deficiencies, we are exploring the importance of PAR signaling in platelets, endothelial cells and other cell types in mouse models of hemostasis and thrombosis, inflammation and other processes. A current emphasis is utilizing advanced light microscopy techniques to visualize the biochemical and cellular events that mediate various stages of thrombus assembly. Early studies show that PAR signaling is unnecessary for formation of an initial juxtamural platelet thrombus but required for enlargment and propagation of such thrombi. Thus different signaling mechanisms may be important at different points in the development of a thrombus, and exploiting such differences may permit the development of safer antithrombotic drugs.

PARs in embryonic development. PAR1 signaling in endothelial cells is important for normal vascular development in the mouse embryo. Efforts to identify the specific endothelial cell behaviors involved as well as the other targets of the coagulation cascade that are important for embryonic development are in progress. PARs, specifically PAR2, also appear to contribution to neurulation. Efforts to determine what PAR2 senses biochemically and physiologically and what it regulates in this context are ongoing. This line of research will reveal new roles for protease signaling.

Sphingosine kinases in development and disease. Sphingosine-1-phosphate acts through G protein-coupled EDG receptors to regulate heart and blood vessel formation in the embryo as well as leukocyte trafficking and other important processes in the adult. The exact sources of S1P and hence when and whether it functions primarily as a hormone or as a paracrine or autocrine factor are unknown. We have generated conditional alleles for the two sphingosine kinases in mice to explore these questions.

Novel roles for G protein signaling. The studies outlined above emphasize that G protein-coupled receptors can play important roles in embryonic development. The ~350 nonodorant G protein-coupled receptors in mice and humans couple through 4 main G protein families, Gs, Gq, Gi, and G12/13. We are ablating G12/13 and Gi signaling in specific cell lineages to probe the roles of these pathways in embryonic development and other processes, then using a candidate approach to identify the receptors and ligands involved.

Title of Research Project: Two main areas:

1. Role of IGF-I and bone development and response to mechanical load.
2. Role of calcium and vitamin D in regulating keratinocyte differentiation.

1. Two main sub projects re IGF-I and bone.
   1. We are exploring the mechanism by which skeletal unloading leads to resistance to the anabolic actions of IGF-I on bone. Data show a clear link to integrin signaling. We are pursuing this using both an unloading model (tail suspension) and a loading model (tibial loading, bone cells on flexible substrate).
   2. Using an IGF-I knockout and a conditional (bone specific) IGF-I receptor knockout animal model we are examining the abnormality in bone development in embryos and the resistance to the enabolic effects of PTH on bone postnatally.
2. Two main sub projects are keratinocyte differentiation.
   1. We are examining the mechanisms by which calcium stimulates keratinocyte differentiation. We have identified key roles for the calcium receptor and for the phospholipase C. We are currently developing epidermal specific knockout models of these two proteins to examine their roles in vivo.
   2. Keratinocytes both produce and respond to the active metabolite of vitamin D, 1,25(OH)2D with changes in keratinocyte differentiation. Our knockout models for the enzyme producing 1.25(OH)2D(1 hydraxylase) and for its receptor (VDR) have abnormalities in the epidermis. The VDR k/o mouse also shows a loss of hair beginning around weaning. We are currently pursing a number of studies investigating the transcriptional activity of VDR, and how this is controlled both in the epidermis and during hair follicle cycling by various co activators and suppressors.

1. Type 2 Diabetes Mellitus
2. Insulin and insulin-like growth factors in cancer
3. Interventions in insulin-resistant subjects.

Title of Research Project:

1. Role of membrane protein PC1/ENPP1 in insulin resistance and Type 2 diabetes.
2. Novel inhibitors of insulin-like growth factor 1 receptors in cancer.
3. Effects of Exercise Training, Chromium, and Lipoic Acid in Insulin Resistance.

Title of Research Project:

1. Clinical:
• Clinical studies in the role of alcohol in Hepatitis C infection; Co-infection of HIV patients with Hepatitis B and C; the role of liver disease in HIV infection. These studies evaluate the role of cofactors such as alcohol, HIV, drug use in outcome of disease and response to therapy.
• Studies on pathophysiology and management of end stage liver disease including autoimmune liver diseases, primary biliary cirrhosis and recurrent disease post liver transplantation.
• Studies on management of end stage liver diseases
2. Translational:
   Host-viral interactions in Hepatitis C and Hepatitis B infection. These projects evaluate clinical outcomes and the role of inflammatory cytokines and their receptors using DNA polymorphism analysis and mRNA gene profiling. We are studying the effect of the host response in induction of disease and response to therapy including the effect of alcohol and HIV co-infection.

