faculty

Neurotransmitter transporters present on the plasma membrane contribute to the clearance and recycling of neurotransmitters and can have a profound impact on the extent of receptor activation during neuronal signaling. Our major research efforts have focused on the structure, regulation and cellular physiology of two families of sodium-dependent neurotransmitter transporters: the biogenic amine and the excitatory amino acid carriers.

Epithelial cells cover external surfaces of organs and line the inner surfaces of organ systems such as the urogenital tract, gut, and blood vessels. We are interested in understanding how these cells use endocytosis to sample their environment and internalize cell-surface receptors, and how mechanical forces affect membrane traffic in the epithelial cells lining the inner surface of the bladder.

As the first step in gene expression, synthesis of mRNA by RNA polymerase II is subject to a multiplicity of controls. Proper regulation of transcription is essential for the normal growth and development of all organisms. The fundamental importance of transcriptional regulation to human health is evident from the large number of diseases, including cancer and AIDS, that result when cellular transcription factors are altered by mutation or when viral proteins commandeer the cellular transcription machinery. Therefore, a detailed understanding of the proteins and regulatory mechanisms that influence the activity of RNA polymerase II, the central research focus of our lab, is a vital step toward understanding the causes of a number of important human diseases.

The goals of my research is to understand the molecular dynamics of biological molecules, using computational models based on the fundamental principles of physical sciences, advanced computational algorithms, and database search and analysis tools.

All secreted proteins, and most that ultimately reside within the cell, must traverse the secretory pathway, a network of intracellular organelles housing the "machines" that help secreted proteins mature.

We are interested in understanding how cells acquire different fates during the course of animal development, primarily in understanding what external signals are required for cells to proceed down specific developmental pathways and how these signals regulate gene expression within these cells to execute these pathways. We use the fruit fly, Drosophila, as a model organism; more specifically, looking at patterning of the legs and wings. Because most of the genetic and molecular mechanisms operating to direct development are shared by all animals from worms to insects to more complex animals such as our selves, it is hoped these studies will have more widespread impact, including understanding of human diseases such as cancer and birth defects.

My laboratory researches three aspects of mammalian development: (1) genomic imprinting, a form of epigenetic inheritance; (2) the etiology of ovarian teratomas, and; (3) the biology of mouse embryonic stem cells.

We have been engaged in the molecular and epidemiologic characterization of the Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8), a new human herpesvirus identified in our laboratory. KSHV is strongly associated with Kaposi's sarcoma, the most common AIDS related malignancy. KSHV serologic studies have demonstrated that seroconversion to KSHV antibody positivity occurs prior to KS onset, primarily in adulthood, and that infection rates follow epidemiologic and geographic risk groups for KS. KSHV is also the cause of two lymphoproliferative disorders: a form of nonHodgkin's B-cell primary effusion (body cavity-based) lymphoma and a subtype of Castleman's disease (MCD).

The development of an organism from a single specialized cell, the fertilized egg, to a multicellular organism with specialized tissues that function together consists of carefully orchestrated events, many occurring simultaneously. The early events must be completed precisely for the later events to occur correctly. Changes in the developmental program can have dramatic effects on the organism as a whole. Sometimes the effects are so drastic that lethality occurs early in the development of the organism. Other times, the effects might not kill the developing organism, but may instead lead to devastating birth defects. A central focus of my lab is to understand how the paraxial mesoderm, which undergoes segmentation to form the somites and ultimately the vertebrae, ribs, skeletal muscle and dermis, is formed and patterned during embryonic development.

The general goal of our neurodegeneration project is to understand the cellular changes that occur in nerve cells that are exposed to oxidative stress. In response to acute injury such as stroke or in many chronic neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, nerve cells are subjected to oxidative stress. Through a better understanding of the biochemical changes that occur in response to oxidative stress in nerve cells we hope to identify molecules and pathways that could be targets for therapeutic intervention. Our laboratory is also interested in prostate cancer and focuses on important components of the tumor microenvironment, (i.e. stromal cells) that provide the support necessary for cancer cells to survive and expand. We have identified one molecular target in stromal cells that plays an important role in the communication between stromal cells and developing cancer cells in the prostate. Our hope is to devise new strategies for limiting the contribution of the tumor microenvironment through detailed molecular studies of prostate stromal factors essential for cancer development and progression. Finally, we examine the cell biology and clinical relevance of glucocorticoid signaling in various models. The basic mechanism of trafficking of the receptor for glucocorticoids (i.e. the glucocorticoid receptor) is examined using state-of-the-art fluorescence microscopy technology coupled with biochemical approaches. In addition, the impact of glucocorticoids on various tissues during development focusing on the brain in model studies in fetal mice and on white blood cells in critically ill children. Since glucocorticoids are a standard course of therapy for premature infants and critically ill children, we hope to provide insights into appropriate treatment strategies when glucocorticoids therapy is indicated.

