The following projects will be offered at the University of Virginia in 2011/2012:
David S. Cafiso, PhD,
Professor of Chemistry
Our lab is involved in the use of magnetic resonance techniques (NMR and site-directed spin
labeling) to determine the molecular mechanisms of membrane transport and the molecular
events underlying membrane fusion.
The prospective student will be studying the structure and structural transitions of a
class of outer membrane bacterial transport proteins that function to bring rare nutrients such
as iron and vitamin B12 into the cell. The student will learn to use both EPR and NMR
methodologies in this work, and will get experience in site-directed mutagenesis, protein purification and
membrane protein reconstitution.
Dr. Cafiso supervised to date two students from Poland, including Damian Dawidowski (UJ), currently
a doctoral student at UVA, Chemistry Department, and Anna Cieslinska (UJ), who obtain Masters
degrees from UJ and UVA, and is a graduate (doctoral) student at Northwesterm University.
Edward Egelman, PhD,
Professor of Biochemistry & Molecular Genetics,
Our laboratory has focused on two main areas: 1) F-actin and complexes of actin with other
proteins; 2) Nucleoprotein filaments formed by RecA/RadA/Rad51/Dmc1 on DNA. The main
tools that we use are electron cryo-microscopy and computed image analysis. Because these filaments are
poorly ordered and have great structural variability, we developed some general methods to be used for
generating there-dimensional reconstructions of helical polymers from electron micrographs. These methods
are now being applied to many other helical polymers in my lab: pili from pathogenic bacteria, filamentous
bacteriophage, bacterial Type Three Secretion System components, and bacterial plasmid segregation
system polymers.
We are looking for students who have some computational experience. Within a one year period, a
student with a reasonable background might be expected to make some significant contribution to one of our
existing projects, leading to a publishable paper. Prior experience with Linux and programming skills would be
very helpful.
Dr. Egelman supervised one doctoral student, Jakub Bielnicki, from Lodz University.
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Marty W. Mayo , PhD,
Professor of Biochemistry and Molecular Genetics
A major focus in biomedical research is to understand how cancer cells invade healthy cells
and metastasize to distant sites. Lung cancer in particular is more prone to aggressive forms of the disease
that are difficult to treat effectively and have a poor clinical prognosis.
Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. The 5-year mean survival for
patients suffering from stage III lung cancer is less than 30%. Poor clinical prognosis for NSCLC is directly
associated with late-stage diagnosis and high propensity for metastasis to liver and bone. For the last several
years our laboratory has been working on a transcription factor called nuclear factor-kappa B (NF-B). NF-B
is a protein complex that is found in all human cells. In non-cancerous cells NF-B is tightly regulated,
however, cancer cells have found ways to elevate the activity of NF-B. Although it is known that cancer cells
enhance NF-B activity, the reasons for this are not fully understood. Our laboratory has discovered that NFB is one of the master-switch transcription factors required to induce phenotypic changes in cells referred to
as epithelial to mesenchymal transition (EMT). EMT is a critical step in cancer metastasis. For the first time,
our laboratory can demonstrate that following the induction of EMT, NF-B is required to orchestrate changes
associated with the induction and maintenance of cancer stem cells. Cancer stem cells are believed to act as
a “seed” which is able to reestablish malignant disease. The overall goal of our laboratory is to understand
transcription and transcription-independent mechanisms by which NF-B induces and maintains cancer stem
cells. Our laboratory uses two dimensional (2D) and 3D tissue culture systems, primary normal and tumor
tissues, and small animal models as methods for studying EMT. Commonly employed techniques in our
laboratory include, Western blot analysis, quantative real-time PCR, Chromatin Immunoprecipitations (ChIP)
and ChIP-sequence, ELISAs, invasion and migration assays, molecular subcloning and site-directed
mutagenesis.
I would prefer to have student who is highly motivated and excited about learning or perfecting molecular
biology techniques. A student who has firmly established wet-bench skills is preferred.
Dr. Mayo supervised Anita Popko from the Technical University of Lodz. Anita co-authored a major
paper, went on to join the lab as a Grad Student and later transferred to Karolinska Institute in
Sweden. Dr Mayo is currently supervising Szymon Szymura, a PhD student from the Jagiellonian
University, and Julia Krupa, a visiting student from the Technical University in Lodz.
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Mark Yeager, PhD, MD,
Professor of Molecular Physiology and Biological Physics
At the basic science level, we are intrigued by
questions at the interface between cell biology and
structural biology: How do membrane proteins fold?
How do membrane channels open and close? How
are signals transmitted across a cellular membrane
when an extracellular ligand binds to a membrane
receptor? How do viruses attach to and enter host
cells, replicate, and assemble infectious particles? To explore such
problems, we use high-resolution electron cryomicroscopy and computer
image processing. With this approach, we can examine the molecular
architecture of supramolecular assemblies such as membrane proteins
and viruses.
In electron cryomicroscopy, biological specimens are quick frozen
in a physiological state to preserve their native structure and functional
properties. A special advantage of this method is that we can capture
dynamic states of functioning macromolecular assemblies, such as open
and closed states of membrane channels and viruses actively
transcribing RNA. Three-dimensional density maps are obtained by
digital image processing of the high-resolution electron micrographs. The
rich detail in the density maps exemplifies the power of this approach to
reveal the structural organization of complex biological systems that can
be related to the functional properties of such assemblies.
Ongoing research projects include the structure analysis of (1) membrane proteins involved in cell-to-cell
communication (gap junctions), water transport (aquaporins), ion transport (potassium channels), and
transmembrane signaling; (2) viruses responsible for significant human diseases (HIV-1, hepatitis B [HBV],
rotavirus, astrovirus); and (3) viruses used as model systems to understand mechanisms of pathogenesis
(arenaviruses, reoviruses, nodaviruses, tetraviruses and sobemoviruses).