Title of Research Project:

1. Transcription factor interactions at the GH promoter -This project analyzes the subnuclear positions and interactions of factors that regulate the transcription of genes in pituitary cells. Aim 1. compare the effects of mutations in specific Pit-1 and C/EBP activities on transcriptional synergy, release of C/EBP binding to -satellite repetitive DNA, and Pit-1 marshaling of C/EBP to euchromatin, Aim 2. define the effects of the Pit-1 and C/EBP mutants on the biochemical interactions and conformations of C/EBP, Pit-1, and target proteins, Aim 3. determine the effects of Pit-1 and C/EBP expression on GH/PRL promoter binding, -satellite DNA binding, and recruitment of co-factors or modified histones to the promoters and -satellite DNA

2. Temporal and Spatial Dynamics of Androgen Receptor Conformation and Interactions in Prostate Cancer Cells -This project developed the low throughput FRET technology for following the temporal and spatial changes in the conformation and interactions of the Androgen Receptor following agonist or antagonist addition. Aim 1. Conformation, dimerization, nuclear transport and activity of flutamide-resistant, AR mutants Aim 2. Conformation, dimerization, nuclear transport and activity of AR in different prostate cancer cell environments. Aim 3. AF-2 interaction with FQNLF in the DHT-induced re-positioning of AF-1 towards the LBD.

3. Novel imaging techniques that compare estrogen receptor structure in tumors and normal tissues of intact living animals -This project uses FRET technology for following in live mouse models, the agonist and antagonist-regulated conformation and interactions of the Estrogen Receptor. Aim 1, Creation of stable cell lines expressing fluorescent protein fusions with ERa Aim 2, FRET imaging in live animal models

4. High Throughput Identification and Characterization of Novel Anti-Prostate Cancer Therapeutics -This pending pilot project is for the very initial stages in the development of the High Throughput Screen of the Androgen Receptor. -Aim 1: creation of constructs and prostate cancer cell lines for AR high throughput AR imaging -Aim 2: multiplex the AR imaging assay -Aim 3: validate the high throughput AR screening protocol, then conduct an initial screen of a chemical library targeted broadly towards kinases.

5. High Throughput Identification of Subtype-Selective Breast Cancer Therapeutics -This pending project is for the development of the High Throughput Screen of the Estrogen Receptor. Aim 1: High throughput protocols will be developed on different ER-positive, Luminal A subtype breast cancer cell lines that respond to, or are resistant to, anti-estrogen treatment. Aim 2: These protocols will be used to screen for compounds and compound combinations with highly selective effects on ER biochemistry, on the structure of ER complexes and on breast cell proliferation. Aim 3: Parallel developments in animal imaging will allow monitoring of the efficacy and bioavailability of the lead compounds on the same processes in live animals. Aim 4: The abilities developed in Aims 1-3 will be applied to studies of HER2 in the ERBB2 subtype.

Title of Research Project:

1. G protein signaling in osteoblasts: These studies focus on the mechanisms underlying the catabolic and anabolic changes in bone that result from specific G protein signals in osteoblasts. Gain of function of specific G protein pathways is achieved by the targeting of Receptors Activated Solely by Synthetic Ligands (RASSLs) to osteoblasts in vivo. Loss of function is produced by induction of G-protein alpha-subunit ablation in vivo. By inducing these changes at different stages of osteoblast differentiation, we are building an integrated model of the role of G protein signaling in controlling skeletal homeostasis.
2. Wnt signaling in osteoblasts: Genetic studies in humans and mice indicate that canonical Wnt signaling in osteoblasts is essential for the maintenance of bone mass. We are carrying out molecular genetic studies to determine the mechanisms underlying this action of canonical Wnt signaling and to determine the relationship of Wnt signaling to the known anabolic effect of intermittent PTH administration. Studies so far indicate that there is convergence in the actions of PTH and Wnt. Elucidation of the basis for this convergence could be useful in the development of new strategies for the treatment of osteoporosis.

Title of research projects:

1. Pathophysiology of hepatic cirrhosis
2. Pathophysiology of intestinal stricture
3. Epidemiology and management of chronic liver disease

Brief description of research project: Molecular and cellular biology techniques are used to examine the pathophysiology of hepatic cirrhosis and intestinal strictures. Meta- and decision analysis, and database investigation are used to examine the epidemiology and management of chronic liver disease.

Title/Description of Research Project: There is a wide scope of possible studies within the interests being pursued in our laboratory such as the relationship of the genotype of the Lp(a) lipoprotein to disease, investigation of the mechanisms of action of gene polymorphisms recently discovered in our collaborations that are associated with increased risk of MI, etc., the molecular speciation of HDL in relationship to disease phenotypes, effects of medications on the distribution and function of HDL molecular species,etc.,etc.

