2013 Summer Plus Projects
The Hedgehog signaling cascade is a developmental cellular signaling pathway that is inappropriately activated in a number of major cancer types. These include cancers of the brain, skin, breast, digestive tract and lung. The goal of our laboratory is to identify novel therapeutic targets in the Hedgehog pathway that will allow us to better treat these cancers. Current projects are focused on two membrane proteins that govern Hedgehog pathway activity. The first is the Hedgehog receptor Patched, a transmembrane protein that inhibits pathway activity in the absence of the activating Hedgehog ligand. The second is Smoothened, another transmembrane protein that transmits the activation signal to the inside of the cell. In the absence of the Hedgehog ligand, Patched inhibits Smoothened signaling by an unknown mechanism. Release of Patched inhibitory activity is THE pivotal point of Hedgehog pathway regulation. As such, elucidation of the molecular events governing this off-on switch is imperative.
We are optimistic that, by understanding the mechanism of how Patched inhibits Smoothened, we will be able to exploit this information to better target Smoothened in cancer. We are currently interrogating several aspects of this question, and have an available student research project to assist in this investigation. The student will study an essential regulatory domain of Patched known as the Sterol Sensing Domain (SSD). It is clear that the SSD is required for Patched to inhibit Smoothened. However, the role of the SSD in Patched-mediated inhibition is not clear. The student will make a series of mutations within the Patched SSD, and assess the ability of these mutant proteins to inhibit Smoothened signaling in a variety of model systems. During the course of these studies, the student will learn a combination of biochemical, cell biological and genetic techniques including DNA and RNA preparation/purification, mammalian cell culture, cellular lysate preparation, immunofluorescence microscopy and in vivo genetic analyses in fruit flies. We utilize the fruit fly Drosophila melanogaster as a model organism because the Hedgehog pathway signaling components are tightly conserved from Drosophila to man. As such, regulatory processes we identify in flies are directly relevant to human disease. Furthermore, the versatility of Drosophila genetics offers a robust system in which to perform rapid in vivo experiments, allowing us to give biological relevance to regulatory processes we identify using in vitro biochemical techniques.
The Dallas lab is focused on improving the outcomes of patient undergoing hematopoietic stem cell transplant (HSCT) using umbilical cord blood (UCB) as an alternative stem cell source. HSCT using stem cells from a human leukocyte antigen (HLA) matched donor is a widely accepted form of therapy for various malignant and non-malignant hematological diseases. However, many patients do not have an HLA-matched donor identified. The use of UCB has significantly increased the donor pool and the number of patients qualifying HSCT. UCB have certain advantages and disadvantages compared to bone marrow or peripheral blood stem cells. Our lab is interested in ex-vivo manipulation and culture of cord blood progenitors that may ultimately have therapeutic potential. Currently, we have three main focuses of the lab.
Understanding the unique immunological interactions and barriers involved in cord blood transplantation. In particular the biology of T cells and antigen presenting dendritic cells. We use unique humanized immunodeficient mouse models, where a human thymic tissue is implanted in the kidney capsule to investigate the immune recovery after transplantation.
Ex-vivo expansion of pluripotent hematopoietic stem cells using the Notch signaling pathway.
Participants of the Rhodes College Summer Plus Program will be actively involved in our laboratory team. Participants interested in the medical field are also invited to observe our clinical activities in the Bone Marrow Transplant Service. In the laboratory, depending on the student’s interest, I have identified few well-defined projects that can be accomplished during this research period. The techniques that will be learned include and not limited to:
Hematopoietic stem cell culture and manipulation
Cord blood hematopoietic stem cell harvest and expansion
Microscopy and imaging of cells culture and tissue
Involvement in murine transplantation and surgery
The emergence of multi and pan drug resistant strains of pathogenic bacteria is an emerging clinical issue that has the potential to significantly impact the healthcare environment. This problem dictates the need to discover and develop novel antibacterial agents. We are seeking an intern to work on the discovery and elucidation of new antibiotic substances from soil actinomycetes, using a novel microbiological screening approach. Trainees will gain experience in microbiology, biochemistry, high throughput screening and chemical isolation.