A rotation in our laboratory will allow students to gain experience in the design of constructs for
expression of membrane proteins in E. coli, Pichia pastoris and SF9 insect cells via baculovirus. Metal
affinity, ion-exchange and gel-filtration chromatography are used for purification. Detergents are screened to
optimize the homogeneity and stability of the expressed protein. Students will receive personal tutorials in
electron microscopy for routine screening of samples by negative-stain EM. Instruction will also be provide for
preparation of frozen-hydrated specimens and the performance of electron cryomicroscopy. Depending on
the duration of the rotation, students will pursue 2D and 3D crystallization trials for higher resolution structure
analysis by electron and X-ray crystallography. Data are analyzed using software packages such as EMAN,
2DX and CCP4.
Ganser-Pornillos, B., Yeager, M. , and Sundquist, W.I. The Structural Biology of HIV Assembly. Curr. Op.
Struct. Biol. 18: 203-217 (2008).
Dryden, K.A., Coombs, K., and Yeager, M. Chapter 1. The Structure of Orthoreoviruses. In: Structure and
Molecular Biology of Segmented Double-Stranded RNA Viruses, Patton, J., Ed. Horizon Scientific Press,
Wymondham, Norfolk United Kingdom, pp. 3-25 (2007).
Yeager, M. and Harris. A. L. Gap Junction Channel Structure in the Early Twenty-first Century: Facts and
Fantasies. Curr. Op. Cell. Biol. 19: 521-528 (2007).
Adair, B.D. and Yeager, M. Electron Microscopy of Integrins. Methods in Enzymol. 426: 337-373 (2007).
Dr. Yeager currently serves as a mentor for Maciej Jagielnicki, Technical University of
Wroclaw.
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Lukas Tamm, PhD,
Professor of Molecular Physiology and Biological Physics
Triggering Neurotransmitter Release at the Neuronal
Synapse.
SNARE proteins are responsible for fast fusion in neuronal exocytosis.
SNAREs are are set up in a trigger-ready configuration before the arrival
of a neuronal stimulus. Upon electrical stimulation, a wave of calcium
arrives at the presynaptic site and the calcium sensor synaptotagmin
and the signaling phospholipid phosphatidyl-inositol-4,5-biphosphate (PIP2) engage with the SNARE fusion
machinery to promote neurotransmitter release within a fraction of a millisecond. In this project, we will
reconstitute neuronal SNARE proteins in supported lipid bilayers together with PIP2 and synaptotagmin and
study their effect on vesicle docking and membrane fusion on the millisecond timescale. The student will
collaborate in a team and will gain experience in membrane protein expression and purification, membrane
sample preparation, and state-of-the-art laser fluorescence (TIRF) microscopy at very high time-resolution
(yes, you will create movies – but they will unlikely play in Hollywood!).
For a primer see:
Domanska, M.K. Kiessling, V., Stein, A., Fasshauer, D., and Tamm, L.K. (2009) Single vesicle millisecond
fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion.
J. Biol. Chem. 284:32158-32166.
Domanska, M.K., Kiessling, V., and Tamm, L.K. (2010) Docking and fast fusion of synaptobrevin vesicles
depends on lipid compositions of the vesicle and the acceptor SNARE complex-containing target
membrane. Biophys. J. 99:2936-2946.
Dr Tamm supervised several visiting students, starting with Marta Domanska, first author of
the publications shown above, who recently completed a doctorate in his laboratory. All other
students have gone on to doctoral programs in several countries.
Salem Faham, PhD,
Assistant Professor of Molecular Physiology and Biological Physics
Membrane proteins currently represent only a small fraction of the structures in the protein
data bank (pdb). That is partly due to the difficulty in obtaining high quality crystals.
Membrane proteins are typically solublized and crystallized in detergent solutions. However,
detergents are often blamed for the poor quality of membrane protein crystals. We have
shown that membrane proteins can be crystallized from a specific lipid/detergent mix that forms bicelles.
Bicelles offer an alternative medium for the crystallization of membrane proteins that holds a lot of promise.
We are interested in the further development of the bicelle method for membrane protein crystallography. This
project will involve cell growth, protein expression, protein purification, and protein crystallization.
This is the first year for Dr. Faham in the program
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Umesh S. Deshmukh, PhD,
Assistant Professor of Medicine
My laboratory is interested in autoimmune disorders, specifically Systemic Lupus Erythematosus (SLE) and
Sjögren’s Syndrome (SS). The main emphasis is to understand how pathogenesis of these diseases is
influenced by the activation of innate and adaptive immunity by microbial agents.
Project 1. Role of molecular mimicry in activation of T cell responses in SLE. Systemic lupus
erythematosus is a complex autoimmune disorder affecting multiple organ systems such as kidneys, skin and
lungs. The presence of autoantibodies reactive against multiple cellular proteins is a hallmark of SLE. We
are interested in understanding how these autoantibody responses are initiated. The initiation of T cell
responses against self-antigens is a critical step in the genesis of autoantibodies in lupus. My laboratory is
investigating the role of molecular mimicry between self-antigens and foreign proteins as one of the
mechanisms for activation of autoreactive T cells. We are using Ro60 as the model autoantigen. Ro60 is
among the first autoantigens to be targeted in lupus, and immune responses against Ro60 are often present
in SLE patients. The current studies focus on the mapping and detailed characterization of HLA-DR3
restricted T cell epitopes on Ro60. Identification of peptide mimotopes will be performed using different
algorithms. The ability of selected peptide mimotopes to induce autoantibody responses against Ro60 will be
investigated in mice transgenic for HLA-DR3. The data generated from this project will demonstrate that
repetitive exposure to different microorganisms shapes the autoreactive T cell repertoire. In a lupussusceptible individual, this leads to initiation of autoimmune responses. This project will aid in identifying
novel environmental risk factors for the development of SLE.