Title/Description of Research Project: To counter the pandemic of obesity and its related illnesses, a molecular genetic approach is taken to identify genes governing neutral lipid metabolism. Large scale chemical mutagenesis in zebrafish has yielded mutants with hepatic steatosis, the inappropriate accumulation of neutral lipids in the liver, an obesity-related illness of significant public health concern. Current projects involve positional cloning and characterizing of these genes. Questions include whether the mutant animals progress through the spectrum of nonalcoholic fatty liver disease, whether they are obese, and what the underlying mechanisms for these phenotypes are. Techniques range from live animal microscopy, to biochemistry, to mammalian cell culture. Spin-off projects include generation of transgenic animals that express fluorescent reporter proteins in adipocytes.

Title of research project/Brief description of project: Brain Aging with HIV
These series of projects address the cognitive consequences of aging with HIV infection. These are all clinical research protocols involving neuropsychological testing, neurological examinations, brain MRI, and laboratory testing. Individuals who work on these projects may learn techniques associated with cognitive characterization of patients and imaging. Opportunities exist to participate in data analysis including imaging analyses.

Title of research projects: Molecular properties of human HDL
Genetic determinants of myocardial infarction
Clinical studies of dyslipidemias

Title of research projects: My research focuses on the population genetics of complex disorders and their related phenotypes in the U.S. understudied populations including Latinos, African Americans and South Asians. We have several ongoing projects that use cutting-edge genetic methods such genome-wide association analysis, admixture mapping and pathway analysis to better understand genetic and environmental risk factors for complex diseases and how interaction between genes and environment can modulate risk for diseases.

Title of research projects: My laboratory studies how regional gene expression across the CNS (including the hypothalamus, frontal cortex, striatum, and cerebellum) changes with ageing, and the behavioral correlates of these changes. Efforts are specifically focused on studies of age-related dysregulation in gene networks governing microglial-based inflammatory and neuronal-based synaptic responses. Gene expression is quantified using both microarray and qPCR-based methods. We analyze both whole tissue and enriched populations of neurons, microglia, and astrocytes separated by FACS. Mouse home cage behaviors (including eating, drinking, movement, and circadian patterning of activity) are measured with a custom platform that acquires data at a high spatial and temporal resolution. Sophisticated computational approaches are then employed to derive complex behavioral metrics from this home cage data. More traditional approaches to mouse behavioral phenotyping are used as needed. We study how interventions thought to improve cognitive reserve, including exercise and environmental enrichment, alter these inflammatory and synaptic processes. We also apply these methods to better understand the basic biology underlying frailty.

Title of research:
My research interests focus on signal transduction and regulation of cellular growth, including T-cell activation, mechanical stimulation of osteoblasts stem cell differentiation and the growth induction of tumors by essential fatty acids. Our interests focus on the initial signaling events, which trigger a cascade of events leading to cell proliferation/differentiation 24-48 hours later. Although our model systems: T-cells, cancer cells, osteoblasts and monocytes are diverse, the signal pathways and gene expression profiles during early activation share common features.

Mechanisms of T-cell activation and monocyte differentiation in microgravity: This investigation explores the mechanism of action for induction of T-cell IL-2R mRNA units after activation. Previous experiments by Dr. A. Cogoli have demonstrated that T-cells are not activated in microgravity with the TCR being the most probable site of gravity dependence. Using qRTPCR, Affymetrix gene array, ELISA and Western blots we have discovered the dependence of several signaling pathways on normal Earth gravity during T-cell activation. Our first manuscript is in press in Cellular Signaling (Impact Factor 5.2), the second manuscript is in revision. The re-flight of our STS-107 Leukin experiment will occur in September 2006 on the Russian Soyuz, where we will have the opportunity to verify our ground RPM data in true microgravity (10-3 -10-6 g). The second study, PKINASE flew in October, 2007 investigates how microgravity affects protein kinase C (PKC) signaling in monocytes and the functional consequences of these changes on downstream signaling events, gene expression and cell fate. When protein kinase C isoforms are activated in normal gravity they translocate to the cell membrane, which is a key step in signal transduction by these kinases. Using Affymetrix gene arrays, we will be able to predict which signal pathways are dysfunctional without gravity. These experiments are essential to our understanding the role of gravity in terrestrial life.

