Laboratory Rotations
PhD PROGRAM IN PATHOLOGY
LABORATORY ROTATIONS
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Faculty |
Projects |
Techniques |
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Berse, Brygida, PhD |
Regulation of gene expression in neurons: 1. Signaling from the TrkA NGF receptor 2. Cross-talk between cytokine and growth factor signaling pathways 3. Effects of DNA methylation on gene expression
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Recombinant DNA techniques Mammalian transfection RNA and DNA isolation RT/PCR, Northern blotting Reporter gene analysis Western blotting Electromobility shift assay |
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Blusztajn, Jan, K, PhD
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We study the mechanisms that regulate the cholinergic neurotransmitter phenotype and our data show that specific bone morphogenetic proteins (BMPs) dramatically upregulate the expression of this phenotype in primary cultures of mouse central nervous system, and in the brain in vivo. The cellular and molecular mechanisms of action of BMPs in inducing and maintaining the neuronal cholinergic phenotype remain unknown and we focus on identifying the intracellular signaling molecules that mediate the induction of the cholinergic phenotype by BMPs, and delineating the promoter regions within the cholinergic gene locus that respond to BMPs, and identifying protein transcription factors that bind to those BMP-responsive regions. In collaboration with investigators at Boston University , Duke University and University of North Carolina we study the effects of perinatal supplementation with a nutrient, choline, on brain development and aging in experimental animals. This research attempts to determine why it is that supplementation with choline during critical perinatal periods in rats and mice causes a long-term facilitation of visuospatial memory which persists until old age. To this end we are utilizing molecular biological, biochemical, neuroanatomical, and behavioral techniques in a highly unified experimental design. Most recently we found that prenatal choline supplementation enhances signaling mediated by MAPK and CREB in the hippocampus, and we have identified a set of genes whose developmental pattern of expression in the hippocampus and cerebral cortex is modulated by prenatal availability of choline. These changes in gene expression may the result of altered DNA methylation that we observe in animals fed choline deficient and/or choline supplemented diets during in utero development. We also study the effects of prenatal availability of choline on mammary carcinogenesis in a rat model. We found that prenatal choline deficiency accelerates tumor growth and we have identified as set of genes whose expression in these tumors is modulated by prenatal choline intake.
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Cell culture: primary neurons, cell lines Intracellular free calcium measurements Brain slices Subcellular fractions Chromatography (HPLC, TLC) Enzyme assays Gel electrophoresis Immunoblotting, immunocytochemistry Gene transfection Promoter/reporter gene analysis Transcription analysis with microarrays
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Bogen, Steven, MD PhD
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Dr. Bogen’s research group bridges academia and industry, including both applied (translational) aims as well as basic science aims that closely tie in with the group’s core capabilities. On the applied (translational) side, our focus is the development of cancer diagnostics technologies for the early detection of, or more accurate diagnosis of, solid tumors. The project furthest along in development is a laboratory testing technology for nationally standardized measurement of Her-2, ER, and PR in the surgical pathology laboratory. These markers are important in breast cancer patient management. That technology, a system of controls and calibrators for quantitative immunohistochemistry, is funded by the National Cancer Institute to enter clinical trials in 2005-06. The team is also working on a solid tumor early detection technology. Solid tumors shed cells at a regular, slow rate. Studies have demonstrated that these cells exist in blood, thereby potentially providing another avenue for early tumor detection. The problem has been to develop a method to isolate and analyze these exceedingly rare cells amongst the millions of red and white blood cells. Last year, we developed a new cell separation technology with high yields of rare epithelial cells in blood. In 2005, we continued the development and validation effort for this cell separation technology. We also initiated a more basic science type of project in 2005, in the area of monoclonal gammopathies. Many B lymphoproliferative disorders and monoclonal gammopathies are believed to be partly triggered by discrete antigens, as part of an immune response. The best example is gastric MALT lymphoma, known to be triggered by H. pylon. The goal of this project is to identify the putative inciting antigen(s) associated with other gammopathies. This project incorporates one of the team’s core technologies – – phage display of random combinatorial peptide libraries. The short-term Aim of this project is to identify the contact sites of monoclonal para proteins from patients with gammopathies, such as multiple myeloma. We will then use a bioinformatic algorithm to query the GENBANK database, to identify homologous stretches to the contact sites amongst known proteins. This project may lead to a better understanding of the etiology of B lymphoproliferative disorders and monoclonal gammopathies. |
Phage display of combinatorial peptide libraries Immunohistochemistry lmmunoassays Cell culture DNA sequencing
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Delalle, Ivana , MD , PhD
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Role of actin capping protein in neurodegeneration. |
Cell culture, RNA interference, GST “pull down” assay, TAP, western blotting
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Murnane, MaryJo, PhD
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Characterization of human primary tumors for proteinase profiles. Defining cancer subtypes and different pathways of cancer progression. Enzyme markers in human tumors. Regulation of proteolytic enzymes by ras.