Project 2. Role of innate immunity activation in Sjögren’s Syndrome. SS is a chronic autoimmune
disorder mainly affecting the salivary and lacrimal glands. A progressive lymphocytic infiltration within these
glands causes glandular destruction. This is responsible for the dry mouth and dry eye symptoms of the
disease. My laboratory is interested in understanding how activation of innate immunity influences the
development of SS. We are using the Toll-like receptor 3 (TLR3) agonist poly(I:C) to activate innate immunity
in NZB/NZW derived strains of mice. Our results demonstrate that poly(I:C) treatment causes acceleration of
SS-like disorder in the NZB/NZW F1 mice. The salivary glands of poly(I:C) treated of mice had severe
lymphocytic infiltration. The mechanisms for this observation will be investigated in details. In our
experimental mouse model system, poly(I:C) causes rapid upregulation of type I IFNs within the salivary
glands, which causes rapid glandular dysfunction. Our studies in interferon receptor knock out mice show
that type I IFNs are directly involved in this process. Paradoxically, systemic treatment of SS patients with
type I IFN, has demonstrated beneficial effects on salivary gland function. Thus, another objective in this
project will be to understand the contrasting mechanisms of IFN action on salivary gland function.
Students with keen interest in immunology, and an enthusiasm to investigate clinical diseases in experimental
mouse models will be preferred. Some experience in tissue culture, animal work and basic immunoassays is
desired. Dominika Nackiewicz, who was a student in this program (2008-2009) is currently working as
research assistant in our laboratory.
Dr Deshmukh currently supervises Agnieszka Szymula, from the Jagiellonian University in Krakow.
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David L. Brautigan, PhD,
Professor of Microbiology and Director, Center for Cell Signaling.
Protein Phosphatase-6 in Cell Cycle, Inflammation and DNA damage
Our goal is to discover how intracellular signaling pathways regulate cell proliferation,
survival/apoptosis and cytokine production in response to stress signals and
infections. This research is relevant to understanding normal physiology as well as the
pathology encountered in human diseases such as cancer, diabetes and
autoimmunity. Our focus is on protein phosphorylation, and especially the enzymes
called protein Ser/Thr phosphatases (the PPP and MPP Families). Genomics has shown that the PPP
enzymes are extraordinarily conserved in all eukaryotes (e.g. mammals, Xenopus, Drosophila, C. elegans, S.
pombe, S. cerevisiae). Humans and yeast have about the same total number of PPP genes, in separate
functional classes (i.e. PP1, PP2A, PP4, PP6). Individual human PPP proteins can substitute in place of their
yeast homologues, but not PPP of other functional classes, showing that individual PPP are functionally
equivalent across evolution, but each class has a distinctive biological role. The conservation across species
allows us to use the results from genetic experiments in various model organisms.
We primarily use cultured cells and combine functional genomics,
biochemistry and cell biology. It is typical for students and fellows to
learn the full array of molecular and cellular techniques while studying
these signaling networks. (e.g. PCR, cloning, mutagenesis, protein
expression and purification, tissue culture, transfections, enzyme
assays, immunoprecipitations, immunoblotting, microscopy, etc).
Protein Phosphatase 6 (PP6) is a distinct member of the protein
Ser/Thr phosphatase PPP family that is the mammalian homologue of
yeast Sit4. The functions of Sit4/PP6 are conserved, because human
PP6 rescues yeast sit4- mutations, whereas other PPP do not. In
yeast Sit4 is genetically linked to cell cycle control. We have found
that PP6 has effects on G1 to S phase progression in human cancer
cells, influencing the levels of cyclin D1 and phosphorylation of Rb (Cell Cycle, 2007). A graduate student is
testing how PP6 regulates levels of cyclin D1 in breast cancer cell lines. Other evidence points to the role of
PP6 in cytokine signaling and pathways leading to activation of NF-kB. PP6 SAPS subunits mediate
association with IkBe and alter the degradation of this regulator in response to TNFa stimulation (J. Biol.
Chem. 2006). Proteomic results using mass spectrometry of immunoprecipitated SAPS complexes revealed a
family of Ankyrin Repeat Subunits (we named ARS) that are functionally equivalent to the SAPS themselves
in siRNA knockdown assays (Biochemistry, 2008). Thus, we propose that PP6 is a trimeric enzyme,
composed of ARS, SAPS and a catalytic subunit (see Figure). We have analyzed the structure of SAPS
subunits using modeling, to predict a helical repeat arrangement, and find charged residues are needed for
PP6 association (BMC Biochem. 2009). Iga Kucharska from Wroclaw Technical University is working on coexpression, crystallization, and solving the structure of the PP6c:SAPS1(PP6R1) complex.
Another protein identified by mass spectrometry was DNA-PK, a kinase activated following damage to DNA
and an initiator of DNA repair by the enjoining (NHEJ) pathway. We found that in glioblastomas (brain tumors)
the PP6 and the SAPS1 subunit we call PP6R1 were recruited into the nucleus and into complexes with DNAPK. PP6 and PP6R1 were both required for the activation of DNA-PK following ionizing radiation (gamma
rays), and for the repair of double strand breaks in DNA, and for the survival of the cancer cells (PLoS One;
2009). Thus, inhibiting this action of PP6 makes cells more sensitive to radiation, and may provide new
therapies to enhance radiation therapy for otherwise incurable brain tumors. We have a project underway that
is looking to express dominant negative forms of SAPS1 to interfere with repair of DNA damage.
Dr Brautigan currently supervises Iga Kucharska, student from the Technical University of Wroclaw.
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Ulrike M Lorenz, PhD,
Associate Professor of Microbiology
Our area of research focuses on the role of phosphatases, and in particular the role of
the tyrosine phosphatase SHP-1, in the regulation T cell development, differentiation
and function (reviewed in Lorenz, U; SHP-1 and SHP-2 in T cells: two phosphatases
functioning at many levels. Immunol Rev. 2009, 228:342-59).
