Rotation projects include development of nanobodies for prevention and treatment of mucosal disease (viral and bacteria).
Endowed Chair in Biochemistry and Molecular Biotechnology I
<Long Text /> Potential Rotation Projects Project #1: Probing the structural basis for cooperativity in a dimeric hemoglobin: Scapharca dimeric hemoglobin is an elegantly simple model system for exploring the structural basis for intersubunit communication. Our analysis to date has established a new paradigm for cooperativity in which tightly bound water molecules are used as sensors for ligand state. In this project, the student will first mutate the gene for the native hemoglobin at a residue that is suspected of playing a role in the intersubunit communication. The mutated protein will then be expressed in E. coli and purified and subjected to functional analysis of oxygen binding. Using conditions already established for the native protein, the mutant hemoglobin will then be crystallized and subjected to preliminary x-ray analysis. This project will provide the student with an introduction to two of the most powerful techniques for investigating the structure and function of proteins: site-directed mutagenesis and x-ray crystallography. Additionally, valuable experience in protein purification will be obtained. Project #2: Structural analysis of the polymerization of sickle-cell hemoglobin Sickle cell disease results from the pathological polymerization of deoxygenated hemoglobin S (ß6E->V) within erythrocytes. This polymerization depends upon a complicated interplay of multiple interactions between tetramers. Our high resolution crystal structure of this molecule has elucidated details of some of the important interactions. Several projects are available for rotation students. These include crystallization of sickle-cell hemoglobin in the presence of an inhibitor to identify its binding site, site-directed mutagenesis of hemoglobin S and crystallization of mutants that show altered polymerization characteristics.
Postdoctoral positions are available to study the relationship(s) between the Rb/E2F proliferation pathway and p53-dependent apoptosis. Areas of investigation include understanding why E2F1 is unique among the E2F family in its strong apoptosis signaling ability and determining the pathways that lead from E2F1 activation to cell death. Fellows will be associated with a highly motivated and interactive group of researchers studying aspects of cell cycle, apoptosis signaling and cancer using cell culture, virology and animal models. If interested, please contact: Timothy Kowalik Department of Molecular Genetics and Microbiology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655 The University of Massachusetts Medical School is an Affirmative Action / Equal Opportunity Employer.
Rotation Projects Identify the region on E2F1 responsible for p53-mediated apoptosis. We know that a transcriptionally functional E2F1 is required for apoptosis signaling. We also know that E2F2 does not induce apoptosis in our system. Therefore, domains on E2F1 and E2F2 will be swapped to determine which E2F1 domain is responsible for apoptosis signaling. Role of nuclear kinases in E2F1 mediated apoptosis. Our recent efforts have revealed that E2F1 expression leads to the phosphorylation of p53 and this modification correlates with apoptosis. There are several candidate kinases that need to be analyzed for their contribution to p53 phosphorylation. A dominant negative approach will be used in this project to identify which kinase(s) contribute to the E2F1 signal to p53. Role of CMV IE proteins in proliferation. We have found that both IE1 and IE2 target different Rb family members. IE2 binds to Rb and IE1 interacts with p107. We now know that IE2 is a strong inducer of S phase but IE1 can only significantly modulate proliferation in cells lacking p53. We are interested in knowing why p53 can prevent IE1 from inducing S phase. Mechanism of p53 accumulation by CMV. It has recently been shown that CMV infection results in an accumulation of p53. We have recently found that IE1 expression also leads to p53 accumulation. We wish to determine the steps leading to p53 accumulation and the consequences of this event.
Focus: Cellular and organismal studies of viral infections
Arthur F. and Helen P. Koskinas Professor in Biochemistry and Molecular Biotechnology
Postdoctoral Fellow Applications are being accepted for one position. A position is available to develop Cys2His2 zinc finger proteins (ZFPs) for the treatment of monogenic disorders. This project will focus on the creation of ZFPs with novel DNA-binding specificity to recognize sequence elements within genomic loci of therapeutic interest. In collaboration with other laboratories, we will evaluate the activity of these ZFP-based gene therapy reagents in vitro and in vivo in relevant disease models. Applicants will also have the opportunity to learn to apply Cas9 and Cas12a editing systems in vivo.
Potential Rotation Projects Improving the Precision of CRISPR/Cas9 nucleases for gene therapy applications: We are working to improve the precision of the CRISPR/Cas9 system to generate nucleases that will cleave at only a single site in the genome. These engineering efforts focus on increasing the DNA-editing precision of Cas9 and using protein engineering to introduce new properties into the nuclease. Much of this work is now transitioning to the use of Cas9 (and Cas12a) protein-RNA complexes. These modified nucleases will then be applied to patient derived cell-culture systems for the targeted repair or inactivation of disease-causing alleles. With the eventual goal of creating therapeutics for Sickle Cell Disease, beta-Thalassemia, HIV, Limb Girdle Muscular Dystrophy, Hermansky Pudlak Syndrome and other monogenic disorders. ex vivo genome editing in CD34+ HSPCs: We are developing improved Cas9 proteins for delivery ex vivo into CD34+ HSPCs for therapeutic application to sickle cell disease and beta-thalassemia. The goal is to modify the hematopoietic stem cells of a patient to complement the loss of function of the beta-globin gene and then return these cells to the patient through an autologous transplant. Development of Cys2His2 Zinc fingers proteins (ZFPs) as targeted therapeutics: We are developing artificial ZFPs for the regulation of target genes to change their gene expression profiles for therapeutic applications.
Potential Rotation Projects A variety of rotation projects are available to study RNP assembly and post-transcriptional regulation during development using quantitative biochemical and modern molecular methods. Project 1: Characterization of the RNA-protein complexes that guide C. elegans early development. Several projects ranging in scope from biochemical characterization of RNA-binding protein complexes to genetic dissection of RNA binding protein function in development are currently available. Contact Sean for more details. Project 2: Post-transcriptional regulation of oligodendrocyte differentiation. Projects are available to monitor changes in gene expression as a function of differentiation with a focus on changes in alternative splicing and mRNA stability. Contact Sean for more details.
Potential Rotation Projects <p >Our laboratory combines genetic and biochemical approaches to investigate the molecular underpinnings of adaptation. Potential rotation projects are available in two general (and partially overlapping) areas: exploring fitness landscapes through systematic approaches and investigating the molecular mechanism of the Hsp90 chaperone in signal transduction. Please contact the lab to discuss specific rotation projects in more detail.
Biochemistry and Molecular Biotechnology