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Spectrophotometer usage Enzyme assay Molecular genetics: –isolation of RNA, DNA –Southern, Northern blots –preparation of cDNA from mRNA3 –cloning, recombinant DNA methodologies Western blotting PCR |
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Sharon, Jackie, PhD
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Cell fusion Cell culture Recombinant DNA techniques including PCR Mammalian cell transfection Western blotting Immunoassays Animal work (mice)
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Slack, Barbara, PhD |
1. Analysis of mechanisms underlying collagen-evoked signaling by the discoidin domain receptor 1 (DDR1), a transmembrane tyrosine kinase that is overexpressed in tumors of the breast, ovary, and brain: We are testing the hypothesis that differential responses of DDR1, a collagen receptor, to collagen type IV (a component of the basement membrane) and collagen type I (a constituent of the stroma) contribute to the development of epithelial cell tumors. 2. Modulation of muscarinic receptor signaling by integrins: We are examining the modulation of G protein-coupled receptor (GPCR) signaling by integrin – extracellular matrix (ECM) protein interactions. We are seeking to identify ECM-dependent mechanisms that regulate nuclear translocation of mitogen-activated protein kinase (MAPK) in response to GPCR activation.
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Cell culture Immunoprecipitation SDS-polyacrylamide gel electrophoresis Western blotting Enzyme assays Measurement of intracellular free calcium
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Faculty in other Departments with Secondary Appointments in Pathology
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Botchkarev, Vladimir, A, MD, PhD Dermatology
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Molecular mechanisms controlling hair follicle growth In health and disease. Our research is focused on four major projects: 1. Delineating the role of BMP-antagonist noggin in the control of hair follicle development and cycling; 2. Analyzing the role of p53 and its target genes in chemotherapy-induced hair loss; 3. Control of apoptosis in autoreactive CD8 cells in autoimmune hair loss; 4. Molecular mechanisms regulating hair pigmentation.
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Generation of transgenic mice, pharmacological manipulation of hair follicle growth in vivo, ex vivo culture of isolated hair follicles and skin explants, isolation of the lymphocyte subsets from skin and peripheral blood, in situ hybridization, double and triple immunofluorescence, multi-color confocal microscopic Western blot analysis, RT-PCR, FACS analysis.
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Cardoso, Wellington , V., MD Medicine
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Control of branching morphogenesis and epithelial cell differentiation during lung development. The overall goal is to Identify and characterize potential regulators of lung morphogenesis, and to study how they interact with the gene network present in the early lung bud. Some regulators we are studying include: Retinoids, sonic hedgehog, hox genes and fibroblast growth factors.
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Manipulations with embryonic lung explants: – organ culture – microdissection/grafting – use of recombinant protein – gene transfer Cell culture RNA-DNA analyses – Northern/Southern blotting – semiquantitative PCR – in situ hybridization Cell proliferation assays lmmunohistochemistry
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Eller, Mark, PhD Dermatology |
Our work focuses on the mechanisms of DNA damage responses to telomere uncapping in mammalian cells and how these responses can be stimulated by DNA oligonucleotides homologous to the telomere 3’ overhang. We are particularly interested in the apoptotic response in cancer cells and how this technology may be therapeutic in the treatment of human cancer.