Fatty acid regulation of gene and protein expression in cancer: We were the first to show that cox-2 is up regulated in prostate cancer and that it has a feed forward mechanism. The current project focuses on the role of the essential fatty acids induction of mRNA, protein synthesis and proliferation in neoplastic tissues. Using RTPCR, ELISA, signal pathway inhibitors, COX-2 inhibitors and fluorescent microscopy we have demonstrated that the essential fatty acids (EFAs) are involved in early gene expression of inflammatory molecules in tumors. Despite the pronounced differences in the mortality rates of the different cultures, latent prostate cancer found at autopsy occurs at the same frequency in Japanese men as in Caucasian males. These accumulating data suggest that the higher intake of dietary fat in Western society compared to other countries may account for the high rate of prostate cancer incidence in the US with African-Americans having twice the rate of the Caucasian male. Over the past 50 years in the U.S., the ratio of dietary intake of -6 FA vs. -3 FA has increased from 1:2 to 25:1; during this same time frame the incidence of prostate cancer has risen. Even in Japan over the last 30 years, the intake of -6 FA has risen to 4:1 from 2:1 (4). In addition, chemokines have recently been linked to angiogenesis and vascularization. Specifically interleukin 8 (IL-8), interleukin-6 (IL-6) and tumor necrosis factor (TNF) have been linked to angiogenesis, vascularization, tumorogenicity and metastases in prostate cancer in vitro and in vivo both in animals and humans. Since elevated levels of chemokines and cytokines are linked with tumor progression, metastases or survival, we hypothesize that patients eating a Western diet with elevated levels of -6 FAs will have higher levels of chemokine and cytokine synthesis and higher incidence of advanced disease. We have found dramatic increases in chemokines with -6 FA both in vitro and in vivo.

Rapid fracture healing by FGF-2: We are testing if growth factors and their anabolic products selectively induce gene expression needed for effective human mesenchymal stem cell (hMSC) migration, proliferation, and wound healing prior to mineralization both in vitro and in vivo. We will also test the hypothesis that delivering growth factors ex-vivo or in an extracellular matrix (ECM), would drastically lower cost of GF treatment in man and allow faster repopulation of the osteoblasts. Finally we will test the hypothesis that combined therapy using ex-vivo expansion of hMSC by growth factors followed by a differentiation treatment just before transplant will result in better vascularizion, greater bone mass and accelerated mineralization of a fracture. Currently we have identified several ECMs and GFs that facilitate fracture repair.

Title of research:
My research deals with the mechanisms of resistance to standard hormone therapy for prostate cancer patients. As a clinical/translational researcher, I conduct clinical trials of new drugs at the phase I and II level. My major clinical trials target the synthesis of androgens by the adrenal gland. Further, I study the interaction of androgen stimulation and signaling by the Insulin like growth factor (IGF) receptor in prostate cancer models. We do preclinical work in the lab that corresponds to our ongoing clinical trials.

1. Mouse models of sickle cell disease.
2. Gene therapy for hematopoietic disease and coronary heart disease.

Title of research:

1. Creation of a mouse model of sickle cell disease:
   1. We are creating several models of sickle cell anemia that mimic the human haplotypes of sickle cell diseases. These haplotypes result in variable clinical severities due to different fetal hemoglobin expression, and will provide good models for testing therapeutic approaches
2. Gene therapy for hematopoietic and coronary heart diseases:
   1. We are studying a mouse model of α- thalassemia. As in humans, the homozygous affected fetuses are lethal in the third trimester of pregnancy. We are using several vectors to transducer hematopoietic cells with the human α-globin gene to rescue these mice.
   2. We are devising new AAV vectors that are much more efficient in tranducing liver and hematopoietic cells to study genetic diseases, such as ornithine transcarbamylase deficiency, that are lethal in the newborn.
   3. We are using gene and cell therapy to express VEGF and other angiogenic factors for the treatment of coronary. Contact: Lisa Woldin 415-476-5841

Title of research project/Brief description of project: Brain Aging with HIV
These series of projects address the cognitive consequences of aging with HIV infection. These are all clinical research protocols involving neuropsychological testing, neurological examinations, brain MRI, and laboratory testing. Individuals who work on these projects may learn techniques associated with cognitive characterization of patients and imaging. Opportunities exist to participate in data analysis including imaging analyses.

Title/Description of Research Projects:

Medication interaction studies -- ongoing studies, have been working for the last 6 years with the HIV-transplant group, and new studies on effects of orange juice on lasix absorption and action, and glyburide activity when given with ciprofloxacin or rifampin to be started in spring 2006

Diet and exercise -- presently starting the "Paleo diet" study -- to see if eating a "Stone Age" diet (high in potassium, fiber, antioxidants, base-producing anions) will improve the ability to exercise or recover from exercise, vascular reactivity, and lipid/glucose profiles. Will soon be starting a study on salt intake and changes in acid/base status.

