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NSF-REU PROJECTS |
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Summaries
of individual projects are listed below alphabetically by
the mentor's last name If you have any questions you may contact the investigator on this list by clicking his/her name |
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Summary of individual projects
Role
of Tubulin, Kinesin and Dynein in Mitochondrial Translocation with Shear-stress
and Hypoxia
Abu-Bakr Al-Mehdi
, MD , Ph.D.
Department of Molecular and Cellular Pharmacology
.Mitochondrial responses to shear-stress and hypoxia in endothelial cells. Mitochondria are motile organelles that exhibit perinuclear, periparasitic, and peripheral clustering with a variety of stimuli, such as hypoxia, viral and parasitic infections, oocyte maturation and fertilization. We postulate that mitochondrial translocation is a response to increased demand for mitochondrial functions (ATP, reactive oxygen species, calcium homeostasis) in a subcellular region. Flow invokes a tangential force (shear-stress) activating the apical domain of endothelial cells resulting in increased demands for mitochondrial presence. On the other hand, hypoxia leads to increased nuclear transcriptional and chromatin remodeling activity requiring ATP. Currently, we are examining the effect of flow and hypoxia on mitochondrial translocation in endothelial cells and its dependence on microtubule based motor proteins, kinesin and dynein, by multidimensional live-cell fluorescence microscopy.
Protein
interactions in mitochondrial morphogenesis
Mikhail Alexeyev, Ph.D.
Department of Cell
Biology and Neurosciences
Mitochondria, formerly thought of simply as powerhouse of the cell, now begin to emerge as “omninvolved” organelles because of their critical involvement in apoptosis and cellular proliferation, as well as their roles in mitochondrial disease and cancer. Mitochondria are dynamic structures, which exist in a perpetual process of fission and fusion. This process is sustained by a balance of pro-fission and pro-fusion proteins. The former group is represented by dynamin-related protein (Drp1) and Fis1, and the latter group is represented by mitofusins Mfn1 and Mfn2. Disruption of this balance may lead to cell death (apoptosis). In fact, mitochondrial fission is one of characteristic features of apoptosis. Although phenotypes of cells that overexpress either pro-fusion or pro-fission proteins are well established, the sequences of protein-protein interactions which govern these processes remain largely enigmatic. The goal of this project is to delineate networks of proteins that interact to effect mitochondrial fission and fusion.
Enzymatic
Protection of the Vitamin Folate
June Ayling, Ph.D.
Department of Molecular and Cellular Pharmacology
One of the critical roles of the vitamin folate is to participate in the biosynthesis of several of the bases used to make nucleic acids. It is also needed to control the levels of the non-protein amino acid homocysteine, too much of which is considered to be toxic. In humans, deficiency of folate is associated with increased risk for stroke, Alzheimer’s disease, birth defects, and other disorders. Recent work in our laboratory as well as others is beginning to reveal that folate is subject to oxidative degradation within cells. The known function of the enzyme dihydrofolate reductase is to catalyze the conversion of 7,8-dihydrofolic acid to tetrahydrofolic acid (a physiologically active form of folate). Normally, this function acts together with the enzyme thymidylate synthase in the production of the DNA base thymidylate which generates dihydrofolic acid as a by-product. In this project the hypothesis will be tested that dihydrofolate reductase also plays a role in the protection of folate against degradation. The overall goal is to understand how an adequate balance of this essential nutrient is maintained.
Role of signaling
proteins in host-parasite interactions
Sailen Barik, Ph.D.
Department of Biochemistry and Molecular Biology
Our laboratory is focused on two major pathogens: Respiratory Syncytial Virus (RSV) and Toxoplasma gondii (T. gondii). While RSV is a negative-strand RNA virus of the Paramyxoviridae family, T. gondii is an Apicomplexan parasite. Together, they cause devastating diseases that claim millions of human lives throughout the world, including the US. In the last few years, through a combination of cell biology, molecular genetics and RNA interference (RNAi), we made major progress in understanding how these human pathogens interact with their host cells and cause the characteristic immunopathology. Our studies focus on the role of critical parasitic and cellular signalling proteins and the mechanism of interaction among them. It is now clear that such signaling events underlie various aspects of the disease process. An aspiring NSF student can choose from a number of ongoing projects, the major ones being: (i) Identify the proteins that function in the RNAi pathways of T. gondii, (ii) Determine the structure and function of Arogonaute, a key player in RNAi and the gene for which was recently identified by our laboratory; (iii) Determine the function and regulation of cytoskeletal proteins in RSV morphogenesis and host-RSV signaling; (iv) Characterize the structure and function of cellular chaperons in pathogenesis.