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Cell culture FACS analysis TUNEL analysis Western blotting Telomere length and integrity analysis Photobiology of UV light Confocal microscopy
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Faller, Douglas, V. MD, PhD Medicine
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A major focus of our laboratory is the study of the basic molecular and cellular biology of virus- and oncogene-transformed cells and tumors. We are involved in determining the mechanisms by which retroviruses and their oncogenes cause tumors, through defining the ways in which oncogenes control host cell gene expression. A special interest of this laboratory involves viral regulation those cellular genes encoding proto-oncogenic molecules and cytokines. We are analyzing the molecular mechanisms by which oncogene-transformed cells become autonomous of growth factor requirements. This work involves the elucidation of growth-factor signal transduction pathways in normal and transformed mesenchymal and lymphoid cells, and study of the ways in which this signaling pathway is disrupted or circumvented in tumor cells. This work has resulted in new information regarding the transduction of growth factor signals by second messenger systems in both normal and transformed cells. My laboratory also studies the role of oncogenes in programmed cell death. A related area of his research is the interaction of retroviruses and the tumors they induce with cellular immune defense mechanisms. The means by which virus- or tumor-specific cytotoxic T lymphocytes, natural killer cells and monocytes recognize and destroy infected cells and tumors is under investigation, as are the molecular mechanisms by which tumors escape from immune surveillance. The mechanisms of aberrant control of Class I Major Histocompatibility Antigen gene expression in oncogene-transformed cells, retrovirus-infected cells and naturally-occurring tumors are being determined. A new transactivation property of murine leukemia viruses has been elucidated, which controls the expression of genes in the host cell important to the leukemogenic process. My laboratory also has a translational research program which develops molecular cancer therapeutics derived from his basic research, and tests them in clinical trials.
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Goukassian, David, A, MD, PhD Dermatology
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1. Age-related decline in DNA repair capacity: the use of short-term human skin organ-culture 2. Prevention of mutagenesis and photocarcinogenesis in repair-proficient and repair-deficient normal and NER knockout mice. 3. DNA oligonucleotide therapy to reduce skin cancer risk. 4. Effect of COX-2 inhibition on photocarcinogenesis in XPA and XPC knockout mice. 5. Effect of small DNA oligonucleotides on the regulation of apoptosis, DNA repair and cell cycle in normal epidermal cells after UV irradiation.
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Tissue culture, FACS analysis (proliferation, apoptosis, gene expression). Conventional and confocal microscopy, immunohistochemistry, ribonuclease protection assay, Electromobility Shift Assay (E2F1, p53, NFkB transcription factors), transient transfections, gene expression in murine and mammalian cells (AdTetOn/Off system), Western, Northern blots, immunoprecipitation, PCR, RT-PCR, routine histology, protein phosphorylation studies, reporter plasmid analyses, several gene knockout and double transgenic murine models (XPA, XPC, p53 and combinations of these gene knockouts with LacZ (mutation frequency sensor gene) evaluation of transdermal penetration.
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Kotton, Darrell N. MD Medicine
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Stem cell biology and Gene Therapy: · The role of bone marrow-derived stem cells in lung injury repair is evaluated in specified mouse models of lung injury. · Embryonic stem cell differentiation into lung epithelial cells: development of an ex vivo platform that models lung epithelial cell development from undifferentiated mouse embryonic stem cells. Regenerative medicine applications for healing the injured lung using lineage-directed embryonic stem cells are also studied. · Gene therapy of lung diseases using lentiviral vectors: lentiviral transduction of hematopoietic stem cells followed by transplantation, or intratracheal delivery of lentiviral vectors is used to overexpress or knockdown genes in mouse models of lung disease. more information available at www.kottonlab.com
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Embryonic stem cell culture Stem cell purification Generation of lentiviral vectors Gene transfer/ gene therapy RNAi-based gene suppression PCR, RT-PCR ELISA FACS Plasmid cloning Histology and immunohistochemistry Mouse dissection Bone marrow transplantation
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Lerner, Adam, MD Medicine