Alternative pre-mRNA splicing is widely used to regulate protein expression by tuning the levels of tissue-specific mRNA isoforms. Yet the molecular codes for these mechanisms and the combinatorial roles of RNA binding proteins are largely unknown. Dr. Grabowski's laboratory utilizes brain region specific exons as model systems to identify the arrangement of sequence motifs that specify exon-skipping patterns. Genome wide analysis is then used to extend these studies to test the generality of the identified motif patterns and to refine the analysis.

The objective of the overall research in the laboratory is centered on achieving as complete a description as possible for the structures of peptides, proteins, nucleic acids and their complexes in solution, principally by NMR spectroscopy. Particular emphasis is being placed on developing approaches that allow the investigation of larger and complex systems as well as increase the precision with which these solution structures can be obtained. Studies aimed at correlating structure and function and experiments investigating protein folding are also carried out. Structures under investigation comprise transcription factor complexes, HIV-1 proteins, and proteins involved in signalling.

Mycobacterium tuberculosis kills more people than any other single infectious agent. Since antibiotics are available and the BCG vaccine is in widespread use, why do nearly three million people die each year from TB? The answer, in part, is that we really don't understand this curious bacterium or what parts of its genetic instructions make this such a deadly pathogen.

1) Proteins have remarkable talents for assembling accurately into complex biologically functional structures. 2) Bacteriophages are the most abundant organisms on our home planet, numbering in excess of 10exp31 individuals.

Research in my lab tries to understand the cellular basis for how the vertebrate embryo is correctly shaped and patterned during the course of vertebrate development. We are particularly interested in the cellular architecture of neural epithelial cells and how changes in cell shape and movement contribute to formation of the neural tube, the embryonic structure that gives rise to the adult brain and spinal cord. This an important biological question as aberrant neural tube patterning and morphogenesis results in human birth defects such as spina bifida, exencephaly, and holoprosencephaly.

When Medicine is confronted with cellular damage as a result of human disease, there is currently little that can be done to repair damaged tissues and organs. Procedures that can replace kidney function in humans are dialysis and organ transplantation, with dialysis therapy difficult for the patient long term and transplantation limited by the availability of donor organs. In order to regenerate compromised tissue in diseased systems, tissue engineering has been sought as a solution. Identifying and subsequently isolating tissue and organ specific stem cells will aid in advancing tissue engineering techniques, by either providing insights into the molecular mechanisms involved in embryonic stem cell plasticity or by coaxing stem cells to develop into specified tissue types. Presently, there is limited knowledge regarding the role of stem cells in kidney development. However, recent evidence reveals that kidney tubule cells regenerate after injury suggesting a self-renewal potential exists in adult kidneys.

The long-range objective of our research is to gain insight into the molecular determinants for high DNA site selectivity by proteins. Our strategy uses the conceptual and analytical methods of biophysics and structural biochemistry in combination with the manipulation methods of synthetic chemistry and of molecular genetics, and emphasizes the concerted use of a number of tools. We are currently combining high resolution "structural-perturbation" methods with isothermal titration calorimetry to obtain complete thermodynamic profiles (i.e., changes in enthalpy, entropy, heat capacity) for protein-DNA complex formation, together with computational modeling to complement our interpretations. Some of our current work focuses on the thermodynamic consequences of molecular strain, experimental measurement of solvation changes upon binding, and the molecular properties of mutant proteins with reduced specificity. Our ongoing principal goal is to adduce general principles applicable to most or all sequence-specific DNA-binding proteins. Our work points to the importance of time-dependent (dynamic) characteristics of protein-DNA complexes, including binding-dependent conformational transitions in the proteins and DNA and configurational (conformational and vibrational) fluctuations in the protein-DNA complexes, so we are pursuing spectroscopic methods (Nuclear Magnetic Resonance and Electron Spin Resonance) that inform us about these dynamics. A profound understanding of these basic physicochemical principles and their applications is what we will ultimately need to develop methods for knowledge-based drug design and design of macromolecules to fulfill specific biological functions.

Membrane proteins are encoded by more than 25% of the genes in typical genomes, many of which have pharmaceutical and/or medical importance because they include structural proteins, channels and receptors that are accessible through the exterior of cells and thus present formidable drug targets.