We use a variety of techniques to study the immune system including transgenic and
conventional and conditional knock-out mouse models, protein chemistry (biochemical determination of
localization, immunoprecipitation, immunoblot), molecular biology, siRNA, cell culture of eukaryotic cells
(primary and cell lines). All of the potential projects outlines below will make use of several, if not all of these
techniques.
There are currently several potential projects that are suitable for a visiting trainee with the VRGT
program.
(I) The process of suppression by regulatory T cells.
Peripheral T cells have historically been categorized as CD4 and CD8 T cells. However over the last 5-10
years, it has become clear that the CD4 T subpopulation are comprised of numerous subsets besides the
well-studies TH1 and TH2 helper T cells. One of these subpopulations, known as regulatory T cells (Treg
cells), is now well recognized as a critical mediator of tolerance and the prevention of autoimmunity.
However, at the same time Treg cells are also inhibitory towards the immune response against tumors,
suppressing both the natural as well as the vaccine-induced response. The goal of our research is to gain a
better mechanistic and molecular understanding of their development and function.
We have recently shown that mice deficient in the tyrosine phosphatase SHP-1 have augmented numbers
of Treg cells (Carter, JD et al. J Immunol. 2005, 174: 6627-38) and that SHP-1-deficient Treg cells display an
increased suppressive activity compared to wild type Treg cells (Iype, T et al. J Immunol. 2010, 185: 61156127). These data suggest a regulatory role of SHP-1 in the development and function of Treg cells. We are
now in the process of identifying intracellular signaling pathways that control the Treg cells differentiation and
activity using a number of different mutant mouse models. Based on preliminary data obtained by our lab, we
hypothesize that SHP-1 regulates the activity of Treg cells via the expression of adhesion molecules. A
visiting trainee could take these studies further.
(II) SHP-1 and its role in conventional T cells
While the critical regulatory role of SHP-1 in T cell signaling has been recognized for several years, the
underlying mechanism is still only partially understood. In preliminary studies, we have identified that T cells
deficient in SHP-1 activity not only hyper-proliferate, but also possess an increased resistance to regulatory T
cell-mediated suppression compared to T cells expressing wild type SHP-1. These phenotypes are
reminiscent of cells lacking the ubiquitin ligase Cbl. Interestingly, we found that SHP-1-deficient T cells, but
not thymocytes, lack Cbl expression despite normal Cbl mRNA levels. Based on these observations, we
hypothesize that SHP-1 regulates Cbl protein levels and thereby influences signaling pathways downstream
of the TCR. A student project could aim to identify the mechanism by which SHP-1 regulates Cbl protein
expression and to determine what signaling pathways are affected by SHP-1 through its regulation of Cbl.
(III) SHP-1 and TH17 T cells
Th17 T cell have recently been identified as a subpopulation of CD4+ T cells that is critical in the host
defense against bacteria, but also involved in the pathogenesis of autoimmune diseases. Our research
focuses on the role of SHP-1 in Th17 differentiation. Based on ongoing in vitro and in vivo studies, we are
hypothesizing that SHP-1 is a negative regulator of Th17 differentiation. Our focus is to identify the signaling
pathways that are regulated by SHP-1. A trainee could participate in these studies by comparing Th17 cell
differentiation and signaling in wild type and SHP-1-deficient mice.
This is the first year for Dr. Lorenz in the program
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Mark D.Okusa, MD,
Chief, Division of Nephrology
Director, Center for Immunity, Inflammation and Regenerative Medicine
Immune Mediated Kidney Injury and Repair
Projection Description: My laboratory is interested in innate and adaptive immunity in
acute and chronic kidney injury. Dendritic cells play an early role in activation of lymphocytes
through antigen presentation of peptides to T cells or glycolipids to natural killer T cells.
Through an understanding of the mechanisms that participate in the early activation and modulation of tissue
injury we have developed pharmacological and cell based approaches to block these pathways. We use a
variety of molecular, cell biological and immunological methods and in vivo models in our studies.
(1) Kidney ischemia-reperfusion injury: In vivo studies are aimed at determining the contribution of
immune cells to ischemia-reperfusion injury and therapeutic strategies to reduce injury following acute kidney
injury with the ultimate goal of bringing novel compounds to clinical trials. Current studies target adenosine 2A
receptors and sphingosine 1 phosphate receptors as potential therapeutic approaches to block inflammation
and tissue injury. These studies have led to a better understanding of the mechanisms of T cell activation by
ischemia-reperfusion and tolerance induction by adenosine 2A compounds.
(2) Diabetic nephropathy: Our approach is to understand the immune mechanisms of injury in diabetic
nephropathy and use novel compounds to reduce functional and morphological consequences of diabetic
nephropathy.
(3) Targeted delivery of novel compounds and small molecules. Using nanotechnology and contrast
enhanced ultrasound we are involved in target specific delivery of novel compounds and specific genes.
Liposomes are nano sized artificial vesicles produced from phospholipids and cholesterol and form a bilayer
sphere. An important property of these lipsomes is that have the ability to trap water soluble and insoluble
substances for their delivery to desired diseased targets.
Methods Description: We use a number of different in vivo models that include genetically deficient mice,
chimeric mice, tissue specific gene knockout mice and transgenic mice. Cell culture, molecular biology,
pharmacology, immunological methods that use Elispot, flow cytometry and confocal microcroscopy are
routinely employed. A new area of investigation is the use of contrast-enhanced ultrasound to study
noninvasively and in real-time renal function and in combination with liposomes the delivery of drugs and
genes to specific targets.