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There are two principle areas of research: Research area #1: 1. My research on human lymphoid malignancies focuses on cAMP and cGMP signaling in B and T cells and the use of family specific phosphodiesterase inhibitors to selectively induce apoptosis in malignant cell populations. We have determined that PDE4 inhibitors induce apoptosis in chronic lymphocytic leukemia cells but not in human mononuclear cells. In addition, we have determined that a new effector protein for cAMP, EPAC, a Rap1 GDP exchange factor that binds to and is activated by cAMP, is expressed and functional in CLL cells but in no other hematopoietic cell examined (ie B cells, T cells, monocytes, neutrophils). Possible projects would be: 1) To assess whether EPAC is potentially a therapeutic target in CLL; i.e. whether it’s function is critical to the growth and survival of CLL cells; 2) To determine why CLL cells differ from other hematopoietic cells with regard to responses to PDE4 inhibitors; 2) To determine how the unique sensitivity of CLL cells to PDE4 inhibitors can be used to best advantage to improve clinical responses to treatment of CLL. Research area #2: 2. We wish to determine what physiologic and pathologic roles a novel Ras superfamily GOP exchange factor, AND-34, plays in breast cancer cells and in lymphoid cells. AND-34 (or its human homologue, BCAR3) induces tamoxifen resistance in breast cancer cell lines when over-expressed. It is phosphorylated after EGF treatment. A possible project would be to identify the tyrosine that is phosphorylated following EGF treatment and establish what effect mutation of this tyrosine residue has on ANO-34’s GEF activity. Other projects include assistance in analyzing knockout mice and development and analysis of cell lines stably expressing inducible siRNA constructs against AND-34.
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Tissue culture, FACS analysis, stable and transient transfections, retroviral infections, Miltenyi magnetic bead purification of primary human hematopoietic cells, realtime PCR, Northerns, Southerns, Western analysis by ECL, immunoprecipitation, recombinant DNA techniques. immunohistochemistry, enzymatic analyses, kinase assays, luciferase assays, “knockout” mouse generation and analysis, stable inducible siRNA cell line generation.
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Ramirez, Maria, PhD Medicine |
Lung Development Analysis of chromatin modifications and DNA methylation of lung specific genes during induction of lung cell fates and initiation of lung development from the embryonic foregut. Analysis of molecular mechanisms involved in cell shape changes during development of lung alveolar epithelial cells.
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• General molecular biology techniques: PCR, Real time RT-PCR, northern and western blots, subcloning. • Culture of primary lung epithelial cells, cell lines, and organ culture. • FACS and cell sorting of GFP positive cells • Immunohistochemistry, in situ hybridization • Laser capture microdissection. • Gene transfection • Lentiviral transduction. • Microarrays • shRNA • Promoter/reporter gene analysis: cHIP analysis, EMSAs. • Transgenic and knockout mice generation and analysis. • DNA methylation analysis |
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Ruenger, Thomas, M., MD, PhD Dermatology |
1. Mutagenesis of longwave ultraviolet light (UVA) in skin 2. Role of a protective cellular DNA damage response on the mutagenic outcome of UVA and UVB exposure 3. Role of the Fanconi anemia/BRCA DNA damage response pathway in the cellular response to ultraviolet light
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Culture of primary human skin cells, muta-genesis assays (hprt assay, shuttle vector-based assays), PCR, DNA sequencing Culture of primary human skin cells, mutagenesis assays, immunoblotting, immunostaining of cells, flow cytometry, siRNA Same as 2) |
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Sherr, David, PhD Public Health (Environmental Health)
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The Sherr laboratory employs state-of-the-art cellular and molecular technologies to research three specific areas of basic and applied science: 1) mechanisms through which environmental chemicals suppress the immune system (the Apoptosis team) <http://www.cireeh.org/pmwiki.php/Main/ATeam>, 2) molecular signaling leading to environmental carcinogen-induced and spontaneous breast cancers (the Breast Cancer team) <http://www.cireeh.org/pmwiki.php/Main/BTeam>, 3) development of vaccines for the treatment of cancer and primary amyloidosis (the Cancer/Amyloid Immunotherapy team) <http://www.cireeh.org/pmwiki.php/Main/CTeam>. The common element in these 3 disciplines is the involvement of an environmental chemical receptor, the aryl hydrocarbon receptor (AhR), in the suppression of the immune system and in maintaining tumor cell growth.
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Cellular immunology, long-term marrow cultures, flow cytometry analysis and sorting, western blotting, molecular biology (cloning, transfections, gene knock-out mice, Northern and Southern blotting, PCR).