Our research is directed toward elucidating the evolution of bacterial genomes, including their size, composition, variability and organization. In other words, why do genomes have the genes that they do? An understanding of the evolutionary process that leads to differences in genomes will shed light on how species themselves differentiate. We take two approaches to understanding how genomes evolve : a computation/theoretical approach, and an experimental approach.

We have been engaged in the molecular and epidemiologic characterization of the Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8), a new human herpesvirus identified in our laboratory. KSHV is strongly associated with Kaposi's sarcoma, the most common AIDS related malignancy. KSHV serologic studies have demonstrated that seroconversion to KSHV antibody positivity occurs prior to KS onset, primarily in adulthood, and that infection rates follow epidemiologic and geographic risk groups for KS. KSHV is also the cause of two lymphoproliferative disorders: a form of non-Hodgkin's B-cell primary effusion (body cavity-based) lymphoma and a subtype of Castleman's disease (MCD).

Molecular Biology of SV40. We utilize SV40 as a model system for understanding molecular events involved in viral productive infection and tumorigenicity. Our studies have focused on the virus-encoded master regulatory protein, large T antigen. Large T antigen controls several aspects of viral infection including DNA replication, transcription and virion assembly. In addition, T antigen is necessary, and in most cases, sufficient for SV40-mediated tumorigenesis.

My laboratory uses zebrafish as a model system to understand the genetic and environmental pathways that shape the developing vertebrate vasculature. Specifically, we are interested in elucidating mechanisms by which congenital arteriovenous malformations (AVMs) form. An AVM is a direct connection between an artery and a vein. In the absence of intervening capillaries, the vein receives blood at very high pressure, which leads to vessel enlargement and weakening. In the United States, the prevalence of brain AVMs is estimated at 0.1 to 0.2% of the population; in approximately one-quarter of these individuals, these lesions will lead to hemorrhagic stroke. AVMs can also form in other organs such as the lung, liver, and gastrointestinal tract, with complications ranging from minor bleeding to ischemic stroke. While it is generally accepted that AVMs form during embryogenesis, the mechanisms by which they form are not understood. The embryonic and genetic accessibility of the zebrafish embryo allows us to literally watch AVMs form in real time, and to elucidate genetic and environmental pathways associated with their formation. By dynamically evaluating AVM formation in live mutant zebrafish embryos, we have uncovered two mechanisms by which embryonic AVMs can form: retention of normally transient venous sprouts that give rise to nascent arteries, and flow-induced arterial enlargement and subsequent fusion with an adjacent vein.

Chromosomes are normally segregated during mitosis with very high fidelity. Loss of individual chromosomes typically occurs at less than one in a hundred thousand cell divisions. This high accuracy is ensured by the spindle apparatus, a transitory microtubule-based structure, which appears and disappears in eukaryotic cells during cell division. Defects in spindle function can lead to a much higher frequency of chromosome loss or nondisjunction. The genomic instability resulting from these spindle defects, may increase the likelihood that the defective cell will become a cancerous cell or lead to birth defects in the next generation. My laboratory is interested in two aspects of the study of cell division in eukaryotes: (1) the study of chromosome segregational defects in oral cancer cells; and (2) the identification of novel meiosis genes.

-Src-family tyrosine kinase-HIV Nef complexes as novel targets for antiretrovral drug discovery
-A novel role for c-Fes/Fps tyrosine kinases as tumor suppressors in colon cancer
-Src-family kinases and embryonic stem cell fate
-Cytoplasmic tyrosine kinases and chronic myelogenous leukemia

Many human diseases find their root cause in dysfunctional cell signaling events. For example, many cancers are linked to inappropriate elevation of Ras-MAPK signaling and downregulation of p53 mediated-homeostasis, resulting in uncontrolled cell growth and the inability to trigger cell death. Thus, understanding how cells balance signal transmission and attenuation has broad implications not only for disease but also normal development.

We are interested in understanding the molecular basis of intracellular protein trafficking events, and focus primarily on clathrin-coated vesicles. These small polyhedral structures were the first transport carriers to be discovered, and are still the prototypical example of coat-dependent cargo sorting into transport vesicles. At the cell surface, endocytic clathrin coats provide a major portal to the interior of most eukaryotic cells and play a pivotal role not only in the internalization of extracellular nutrients, but also in modulating the density and activity of important membrane proteins, like signaling receptors. A good example of this is the neurological synapse.

The focus of our laboratory is the regulation of apical membrane traffic in polarized epithelial cells.