Title of Research Projects:

Title of research project 1: Regulation macrophages and microglia by TREM-2
Brief description of research project 1: We cloned mouse TREM-2, a receptor on activated macrophages and microglia, and we demonstrated that it recognizes a broad range of bacteria as well as endogenous ligands on brain cells. Our recent studies have found that TREM-2 can augments some macrophage responses to bacteria but downregulates others. Our current studies seek to define the role of TREM-2 in pathogen recognition by bacteria and in the recognition of self-ligands by microglia.

Title of research project 2: TIM-2 is a receptor for H-ferritin
Brief description of research project 2: We recently found that the mouse TIM 2 surface protein is a receptor for H ferritin (the first receptor identified for H ferritin. We found that TIM-2 is expressed in the liver and kidneys and on activated B cells. Our current studies seek to define the role of this receptor in regulating B cell function. We also seek to clone the human receptor(s) for H ferritin.

Title of Research Project: Small molecule therapies of membrane transporter diseases.
Brief Description: Projects available depending on research interests. See www.ucsf.edu/verklab. Projects include identification and testing of small molecule therapies for membrane transporter diseases; phenotype analysis of transgenic mouse models.

Title of Research Project: Identification of new genes involved that control acid excretion.
Brief Description: Use of RT-PCR and yeast 2-hybrid to identify new genes involved in the control of kidney and osteoclast acid secretion.

Title/Description of Research Project: Two projects are currently active. In one, we are studying how melanocyte stimulating hormones (MSH's) affect blood pressure. These are peptides with α-, β-, and γ- primary structure which share a core heptapeptide sequence; they are all derived from the prohormone proopiomelanocortin, and they all are secreted by the pituitary and circulate in plasma. However, they also serve important functions in the CNS. They increase sodium excretion by the kidneys, and the plasma concentration, and pituitary content, of γ-MSH increase when rodents are placed on a high sodium diet. Deficiency of γ-MSH causes marked salt-sensitive hypertension, which can be rescued by administration of very small amounts of the peptide directly into cerebrospinal fluid. We are following up on the mechanisms involved in the salt sensitivity, and it relationship to our recent observation that mice and rates with γ-MSH deficiency develop insulin resistance as well as hypertension when exposed to the high sodium diet.

The other project relates to the observation that states of pathological sodium retention--liver disease, nephrotic syndrome, and congestive heart failure--are accompanied by resistance to the renal actions of atrial natriuretic peptide (ANP) to increase sodium excretion. We have found that there is upregulation of a phosphodiesterase (PDE) enzyme in kidney which hydrolyzes the cGMP formed when ANP interacts with its renal receptors. This upregulation thus contributes to ANP resistance. We have observed that normal pregnancy in rats is also characterized by ANP resistance and an increase in PDE expression. This presumably allows for the large plasma volume expansion which characterizes normal pregnancy. We are currently seeking the signal(s) which tell the pregnant rat kidney to upregulate its PDE activity.

Both projects will expose the interested participant to studies in intact animals (rats, mice), to techniques of physiological measurement of blood pressure, heart rate, and renal function, and to certain techniques of molecular and cell biological research.

Title of research:
My research deals with the mechanisms of resistance to standard hormone therapy for prostate cancer patients. As a clinical/translational researcher, I conduct clinical trials of new drugs at the phase I and II level. My major clinical trials target the synthesis of androgens by the adrenal gland. Further, I study the interaction of androgen stimulation and signaling by the Insulin like growth factor (IGF) receptor in prostate cancer models. We do preclinical work in the lab that corresponds to our ongoing clinical trials.

1. Transcription factor interactions at the GH promoter -This project analyzes the subnuclear positions and interactions of factors that regulate the transcription of genes in pituitary cells. Aim 1. compare the effects of mutations in specific Pit-1 and C/EBP activities on transcriptional synergy, release of C/EBP binding to -satellite repetitive DNA, and Pit-1 marshaling of C/EBP to euchromatin, Aim 2. define the effects of the Pit-1 and C/EBP mutants on the biochemical interactions and conformations of C/EBP, Pit-1, and target proteins, Aim 3. determine the effects of Pit-1 and C/EBP expression on GH/PRL promoter binding, -satellite DNA binding, and recruitment of co-factors or modified histones to the promoters and -satellite DNA

2. Temporal and Spatial Dynamics of Androgen Receptor Conformation and Interactions in Prostate Cancer Cells -This project developed the low throughput FRET technology for following the temporal and spatial changes in the conformation and interactions of the Androgen Receptor following agonist or antagonist addition. Aim 1. Conformation, dimerization, nuclear transport and activity of flutamide-resistant, AR mutants Aim 2. Conformation, dimerization, nuclear transport and activity of AR in different prostate cancer cell environments. Aim 3. AF-2 interaction with FQNLF in the DHT-induced re-positioning of AF-1 towards the LBD.