Carboxylesterases (CE) are ubiquitous enzymes responsible for the hydrolysis of esterified xenobiotics, including numerous clinically used drugs. This includes the anticancer agents CPT-11(irinotecan) and capecitabine, as well as commonly prescribed drugs such as Lunesta, Ramipril and Tamiflu. We have undertaken a comprehensive evaluation of the biology of human CEs and have purified both isozymes to homogeneity. Using these reagents, we have identified numerous classes of specific CE enzyme inhibitors, and demonstrated that these small molecules modulate drug metabolism. This can result in the loss of efficacy of the drugs and could also potentially increase their toxicity. Using QSAR analyses, we have identified a pharmacophore that accurately predicts the inhibitory properties of such compounds. We propose therefore to search natural product databases with this pharmacophore to identify novel compounds that inhibit CEs.
This project will therefore combine computational, biochemical, and cell culture approaches to identify and validate such molecules. Furthermore, if natural product sources of such inhibitors are available, we will use chromatographic and physico-chemical approaches to purify and isolate these inhibitors. In addition, if necessary, medicinal chemistry will be employed to synthesize bona fide compounds to provide complete biochemical and chemical characterization. All techniques are currently used or available in the Potter lab, or within the Department of Chemical Biology and Therapeutics, and we do not anticipate any technical issues that cannot readily be overcome. As always, all data will be published in peer reviewed journals with an emphasis on the Rhodes student generating the drafts and revisions of these documents.
The goal within my lab is to educate these student in a manner that allows them to research the problem and then attempt to successfully complete the task. Clearly all of my lab and myself are available for advice, but I believe that the learning process also requires a few mistakes. Ultimately, we seek to provide a challenging environment which provides the candidate with all of the necessary expertise and tools to generate high quality data, and a complete understanding of how biomedical research is conducted.
Research in the Rivas Laboratory focuses on the field of natural product synthesis with an emphasis on the development of catalytic asymmetric methods that facilitate the construction of complex molecules of promising pediatric therapeutic value. To expedite our drug discovery work, we also conduct bioactive-guided terrestrial natural product isolation and chemical structure elucidation in collaboration with the Natural Product Museum of Paraguay. Pediatric malignancies such as glucocorticoid resistant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia AML are among our priorities.
Current areas of research in the Rivas laboratory include total synthesis of sesquiterpenoids and the development of catalytic systems for enantioselective cycloaddition reactions. In parallel to our synthetic work, we conduct both biochemical and cellular assays in the lab. Our research integrates the critical areas of chemical synthesis, biochemistry and molecular biology.
Learn to design and carry out synthesis of novel chemical entities for drug discovery through innovative chemical strategies
Learn how to handle highly sensitive, hygroscopic, and air sensitive reagents
Learn to purify reaction mixtures using modern techniques such as HPLC, Isolera purification system as well as standard methods (thin layer chromatography, column chromatography, ion exchange chromatography
Learn how to use Chem. Draw Ultra, SciFinder, ISIS Draw, and Excel for chemists
Ability to conduct and/or interpret 1D NMR, 2D NMR, IR/UV, and Mass Spectroscopic Data
Learn to critique, present, and write scientific articles
Learn to work in a collaborative multi-disciplinary environment
The student will understand the process of drug discovery from compound lead to product optimization, and be able to conduct independent and original research. He/she will have the opportunity to present their work at either local or national meetings and publish their results in peer reviewed journals.
Jinghui Zhang, PhD
My research interest has been in the development of highly accurate and sensitive computational methods for analyzing large-scale cancer genomic data, especially in the area of detecting and analyzing genetic variations and somatic alterations including sequence mutations, structural variations and copy number variations. Building a comprehensive error model and developing cross-platform integrated analysis methods have been the unique approach that we take to identify critical genetic alterations that affect cancer initiation or progression. Development of visualization tools for viewing high-throughput integrative genomic data is another key interest as these tools are indispensable for gaining understanding of the experimental data and for reviewing the accuracy of the computational results obtained from the astronomical volume of the genomic data generated with the current technology.