Clinical Problem: Acute kidney injury is a burgeoning problem. Based upon the National Health Statistics
and National Hospital Discharge Survey, between 1979 and 2002 there has been an increase in
hospitalization for acute kidney injury (AKI) from 35,000 to 650,000 cases per year. Overall mortality has
been reported to be 40-60% in critically ill patients. The estimated annual health care expenditures attributed
to hospital-acquired AKI exceed $10 billion Furthermore there is recognition that there is an increase in the
end stage renal disease (ESRD) population due to AKI. In patients suffering from AKI, 13.4% of patients (or
30% of patients with AKI superimposed on chronic kidney disease) will progress to ESRD in 3 yrs. Thus to
overcome barriers to successful treatment of AKI, well designed clinical trials will need to be based upon a
precise understanding of the molecular, cellular and immunological basis of AKI. My laboratory focused on
defining critical pathways of early activation of innate immunity and identifying novel therapies.
Student Objectives:
1. To learn basic immunological and/or advanced imaging methods
2. To apply methods in the study of relevant kidney diseases.
3. To develop skills in scientific principles, writing manuscript, and data presentation
Readling List from the Okusa Laboratory:
1. Jo SK, Rosner MH, Okusa MD. Pharmacologic Treatment of Acute Kidney Injury. Why Drugs Haven’t:
Why Drugs Haven't Worked and What is on the Horizon. Clin J Am Soc Nephrol 2007;2:256-365.
2. Li L, Huang L, Vergis AL, et al. IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil
migration in mouse kidney ischemia-reperfusion injury. J Clin Invest 2010;120:331-42.
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3. Bajwa A, Jo SK, Ye H, et al. Activation of Sphingosine-1-Phosphate 1 Receptor in the Proximal Tubule
Protects Against Ischemia-Reperfusion Injury. J Am Soc Nephrol. 2010 Jun;21(6):955-65
4. Li L, Okusa MD. Macrophages, dendritic cells, and kidney ischemia-reperfusion injury. Semin Nephrol
2010;30:268-77.
5. Bajwa A, Kinsey GR, Okusa MD. Immune Mechanisms and Novel Pharmacological Therapies of Acute
Kidney Injury. Curr Drug Targets 2009.
6. Kinsey GR, Li L, Okusa MD. Inflammation in acute kidney injury. Nephron Exp Nephrol 2008;109:e102-7.
7. Li L, Okusa MD. Blocking the Immune respone in ischemic acute kidney injury: the role of adenosine 2A
agonists. Nature Clinical Practice Nephrology 2006;2:432-44.
8. Kinsey GR, Sharma R, Huang L, et al. Regulatory T Cells Suppress Innate Immunity in Kidney IschemiaReperfusion Injury. J Am Soc Nephrol 2009;20:1744-53.
9. Li L, Huang L, Sung SJ, et al. NKT cell activation mediates neutrophil IFN-gamma production and renal
ischemia-reperfusion injury. J Immunol 2007;178:5899-911.
This is the first year for Dr. Okusa in the program
Loren D Erickson, PhD,
Assistant Professor of Microbiology
Immunology/Cell differentiation Research Project
Research Summary: The immune system is comprised of multiple cell types that provide
the host with protection against infectious diseases. Humoral immunity is provided through
the production of specialized proteins called antibodies that are produced by B cells in
response to infection. Antibodies circulate throughout the bloodstream, specifically recognize and bind to the
invading pathogen, leading to the pathogen’s demise. My laboratory is interested in the cellular and molecular
signals that control B cells to produce antibodies. A hallmark of this antibody-based protection is the capacity
of specialized B cells called plasma cells (PC) to live for years continually producing antibodies. These longlived PCs are the underlying basis for vaccines to establish long-term immunity. However, the prolonged
survival of PCs is a significant problem in diseases where PCs function abnormally, such as in the antibodymediated autoimmune disorder systemic lupus erythematosus (SLE). The mechanisms controlling the
longevity of PCs are not well understood. These are fundamental issues relevant not only for development of
antibody protection, but may also lead to new insights into vaccine design as well as the processes controlling
pathogenic PCs in SLE.
Research Project: The decision of a B cell to commit to a PC fate is terminal – in other words, once a PC is
made it will survive for years performing its function of secreting protective antibodies. Thus, multiple
checkpoints are embedded in the immune system to guarantee that none of the B cells that decide to become
a PC will produce antibodies that target and destroy host tissue. However, antibodies can be generated that
recognize self proteins and lead to autoimmunity. SLE is one such autoimmune disease that is mediated by
self-reactive antibodies produced from PCs. How the immune checkpoints fail to eliminate autoreactive PCs is
unclear. The overall aim of this research project is to test the role of the cytokines BAFF, IL-6, and CXCL12 in
the abnormal survival of self-reactive PCs in SLE using mouse models of autoimmunity. This project will use a
combination of recombinant antagonists and knockout mice to manipulate expression of BAFF, IL-6, and
CXCL12, or their receptors. Findings from these studies will have significant impact on both the etiology and
treatment of antibody-mediated autoimmune diseases.
Expected Skills: The candidate should have a working knowledge of basic cell- and molecular- based
assays such as ELISAs, cell culture, RT-PCR, and Western blotting. A working knowledge of flow cytometry is
highly preferred since this project involves multi-color flow cytometric analysis and cell sorting. The capacity to
handle mice is a requirement for this project. A basic understanding of immunology is preferred, but not
required.
This is the first year for Dr. Erickson in the program
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Adrian Halme, PhD,
Assistant Professor of Cell Biology
Investigating the role of endocrine signals in tumor development and
tissue regeneration.
In the 1860’s, pathologist Rudolph Virchow proposed that tumors arise at sights of
persistent inflammation and tissue damage. However, it is only more recently that we
have we come to recognize that tumors often behave as “wounds that never heal”
exhibiting many of the same responses that are observed in damaged tissues that are
undergoing repair. An overarching interest of our research group is to understand the relationships between
tissue regeneration and tumor growth. In the fruit fly, Drosophila melanogaster, we can produce both
regenerative growth and tumor neoplasia in the imaginal tissues – the larval precursors to adult tissues.