3. Novel imaging techniques that compare estrogen receptor structure in tumors and normal tissues of intact living animals -This project uses FRET technology for following in live mouse models, the agonist and antagonist-regulated conformation and interactions of the Estrogen Receptor. Aim 1, Creation of stable cell lines expressing fluorescent protein fusions with ERa Aim 2, FRET imaging in live animal models

4. High Throughput Identification and Characterization of Novel Anti-Prostate Cancer Therapeutics -This pending pilot project is for the very initial stages in the development of the High Throughput Screen of the Androgen Receptor. -Aim 1: creation of constructs and prostate cancer cell lines for AR high throughput AR imaging -Aim 2: multiplex the AR imaging assay -Aim 3: validate the high throughput AR screening protocol, then conduct an initial screen of a chemical library targeted broadly towards kinases.

5. High Throughput Identification of Subtype-Selective Breast Cancer Therapeutics -This pending project is for the development of the High Throughput Screen of the Estrogen Receptor. Aim 1: High throughput protocols will be developed on different ER-positive, Luminal A subtype breast cancer cell lines that respond to, or are resistant to, anti-estrogen treatment. Aim 2: These protocols will be used to screen for compounds and compound combinations with highly selective effects on ER biochemistry, on the structure of ER complexes and on breast cell proliferation. Aim 3: Parallel developments in animal imaging will allow monitoring of the efficacy and bioavailability of the lead compounds on the same processes in live animals. Aim 4: The abilities developed in Aims 1-3 will be applied to studies of HER2 in the ERBB2 subtype.

Title of Research Project: The Tumor Microenvironment in Mice with Lung Adenocarcinoma.
Brief Description: We use a mouse model of lung adenocarcinoma to study the tumor microenvironment. These studies include understanding the inflammatory cells that may promote malignant progression directly or indirectly by influencing the tumor andiogenic vasculature. The inflammatory cells we focus on are mast cells, neutrophils and macrophages, We also study specific angiogenic proteins expressed by both the vasculature and bye the inflammatory cells in the tumor stroma. The long-term goal of our project is to design specific antitumor therapies.

Title/Description of Research Project:
The Y chromosome is a man-only and the smallest chromosome of the human genome. The Y-genes are involved in male sex differentiation; spermatogenesis and various less understood sexual dimorphisms (such as in brain development). Dysfunctions of Y-specific genes, under certain conditions, lead to oncogenesis, such as the testis and prostate cancers, and sex-biased disease phenotypes, such as autism spectrum disorder (with a manifestation ratio of 4:1 between boys and girls). Our research focuses on two aspects of these processes, i.e. sex determination and oncogenesis. We use advanced technologies, including molecular genetics, proteomics, genomics, microarray analysis, bioinformatics, chromatin biochemistry and transgenic mouse strategies, to study 1) how the testis differentiation is switched on during embryogenesis, and 2) when dysregulated, how the Y chromosome genes contribute to male-specific cancers.

Recent exploratory projects include 1) evaluation of cell penetrating peptides as therapeutics in neurodegeneration and cancers; and 2) epigenetic effects of Y chromosome genes in sex-biased diseases, such as autism and HirschsprungÕs disease.

Title/Description of Research Projects
A series of studies on the influence of "Paleolithic-type" diets on:

1. lipid and glucose metabolism in healthy subjects and those who are insulin resistant - one project in type 2 diabetics, another in women with PCOS
2. exercise and vascular physiology in healthy sedentary controls and athletes

1. pharmacokinetics of antiretrovirals and immunosuppressant interactions
2. adverse effects of disease and medications on body composition and bone, and the cardiovascular system

Title/Description of Research Projects:
The general focus of our laboratory has been innate immunity, where we have focused on the study of cell surface receptors that activate or inactive macrophages and/or natural killer cells. This led us to also study microglia, which are the innate macrophages in the brain. Also, in studying receptors on the surface of cells we discovered the first known receptor for H-ferritin in mice, and we have recently discovered in human cells that the receptor for transferrin also binds to H-ferritin. We are currently pursuing studies in each of these areas.