In the last three years, my lab has focused on developing highly accurate methods for analyzing whole-genome sequencing data for the St. Jude/Washington University Pediatric Cancer Genomic Project (PCGP). Our work has unveiled genetic landscape of T-ALL, a novel drug target for retinoblastoma, subtype-specific mutation profile for medulloblastoma, association of age at diagnosis and ATRX mutations in patients with neuroblastoma and recurrent mutations of Histone H3 in high grade glioblastoma. Our algorithm for detecting structural variations in the cancer genome, CREST, is shown to improve the sensitivity by 70% compared with existing approaches while maintaining a 80-90% validation rate. Currently we are focusing on developing new approaches for applying genomic analysis for clinical diagnosis and prognosis for pediatric cancer patients and for developing integrative analysis of genomics, expression, epigenetics and proteomics data. Specific projects for Rhodes Summer Plus program including data analysis for clinical sequencing, developing novel approaches for understanding somatic mutations in non-coding regions and integrating epigenetic landscape with genetic landscape for understanding the initiation and progression of pediatric cancer, comparison of pediatric cancer with adult cancer.
Specific skills required: basic understanding of molecular biology or genetics, training in at least one programming language (C/C++, Perl or Java) and exposure to Linux shell, statistical analysis skills (experience with R or MatLab is highly appreciated), quick learner, attention to details with a focus and dedication to research. Students will be working with Ph.D-level senior scientists in the lab to acquire analytical skills for these challenging projects.
Molecular Studies of Pancreatic Cancer
Pancreatic ductal adenocarcinoma is one of the deadliest forms of cancer. This year, about 32,000 Americans will be diagnosed with pancreatic cancer and almost all of them will die within 3-6 months. The main reasons for the severity of the disease are late diagnosis and excess of connective tissue within the tumors, making them inapproachable to chemotherapeutic drugs. The early molecular events that lead to oncogenic transformation in the pancreas are poorly understood, and uncovering them will provide valuable information for monitoring and drug selection.
We investigate molecular processes involved with pancreatic cancer initiation and progression, using in vivo and in vitro approaches. The interested student will use the following techniques to characterize the initiation and progression of pancreatic cancer in mouse models of this disease:
Sectioning paraffin embedded pancreatic tumors and staining the sections using histologic techniques or specific antibodies.
Isolating RNA from pancreatic tissues and using quantitative real time PCR techniques to analyze gene expression.
Performing Western immunoblot analyses of primary cultured pancreatic cancer cells and whole pancreatic tissues, to identify defects in signaling pathways.
The Clements Laboratory investigates the molecular mechanisms that specify hematopoietic stem cells (HSCs), which are the therapeutic component of bone marrow transplants. During embryonic development, HSCs come from endothelial cells in the primitive dorsal aorta, thus development of the primitive vasculature and the hematopoietic system are deeply intertwined. It is currently impossible to make HSCs or vasculature from pluripotent stem cells in vitro, but understanding the normal molecular regulation during development might allow us to recreate these events in a petri dish. In vitro synthesized HSCs and vasculature would permit generation of perfectly immune-matched tissues for transplantation therapies in, for example, treatment of leukemia or ischemic disease. The Clements lab takes advantage of the powerful experimental capabilities of zebrafish to understand the embryonic specification of HSCs and early vasculature.
Zebrafish develop external to the mother and are transparent during the period when vessels and HSCs first appear. In combination with fluorescent transgenes expressed specifically in developing tissues, it is possible to microscopically examine the effects of gene perturbation in living embryos in real time. Gene knockdown can be rapidly accomplished by injection of antisense morpholino oligonucleotides at the 1-cell stage. The zebrafish genome has been nearly completely sequenced, and a large database of genes expressed in tissues that give rise to axial vessels—but of unknown function--is available. We are selecting candidate vascular-expressed genes that will be targeted for knockdown to determine their function during patterning of the vessels and HSC specification. Lab members employ bioinformatics, fluorescence microscopy, molecular biology, microinjection, whole-mount in situ, and many other techniques to dissect regulatory mechanisms.