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Spira, Avi , MD Medicine
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Our laboratory research interests focus on applying high-throughput genomic and bioinformatic approaches to the study of lung cancer, the leading cause of cancer death in the US . While cigarette smoking is the major cause of lung cancer, only 10-20% of heavy smokers develop lung cancer. Because of the lack of effective diagnostic biomarkers and the inability to identify which current and former smokers are at the greatest risk for developing cancer, lung cancer is most often diagnosed at a late stage after it has spread. The primary research focus of our lab is to determine how cigarette smoking affects intra-thoracic (lobar bronchi) and extra-thoracic (buccal and nasal mucosa) airway epithelial cell gene expression and to use this information to develop a non-invasive biomarker for lung cancer that can identify that subset of smokers who have, or are at risk for developing, lung cancer. We have also begun to explore how this molecular “field of injury” in the airway of smokers reflects information about the perturbation of specific oncogenic pathways within an individual, potentially allowing personalized genomic approaches to chemoprophylaxis and therapy. This disease-specific airway “field of injury” concept is also being extended to identify biomarkers for and pathways perturbed in Chronic Obstructive Lung Disease (COPD), a smoking-related pulmonary disease that represents the 4th leading cause of death in the United States . Finally, we are interested in understanding the underlying mechanisms of smoking-related disease risk and are seeking to identify DNA polymorphisms and regulatory micro RNA changes that are predictive of the gene-expression changes we’ve identified as hallmarks of the disease-specific field of injury. Please visit us at www.pulmonomics.com for additional information.
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DNA microarrays SNP arrays microRNA arrays Real time PCR Northern Blotting Real-competitive PCR (MADLI-TOF Mass spectrometry-based gene expression assay) Tissue and serum proteomics using Tandem Mass Spectrometry Cell culture
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Thiagalingam, Sam, PhD Medicine
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Our major research focus continues to be on the use cancer genomics and biology, employing primarily breast and colon cancers as model systems. We hope to contribute to the elucidation of the multi-modular molecular network (MMMN) cancer progression models as the road map to dissect the complexity inherent to cancer through these studies. Our approach to achieve this overall goal is to undertake research under the following topics: (i) The Smad signaling connection to colon cancer metastasis; (ii) The Smad signaling connection to breast cancer metastasis/bone metastasis; (iii) Development of therapeutic approaches to breast cancer by targeting TGF-beta signaling events; and (iv) hBub1 is a suppressor of p53 mediated cell death. Recently, we have also become interested in the role of epigenetics in the pathogenesis of major psychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BD). Because of the overwhelming evidence for the role of environmental factors in the presentation of the major psychiatric disorders, we hope to decipher a direct correlation between the altered epigenome and SCZ and BD. |
-Standard recombinant DNA methods. -Cell culture (cell lines and primary tumors). -Immunoprecipitation and Immunohistochemistry. -Western blotting. -Assays for apoptosis. -Protein kinase and other enzyme assays. -Cell cycle profiling using FACS analysis. -Immunofluorescence microscopy for sub-cellular localization of proteins of interest. -Metastases and invasion assays. -The use of RNAi to knock down genes of interest. -Using orthotopic xenograft mouse models to investigate metastasis and drug therapy. -Methylation Specific PCR (MSP) to determine epigenetic gene silencing by DNA hypermethylation. – Development and application of a novel immunoprecipitation coupled PCR based method known as “methylated CpG island capture, selection and amplification” (MCCSA) to isolate methylated sites in cancer and schizophrenia. -Mutation screening methods. -Targeted Expressed Gene Display (TEGD). |
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Yaar, Mina, MD Dermatology
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1. Mechanism of telomere overhang-induced apoptosis in malignant cells including breast cancer cells and pancreatic cells. 2. Evaluating pathways that induce pigmentation in skin-derived melanocytes including bone morphogenetic proteins and UV irradiation. 3. Mechanism of p75NTR-induced apoptosis in Alzheimer’s disease
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Cell culture, Western and Northern blot analysis, Plasmid propagation, RT-PCR, animal experiments, tranfection and promoter analysis
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9/13/07