Therefore, this experimental model can provide important insights into the fundamental relationship between
regenerative and neoplastic growth. In our experiments, we have observed that regenerative and neoplastic
growth share very similar features. In particular, we have been focused on how both these types of growth are
regulated by systemic endocrine signals that regulate normal tissue development, growth, and metabolic
activity.
Recently, we have observed that experimentally induced tumors in the fruit fly, Drosophila melanogaster, only
begin to appear at a specific stage of development. The appearance of these tumors coincides with the
activity of a steroid-hormone, ecdysone. Therefore, a project is now available in the lab to examine the role of
ecdysone and other endocrine signals in regulating tumor development. By manipulating hormone levels in
developing animals, and employing genetic tools that allow us to alter the response of cells to hormone
signals, we will examine specifically how endocrine signals regulate tumor development. Using genetic tools
available in Drosophila along with antibodies and transgenic reporters that allow us to examine signaling
activity in situ, we will identify the molecular pathways that trigger tumor growth and determine how these
pathways respond to hormone signals. Whole-genome expression analysis will also be used to identify
transcriptional pathways regulated by hormone signaling in normal tissues and tumors. Finally, we would like
to examine whether hormone signaling regulates tissue regeneration. To do this, we have several different
genetic models for tissue regeneration that will allow us to examine the impact of hormone signals on
regenerative growth.
This project will involve both molecular analysis and genetic approaches in Drosophila. Therefore, we are
looking for a student with some molecular biology experience and an understanding of basic Mendelian
genetics. Drosophila is a very easy organism to learn to manipulate experimentally and has a short generation
time, allowing one to generate recombinants in a matter of weeks. Therefore, during the course of this project
the student will gain substantial experience in using genetic tools to address problems in developmental
biology, endocrine signaling, tumor biology, and tissue regeneration.
This is the first year for Dr. Halme in the program
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Jung-Bum Shin, PhD
Assistant Professor of Neuroscience
Hearing loss is America’s leading disability, affecting 28 million people of all ages. The cells that mediate our
senses of hearing and balance, so-called sensory hair cells in the inner ear, are easily affected by
environmental factors such as aging, noise, certain antibiotics and anti-cancer drugs. It is known that the
aforementioned insults lead to the generation of radicals and reactive oxygen species in hair cells that are
especially harmful for proteins. The goal of this project is to uncover how hair cells protect their protein
complement against oxidative damage. Understanding the endogenous, protective mechanisms of hair cells
will guide possible strategies to prevent and repair hearing loss.
Specifically, we will use a combination of molecular biology, protein chemistry, confocal microscopy, FACS
(fluorescence activated cell sorting) analysis and mass spectrometry to study how hair cells maintain their
protein homeostasis by regulating the synthesis, modification and degradation of their proteins. Animal
models used in the lab include mouse, rat and chicken.
A typical experiment might consist of, but is not restricted to the following activities: 1) microdissection of mice
and chicken to remove the inner ear tissue, 2) establishment of inner ear organ explant cultures and 3) using
microscopy, FACS and mass spectrometry to test the effects of various compounds on hair cell proteins and
hair cell survival.
I would like to welcome a dedicated student who has experience with standard laboratory practices.
Microdissection is a major part of this project. Although prior dissection experience is greatly appreciated, it is
not required.
This is the first year for Dr. Shin in the program
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Kenneth M. Tung, MD,
Professor of Pathology
The immunoregulation of CNS injury and sperm autoimmunity by regulator
of T cell response.
This is a collaborative project between the laboratories of Ulrike Lorentz, Jonathan Kipnis and ours. Ulrike
Lorenz’s research focuses on SHP1, a phosphatase that negatively regulates the response of T cells and
myeloid cells. Global SHP1 knockout mice have the motheaten phenotype with early fatality. To study the
effect of SHP1 exclusively on effector and regulatory CD4 T cell (Treg) response, she has generated CD4 T
cell specific knockout mice lacking SHP1 and accrued synthetic inhibitory molecules of SHP1, and showed
that SHP1 strongly controls Treg and effector CD4 T cell function in vitro. In the past few months, she has
joined force with Kenneth Tung and Jonathan Kipnis in a 3-way collaboration to study how SHP1 influences
CD4 T cell response in vivo, in disease context including T cell response injury and in autoimmune disease.
Kenneth Tung studies how Treg function to maintain physiological tolerance. Using unilateral vasectomy as a
local danger signal, his laboratory recently showed that Treg prevent autoimmune disease induction by the
innate responses (danger signal) emanating from the injured epididymis. Working with the Lorenz lab, they
have also shown that autoimmune disease progression is associated with resistance of effector T cells to
suppression by Treg, and this is associated with downregulation of cbl, a ubiquitin ligase controlled by SHP1.
Jonathan Kipnis is a neuroimmunologist who studies the interaction between immune cells and CNS function;
he also studies the effect of immune cells on physical and ischemic injury of the CNS (3). He has recently
used the SHP1 knockout mice to document SHP1 regulation of neuronal survival and effector T cell function
in CNS injury.
As an overall hypothesis, we propose that SHP1 signaling, by regulating cbl expression and function, either
suppress an overall immune response by enhancing Treg function, or promoting effector T cell response by
enhancing the resistance of effector T cells to suppression by Treg. We further hypothesize that the net in
vivo outcome of this divergent T cell response to SHP1 signaling is context dependent. In this project, we will
use critical reagents to investigate this hypothesis using unique animal models of CNS injury and testis
autoimmunity.
To facilitate this multidisciplinary research project, we wish to recruit a highly motivated individual with
knowledge and interest in fundamental immunology and biochemistry of cell signaling.