Magnetic resonance imaging (MRI) can produce images non-invasively that reveal the morphology, metabolism, and function of internal tissues or organs. MRI offers many ways to manipulate and quantify the image contrast which is the basis of ongoing research of the MR Physics Lab. We focus on the development and application of novel and advanced imaging tests that potentially replace existing, suboptimal diagnostic tests which, for example, apply radioactive substances or are invasive. The team works on MR methods to acquire functional information from different organs in patients with sickle cell disease (SCD) and cancer. We want to provide physicians with the most accurate and effective tools for non-invasive assessment of disease (e.g., organ vitality/damage) and treatment outcome and are currently working on the following main projects:
(a) Non-invasive quantitative assessment of liver iron content in patients with transfusional iron overload. This is a problem in patients with SCD and blood cancers and can lead to iron related toxicity and death. We are working on an accurate MR test that is applicable to patients with extremely high liver iron content and will calibrate our imaging method with biopsies from an ongoing patient study.
(b) Use of magnetically labeled targeted nanoparticles with and without a therapeutic payload. The MR relaxivity properties of these nanoparticles will be characterized in vitro and in vivo. The nanoparticles are also needed as calibration standards for iron quantification.
(c) Whole body diffusion imaging (WB-DWI). WB-DWI is an emerging method with already demonstrated clinical impact on bone marrow involvement, tumor staging, and screening. The project involves testing of different WB-MRI imaging strategies in healthy volunteers and pediatric patients. The best method for certain patient groups and clinical applications needs to be identified. For the patient scans techniques of co-registration with images from other modalities (e.g., PET, MIBG) have to be investigated. In addition, criteria for suitability of WB-DWI as a cancer staging method have to be developed through comparison with gold standard methods.
The Rhodes Summer Plus Student would work on a project related to one of these areas, depending on personal preference. All of these projects encompass learning how to operate an MRI scanner, how to run quantitative MR analyses, and how to interpret the data. These projects would provide an insight into typical work of a physicist/biomedical engineer specialized in MRI research.
The interested student will work as member of a multi-disciplinary team consisting of MR Physicists, Radiologists, Hematologists, Oncologists, Biomedical Engineers, and Computer Scientists in the Department of Radiological Sciences at St. Jude Children’s Research Hospital. The student will be given the opportunity to work on an independent research project and summarize the results as a conference abstract and manuscript
Radiation therapy is the single most effective agent in the treatment of children’s cancer, but it carries a risk of side effects that must be both minimized and understood. Research within the Radiation Oncology group of the Developmental Biology and Solid Tumor Program is focused on maximizing cure while minimizing side effects. This is accomplished by successfully integrating the latest in cancer treatment technology into the management of children with radiation, from photon to now cutting edge proton radiation therapy. Our portfolio of clinical trials provides ample hypothesis driven data, aiming to integrate modern PET and MR imaging into the radiation treatment paradigm as well as define the functional outcomes of patients treated with these approaches.
Students will be given their own independent project as part of this on-going research. They will be immersed in our clinic and the research that comes out of our daily activities. Students will learn how the scientific method can be applied to clinical research and how this work relates directly back to the patients under treatment. We anticipate that most students interested in working within our group would be prospective medical students or medical physicists.