Title of research project/Brief description of project: The identification of molecular sub-phenotypes of asthma and COPD:
These studies are funded by:

• An R01 from the NIH/NHLBI, entitled "Molecular Phenotyping of Asthma" (R01 HL-095372) which has applied genomic analyses to airway samples from patients with asthma to distinguish Th2-driven and non-Th2-driven sub-phenotypes of asthma that have distinct clinical, pathological and treatment-related characteristics.
• A seven-year NIH contract to apply similar methods to study COPD as part of the NHLBI Spiromics Project (N01 HR-08-08). The goal of the Spiromics project is to identify molecular phenotypes of COPD and to develop intermediate outcome measures for clinical trials.
• Mechanisms of airway inflammation and remodeling in lung diseases:
  These studies apply innovative methods for quantitative morphometry and gene expression analyses to human tissue samples obtained at fiberoptic bronchoscopy. In work to date, we have applied these methods to study mechanisms of disease in asthma, COPD and sarcoidosis. Ongoing work in this area is supported by the NIH (R01 HL-095372). Our approaches combine the application of gene expression profiling methods (microarrays, qPCR, laser capture microdissection) and design-based stereology (for quantitative measurement of tissue remodeling in human samples).
• Sample analysis and support for Clinical Trials:
  Finally, the Woodruff Laboratory supports sample analyses for clinical trials in asthma and COPD in both NIH and industry-supported studies. These sample analyses include measurement of changes in airway remodeling in response to specific therapeutic interventions, assessment of inflammation and the application of biomarkers to enhance the interpretation of clinical trials.

Title/Description of Research Projects:
"Invaders" from the environment (eg, viruses, bacteria, cigarette smoke, allergens, occupational irritants) invade the host after deposition on the airway epithelial surface. The epithelium responds to these invaders by intercepting the signals and developing a series of defensive responses that include leukocyte mobilization, the production of antibacterial peptides, mucin production (to aid in trapping and clearing of foreign particulates). These so-called Òinnate immune responsesÓ also include healing of wounded epithelium via epithelial proliferation and production of new blood vessels (angiogenesis). Normally, these host responses prevent invasion with few symptoms. However, occasionally overexuberant inflammatory responses occur, leading to chronic inflammatory diseases [eg, asthma, chronic obstructive pulmonary diseases (COPD), cystic fibrosis] or overexuberant proliferative responses (cancer). Over the past several years, my laboratory has discovered a series of signaling pathways responsible for multiple epithelial defensive responses. We have developed therapies for blocking the exuberant responses, which we believe to be important in the pathogenesis of chronic airway diseases.

Title of Research Project: Molecular Epidemiology Studies of Pneumocystis Pneumonia
Brief Description: Dr. Huang's main clinical and clinical research interests are in HIV-associated pulmonary disease and especially Pneumocystis pneumonia (PCP). He has collaborations with researchers at the National Institutes of Health, the University of Cincinnati, Makerere University (in Kampala, Uganda), the University of Pittsburgh, and Yale University as well as independent studies on PCP and ICU outcomes among HIV-infected patients. Current research studies include:

• Development and validation of several new molecular applications to PCP, including the use of a quantitative PCR assay on oropharyngeal wash specimens to diagnose PCP and development of Pneumocystis antibody assays to study Pneumocystis epidemiology and transmission.
• Comprehensive molecular-epidemiology study to address the question of whether PCP in humans results from person-to-person transmission (as has been convincingly demonstrated from animal-to-animal laboratory studies) and whether disease results from reactivation of latent infection or from recent exposure and infection.
• Prospective cohort study of the incidences, persistence, and consequences of Pneumocystis colonization both for the individual under study as well as for the potential as a reservoir for the organism.
• Prospective cohort study of the incidence and persistence of Pneumocystis colonization among health care workers (HCW).
• Creation of an international clinical research network to study HIV-associated pulmonary diseases worldwide.

Title of Research Project: Clinical and also lab based studies of acute lung injury.
Brief Description: Studies of biological markers in plasma and edema fluid of patients with acute lung injury or more basic lab.

Title/Description of Research Projects:
Translational, laboratory-based research projects focused on the electrophysiological properties of the paracellular pathway in epithelia, transcriptional regulation of tight junction constituents, cell signaling, and protein trafficking in cells, tissues, and clinical specimens.

Regulation of alveolar barrier function by claudins
Alveolar barrier repair following injury
Role of junctional proteins in epithelial cell migration

Title/Description of Research Projects:
My research activity encompasses both clinical and bench research into the mechanisms of diseases of the airways and, consequently, much of it falls under the rubric of "translational research". In these studies, I am interested in understanding the mechanisms of inflammation, airway remodeling, and airway hyper-responsiveness in asthma and chronic obstructive pulmonary disease. These studies are performed in the Airway Clinical Research Center here at UCSF and in the General Clinical Research Unit at the UCSF Parnassus campus where I am an investigator. A major focus of my recent work has been gene expression profiling in tissues obtained at fiberoptic bronchoscopy. Recent applications have included studies of airway smooth muscle structure and phenotype in airway diseases and studies of alveolar macrophage activation in smoking related lung disease. From the purely clinical research perspective, I am a Co-investigator in the NIH/NHLBI COPD Clinical Research Network which is currently designing protocols for multicenter-clinical trials in the therapy of COPD.