This is the first year for Dr. Tung in the program
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Jay Hirsh, PhD
Professor of Biology
My laboratory studies the action of light on fruit fly behavior, and how neurotransmitters such
as dopamine and serotonin affect such behaviors. We study these effects in very dim light,
where photons are the limiting factor. We have recently shown that fruit flies are far more
sensitive to light than previously appreciated. When assaying either entrainment of the circadian clock, or the
direct effect of light to stimulate locomotion, an activity called behavioral masking, flies respond at light levels
far lower than humans can sense (Hirsh et al., 2010). Intriguingly, these distinct behavioral responses have a
distinct dependence the neurotransmitter dopamine: circadian entranment to dim light requires dopamine, but
behavioral masking does not. This implies distinct neuronal pathways leading to each behavior.
These assays of dependence on dopamine take advantage of a clever tissue-specific rescue of the
tyrosine hydroxylase gene, encoding the rate limiting step in dopamine biosynthesis, by our colleagues in
Paris (Riemensperger et al., 2010). This rescue is designed to occur robustly in the hypoderm, where
dopamine has a vital role, but not in the adult brain, resulting in viable and healthy flies without detectable
dopamine in the adult brain.
Potential projects:
1.
My current Masters student, Karol Cichewicz, is making flies lacking brain dopamine via an alternative
method that will make it feasible to rescue dopamine production in selected brain neurons. With this
methodology, it will be possible to determine behavioral roles of very small dopamine neuron subsets, working
towards the developing a circuit-level view of dopamine function in the brain.
2.
As a complementary technique, we have found developmental effects of RNAi-based inhibition of
dopamine receptor expression in neuronal subsets. The most interesting data to date indicate involvement of
the fly insulin signaling system in dim-light behaviors. This is an open-ended project that will expand our
knowledge of insulin signaling, an important and evolutionarily conserved pathway.
3.
We have data indicating that the extreme light sensitivity of our flies results from the flies having a
system to store photon information over a time period of hours. We know little of how this works, but we have
the genetic tools to dissect what parts of the brain are involved, and what signaling systems are required. We
expect that this system will share components with other behavioral processes that require keeping track of
time, such as learning and memory.
Hirsh, J., Riemensperger, T., Coulom, H., Iche, M., Coupar, J., and Birman, S. (2010). Roles of dopamine in
circadian rhythmicity and extreme light sensitivity of circadian entrainment. Curr Biol 20, 209-214.
Riemensperger, T., Isabel, G., Coulom, H., Neuser, K., Seugnet, L., Kume, K., Iche-Torres, M., Cassar, M.,
Strauss, R., Preat, T., et al. (2010). Behavioral consequences of dopamine deficiency in the Drosophila
central nervous system. Proc Natl Acad Sci U S A.
This is the second year for Dr. Tung in the program; he is currently serves as a mentor for Karol
Cichewicz, a student from UJ.
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Bettina Winckler, PhD, Associate Professor of Neurosciences
Neurofascin accumulation at the axonal initial segment is promoted by
neurotrophin signaling and doublecortin (DCX).
Proper functioning of neuronal circuits depends critically on the correct wiring of
large numbers of neurons. In addition, the electrical properties of the neurons in the
circuit determine the ultimate output from the circuit. The electrical properties of a
neuron are determined by the types of channels present, and by the distribution, and
abundance of the channels in the neuronal plasma membrane. Changes in channel/receptor distribution and
abundance can therefore lead to malfunctioning circuits. Several disorders are thought to be associated with
changed receptor distribution or density, including neuropathic pain,
schizophrenia, myasthenia gravis, epilepsy, and multiple sclerosis.
There are several subcellular locations in a neuron that contain high
concentrations of channels and these domains are particularly
important for determining the electrical properties of the cell.
Synapses are one such place and much effort is directed at
understanding their assembly and regulation. The axon initial
segment (AIS), on the other hand, is an understudied domain
despite its crucial importance in influencing neuronal excitability.
Large numbers of voltage-gated sodium channels are clustered at
the AIS, making it the spike initiation zone and the “gatekeeper” for
action potential firing. The AIS is also the site of a large number of
inhibitory synapses that modify its excitability. Loss of these AISresident GABAergic inputs might lead to serious disorders, such as
epilepsy and schizophrenia. This project in my lab aims at
understanding the cellular and molecular mechanisms that lead to
assembly of the AIS-resident cell adhesion molecule neurofascin.
This work has important implications for both neurodevelopment of functional domains in neurons, as well as
for disease states in which these domains fail to assemble properly or fail to be maintained.
Current models of how the axon initial segment assembles are solely based on steady-state analysis.
While important insights have been gained from steady-state analysis, new approaches are necessary in
order to elucidate the cellular pathways and molecular mechanisms responsible for proper assembly of the
axon initial segment. The student will therefore combine steady state analysis with kinetic analysis,
endocytosis assays, interference approaches, and live imaging to study the dynamics of axon initial segment
assembly. Background in cell and molecular biology is desirable.
Dr Winckler currently supervises Kamil Kruczek from the Jagiellonian University.
14
Jochen Zimmer, D.Phil,
Assistant Professor of Molecular Physiology and Biological Physics
Mechanism of Biosynthesis and Membrane Translocation of Hyaluronan
Hyaluronan (HA) is an extracellular polysaccharide that is ubiquitously expressed in
vertebrates and has a profound effect on a broad range of physiological processes, ranging
from cell differentiation and proliferation to adhesion and mobility. Altered expression levels
of HA are involved in a number of malignant transformations, including breast, colon, prostate and lung
cancer. Due to its high biocompatibility, the HA polymer also finds a broad spectrum of biomedical
applications, e.g. in the treatment of arthritic joints, in eye surgery, and drug delivery.
The linear HA polysaccharide is synthesized at the cytoplasmic side of the cell membrane by a
membrane embedded, processive glycosyltransferase (the hyaluronan synthase) and can grow to several
microns in length. However, it is completely unknown how this hydrophilic polymer is transported to the
outside of the cell, where it performs its biological function. Our mission is to determine, on a molecular level,
how HA is synthesized and translocated across the cell membrane.