Regulation of dynamic protein complexes by prolyl isomerases
Intrinsically disordered proteins (IDPs) play important roles in signal transduction and other regulatory processes in cells despite their lack of a unique structure. According to the structure/function paradigm, IDPs are expected to become folded in their functional state, e.g. upon interaction with binding
partners. However, it has recently been shown that some IDPs remain significantly disordered even in
complex and that this disorder is functionally important. Ash1, a yeast protein involved in mating type switching, whose protein levels are tightly controlled, interacts with the substrate recognition subunit, Cdc4, of its ubiquitin ligase via multiple binding motifs called Cdc4 phospho degrons (CPDs). These CPDs contain pThr/pSer – Pro sequences. Each degron interacts with the one binding site in Cdc4, one at a time, in a dynamic equilibrium giving rise to a ‘fuzzy’ complex. ‘Fuzzy’ complexes represent a new paradigm of protein/protein interactions and are thought to allow tight regulation. The affinity of the Ash1/Cdc4 complex depends on the number of phosphorylated degrons and hence provides a sensitive readout of kinase activity in the cell. Only the trans prolyl conformation of each phospho degron can interact with Cdc4. Hence, prolyl isomerases such as Pin1 may serve as an additional means of modulating the affinity. We will test this hypothesis by carrying out Ash1/Cdc4 binding experiments in the absence and presence of Pin1 to test its effect on the affinity of this dynamic complex
We produce the proteins recombinantly and study them in vitro using biochemical and biophysical techniques, in particular NMR spectroscopy. NMR spectroscopy is a powerful technique that reports on the flexibility, structural features and binding properties of even disordered proteins with atomic resolution. We hope to elucidate benefits of disorder in protein complexes and enhance our understanding of the physical basis of interactions in signal transduction. We may thereby shift the current view of predominantly rigid complexes to more dynamic alternatives and find new targets for drug discovery.
Recombinant protein expression in bacterial culture
NMR and fluorescence binding experiments
In our laboratory, biochemical, biophysical and structural methodologies are used to investigate the molecular mechanisms underlying the specific regulatory and targeting interactions of the intracellular signaling pathways. Expertise in the laboratory in which trainees will receive instruction includes protein chemistry, protein expression and purification, systems biology, and structure-based drug discovery.
Currently, the WNT pathway is one of the major focuses in our laboratory. Wnt signaling plays an important role in embryonic development and in regulation of cell growth. Inappropriate activation of the Wnt pathway has been shown to lead to several cancers, including pediatric cancers. Riming to develop potential novel pharmaceutical agents, we have taken a systems biology approach and are working on development of small-molecule inhibitors that target different protein–protein interactions alone the Wnt signaling pathway.
Mechanism of IgM assembly into pentameric structures
Concept: Vertebrates respond to foreign antigens by producing antibodies, which can provide long term protection to pathogens. The initial antibody response to immunization is predominantly IgM, and further boosts with antigen elicit a response that is a mixture of various IgG antibody subclasses. It has been calculated that an IgM producing plasma cells makes up to a thousand antibody molecules per second. Secretory IgM is usually assembled into pentamers that are composed of five IgM heterodimers (H2L2)5 and a single J chain, which are held together by 100 disulfide bonds. The IgM pentamers are post-translationally modified by 50 N-linked glycans that serve to protect and stabilize the antibody in the blood stream. This requires that 100,000 disulfide bonds must be made and 50,000 carbohydrate moieties be added each second!! Thus the plasma cell can be considered a highly efficient factory for producing antibodies. Accordingly, the cell has a dedicated quality control machinery to ensure that only properly processed antibodies reach the cell surface, while those that fail to mature properly must be identified and targeted for degradation. This discrimination is absolutely critical to the proper functioning of the immune system.
There has been a great deal of progress in the past few years in identifying components of the cellular quality control machinery, but our understanding of the characteristics or features on nascent antibodies that form the molecular basis of recognition by the quality control system remains very poorly understood. Our goal is to scrutinize features on the various portions of the antibody molecule to understand which regions play critical roles in the assembly process. In this particular project, we will focus on the role of the terminal domain in pentamerization of IgM antibodies. There are data showing that in the absence of J chain synthesis, IgM incorrectly assembles into hexameric structures and other data to argue that the terminal region of the protein plays and important role in assembly. We will construct a number of vectors that encode isolated regions of the protein and express them in cells. The assembly will be monitored by several methods and the role of various components of the quality control machinery in this process will be determined.
Project: The published data lead us to take a much more detailed look at the effects of J chain, glycosylation, and ERGIC/ERp44 on polymerization in cells using simplified IgM heavy chain constructs. To do so, we propose the following experimental plan:
- Express various constructs in cells
- Produce point mutants (C575S/A, S565F/A, tail piece variants)
- Obtain J chain cDNA
- Express various constructs in cells
- examine assembly and secretion
- co-express J chain, examine assembly and secretion
- knock-down ERGIC and ERp44 (alone and together), examine assembly and secretion