Title/Description of Research Projects:
Our recent studies indicate that epithelial cells in the alveolar compartment of the lung undergo a mesenchymal transition (EMT) during fibrogenesis in vivo and that this process is critically regulated by cellular interactions with the baseline and provisional extracellular matrices that develop during injury. Similarly, during the development of emphysema there is accelerated epithelial apoptosis. We are currently interested both in dissecting mechanisms for these processes in model systems and in developing clinical biomarkers that could be used in patients to track these processes during disease progression. Two current clinical projects in which a resident with limited time could actively participate are:

Biomarkers of IPF progression. We ae currently establishing a database of all ILD subjects referred to the ILD practice that includes DNA, serum/plasma, and RNA collected on all new subjects. We now need to obtain one year follow-up specimens on these patients (> 125) and begin to examine an initial set of biomarkers. A resident taking on this project, and supported by lab personnel, could use this as a beginning study for subsequent studies as a clinical fellow examining disease progression and therapeutic responses in ILD patients.

Diagnostic Signature of Lung Proteolytic Activity in Emphysema. We have preliminary data examining the proteomic profile of bronchoalveolar lavage (BAL) fluid from mice susceptible to or resistant to emphysema because of targeted deletion of an elastolytic protease, cathepsin S. We also have comparable samples from nonsmokers and smokers and have tentatively identified protein fragments in the BAL that appear to track with protease activity. A resident could undertake biochemical validation of the specificity and sensitivity of a proteomic signature in human BAL fluid and then use a validated marker to examine sera/BAL from early-onset emphysema patients. A validated biomarker for disease progression currently limits all contemplated clinical trials for intervention in COPD.

Title/Description of Research Projects:
The general focus of our laboratory has been innate immunity, where we have focused on the study of cell surface receptors that activate or inactive macrophages and/or natural killer cells. This led us to also study microglia, which are the innate macrophages in the brain. Also, in studying receptors on the surface of cells we discovered the first known receptor for H-ferritin in mice, and we have recently discovered in human cells that the receptor for transferrin also binds to H-ferritin. We are currently pursuing studies in each of these areas.

Sub-Specialty/Research Focus

Osteoimmunology
Bone Biology
Immunology
Rheumatology

Title/Description of Research Projects

Our laboratory is interested in the role of innate immune receptors in the regulation of differentiation and function of innate immune cells in normal and pathological states. We have examined innate receptor regulation in natural killer cells for a number of years and recently have focused on the role of innate receptors in the regulation of osteoclasts. These more recent studies focusing on "Immunoreceptor Regulation of Osteoclasts" are now considered part of the evolving field of osteoimmunology, which examines the interactions between the immune system and bone. Osteoclasts are specialized bone resorbing cells that form from myeloid precursor cells, and express a repertoire of innate immune receptors that are critical for osteoclast development and function. Abnormal bone remodeling, secondary to increased osteoclast maturation or activation, contributes to bone destruction in osteoporosis, rheumatoid arthritis and bony metastases. We are currently working to 1) define receptors that mediate activation and inhibition of osteoclast function 2) examine the roles of specific signals in osteoclastogenesis 3) examine the roles of receptors and signals in mouse models of inflammatory bone loss. We are also interested in further defining circulating osteoclast precursors in human disease states and will begin by examining circulating OC precursors in RA patients. The goal of this more translational study will be to specifically address the relationship between circulating osteoclast precursors, disease activity, development of erosions and responses to therapy.

Title of Research Projects:

Title of research project 1: Regulation macrophages and microglia by TREM-2
Brief description of research project 1: We cloned mouse TREM-2, a receptor on activated macrophages and microglia, and we demonstrated that it recognizes a broad range of bacteria as well as endogenous ligands on brain cells. Our recent studies have found that TREM-2 can augments some macrophage responses to bacteria but downregulates others. Our current studies seek to define the role of TREM-2 in pathogen recognition by bacteria and in the recognition of self-ligands by microglia.

Title of research project 2: TIM-2 is a receptor for H-ferritin
Brief description of research project 2: We recently found that the mouse TIM 2 surface protein is a receptor for H ferritin (the first receptor identified for H ferritin. We found that TIM-2 is expressed in the liver and kidneys and on activated B cells. Our current studies seek to define the role of this receptor in regulating B cell function. We also seek to clone the human receptor(s) for H ferritin.

Title of Research Project: Repetitive loading in degenerative joint disease.
Brief Description: Collect and analyze data from an in vivo animal model for repetitive finger joint loading, and prepare & publish manuscript of results and conclusions. Note: Office and laboratory are located in the East Bay at the Richmond Field Station. Contact information: kbking@itsa.ucsf.edu 5110-231-9448