In order to identify the essential components for HA synthesis and translocation, we reconstituted the
HA synthesis and translocation reaction in vitro from purified components. Our assay demonstrates that the
hyaluronan synthase catalyzes the synthesis and translocation of the HA polymer across the lipid bilayer.
Next, we would like to map the path of the HA polymer through the synthase by using a site directed crosslinking approach. By incorporating UV-inducible cross-linkers into the sequence of the hyaluronan synthase,
we will be able to covalently link HA to the synthase during translocation. By probing several positions within
the membrane spanning region of the synthase, we shall be able to determine the path of the HA polymer
across the membrane.
A second aim is to determine the structure of the hyaluronan synthase by X-ray crystallography, which
is a prerequisite for addressing mechanistic aspects of the translocation reaction. To this end, we have
already established an expression, purification and crystallization protocol for a bacterial hyaluronan synthase
and are currently exploring strategies to crystallize the synthase in complex with HA.
The laboratory utilizes a broad range of molecular biology techniques to address fundamental
biological problems. Our daily routine includes the expression and purification of membrane proteins, the
functional characterization of the proteins in vitro, as well as protein crystallization and structure
determination. This multi-disciplinary approach offers attractive projects for students with an interest in
biochemistry, molecular-, or structural biology.
This is the first year for Dr. Zimmer in the program
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The following projects may be offered at the University of Virginia in 2011/2012,
pending the clarification of funding prior to March 2011:
Dr. Lucy. F. Pemberton ,
Associate Professor of Microbiology
Center for Cell Signaling
Our lab is interested in how assembly and disassembly of chromatin regulates gene
expression, replication and DNA repair. Correct regulation is fundamental to cellular
processes such as cell division, differentiation and development, and misregulation can
lead to genomic instability and ultimately cancer.
Histones are synthesized in the cytoplasm and imported into the nucleus. Our lab uses the
model system S. cerevisiae and focuses on the early events of chromatin assembly, including understanding
how nuclear import helps coordinate the assembly of chromatin. We are interested in questions such as how
histones are imported into the nucleus and which transport factors and histone chaperones are important in
this process. How do the histone chaperones coordinate the assembly of histones onto DNA and how does
this regulate replication and transcription? Lastly, we are also trying to determine what role histone
chaperones and the nuclear transport factors play in the removal of histones from chromatin?
We have several projects regarding the function of the histone chaperone Nap1p. Nap1p is part of an
evolutionarily conserved superfamily of proteins. Human cells have 4 Nap1 proteins, the SET protein and the
TSPY and TSPYL protein families. Different members of this superfamily have been shown to be upregulated
or mutated in various cancers such as leukemias and gonadoblastoma, These proteins likely have a
developmental role, as in mice loss of a neuronal-specific member of the family is embryonic lethal, and
mutant embryos show overproliferation defects in neuronal tissues. We want to understand the mechanisms
by which Nap1p is recruited to the promoters of different genes and its function there. We also want to
determine how Nap1p mediates the exchange of variant histones with chromatin. This project will use a range
of cell biological and biochemical techniques, as well as some yeast genetics.
Recent Publications
Del Rosario B.C and Pemberton, L.F. (2008) Nap1 links transcription elongation, chromatin assembly and
mRNP biogenesis. Mol. Cell. Biol. in press (epub.
Calvert M, Keck K, Ptak C, Shabanowitz J, Hunt D and Pemberton, L.F. (2008). Phosphorylation by CK2
regulates Nap1 localization and function. Mol. Cell. Biol. 28: 1313-1325.
Blackwell Jr, J.S., Wilkinson, S.T., Mosammaparast, N. and Pemberton, L.F. (2007) Mutational Analysis of H3
and H4 Amino Termini Reveals Distinct Roles in Nuclear Import. J. Biol. Chem. 282: 20142-50.
Mosammaparast, N. Del Rosario, B. D, and Pemberton, L.F. (2005) Modulation of Histone Deposition by the
Karyopherin Kap114. Mol Cell Biol. 25: 1764-1778.
Dr. Pemberton supervised previously Kamila Grabowska from the Technical University of Lodz and
currently serves as a mentor of Magda Wegrzynska from the Jagiellonian University
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Paul Adler, PhD
Professor of Biology
Cells in many tissues display polarity within the plane of the tissue. This is often
called planar cell polarity (PCP) and research in this laboratory focuses on the
genetic, cell biological and molecular basis for PCP. We discovered a genetic
regulatory pathway for PCP in the Drosophila model system and has been found
to be conserved in vertebrates including humans. Mutations in PCP genes have been linked to a
failure in neural closure, polycystic kidney disease, hearing and balance problems and heart and
lung developmental defects. The pathway consists of a regulatory hierarchy, with the fz-like PCP
genes being upstream of the planar polarity effector (PPE) genes which are in turn upstream of the
mwh gene. The proteins that are encoded by pathway genes all accumulate asymmetrically in
epithelial cells and this is thought to be essential for their function. There is evidence that direct
protein:protein interactions between proteins of the pathway are important. A major goal of the
laboratory is to determine the stoichiometry of proteins in the PCP complexes in their endogenous
cells. This will be approached using trangenically encoded flourescent proteins and quantitative
confocal microscopy.
A student working on this project will construct plasmids that encode fusions of various PPE proteins
to either CFP or YFP. Transgenic flies will be generated using the phiC31 site specific
recombination system for integration. The transgenic lines will be characterized to insure the fusion
protein is active (we have good reason to expect this to be the case) and then confocal microscopy
will be done on pupal wings from animals that carry two different transgenes. Flouresence ratios will
be determined and used to estimate protein stoichiometry.
A new student should have some experience in basic DNA technology as this will allow him/her to
get started on their research in a timely manner. Drosophila genetics and confocal microscopy can
be picked up as the project proceeds.
This is the first year for Dr. Adler in the program
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