Developmental
Regulation of Heat Shock Protein Expression in Marine Invertebrates
Anne Boettcher, Ph.D.
Department of Biological Sciences
Developmental regulation of heat shock protein (HSP) expression has been reported for insect, plant, teleost, avian, and mammalian species. In many cases, significant changes in expression occur during metamorphosis and may be linked to cellular differentiation during this process. HSPs are molecular chaperones that aid in the proper folding of proteins and are known to play a role in normal cell processes and in response to environmental stress. Two types of HSPs have been identified, constitutively expressed forms which may support translocation of newly synthesized polypeptides and stress inducible forms which correct misfolded proteins in the cells. Despite reports from diverse taxonomic groups, little is known about developmental regulation of HSPs in marine invertebrates. The proposed study will examine variation in expression of HSPs during early development in several marine invertebrates. The impact of environmental stress on these expression patterns will also be examined. The REU student funded on this project would gain both field and laboratory experience. The primary techniques used include gel electrophoresis and immunoassays.
Proteins
required for acid resistance in Escherichia coli
John W. Foster, Ph.D.
Department of Microbiology and Immunology
How microbes cope with acid stress remains a fundamental question of biology with important medical significance. All intestinal pathogens encounter extreme acid stress upon ingestion, moving from the safe, neutral pH of food or water into the harsh, pH 2 milieu of the stomach. This is a five-log increase in acidity that destroys most bacteria and thereby protects the host from infection. Yet commensal and pathogenic strains of E. coli have developed elegantly regulated acid resistance mechanisms that enable survival in stomach acid. This project will focus on identifying proteins involved in providing that acid resistance. Mutagenic procedures will be used to identify mutants defective in acid resistance and the proteins involved will be identified by cloning. The project will then focus on potential protein-protein and protein-DNA interactions required to achieve acid resistance. The outcome of these studies will provide insight into bacterial physiology under extreme environmental stress.
Proteome analysis
by mass spectrometry
Lewis Pannell, Ph.D.
Mitchell Cancer Research Institute and Department of Biochemistry
The analysis of proteins is a complex procedure as they may be modified extensively from what is predicted by genetic analyses. In addition, the levels of protein found in systems does not necessarily follow that predicted by gene expression. The proteomics research facility develops methods for the rapid identification and analysis of proteins isolated from, or secreted by cells. This included methods to quantify the relative expression levels of proteins. The summer projects are focused on the improvement of proteomic technologies, especially with the use of mass spectrometry. Previous applicants have looked at post-translational modifications of proteins, methods for capturing secreted proteomes by growing cells in beads in suspension and antibody isolation for mass spectrometry analyses. While the equipment is housed in the Cancer Institute, projects and applications are applicable to multiple research fields and currently involve Biology and Chemistry Departments as well as the College of Medicine. In summer interns get a wide variety of training in a filed that is much in demand and are given their own research project(s) to pursue.
Protein
Interactions of Molecular Chaperones in Transcription Regulation
Rajeev Samant, Ph. D.
Mitchell Cancer Research Institute and Department of Pharmacology
We recently discovered that MRJ (a heat shock
protein/chaperone with J domain) binds to BRMS1, a protein which suppresses
metastasis (spread of cancer to distant sites) of human breast carcinoma. While
unraveling the mechanism this suppression phenomenon, we found. “J” domain is
implicated in regulation of the tumor suppressor gene retinoblastoma (RB). It
stops RB function leading to uncontrolled cell division and formation of tumor.
Since BRMS1 binds to MRJ, we propose that it stops MRJ from inhibiting RB
function, thus allowing proper cell cycle control. This may activate the
apoptosis (cell death) machinery, leading to destruction of cancer cells.
Objective:
Elucidate the members of the MRJ-BRMS1 protein complex and investigate its
role in metastasis suppression. The goals of this project are
1. Determine the members of RB-E2F pathway that bind to MRJ
2. Analyze the effects on cell cycle and apoptosis.
Approach:
Aim1:-Use FPLC (Fast Protein Liquid Chromatography) to isolate high
molecular weight protein complexes containing MRJ.
Establish the identity of the proteins associated with MRJ and BRMS1 using Mass
spectrometry and Western blotting.
Aim 2: Cells constitutively expressing MRJ will be compared with the
parental lines for cell cycle using propidium iodide staining followed by flow
cytometric analysis. Apoptosis will also be evaluated by AnnexinV staining and
TUNEL
Proteomic analysis of host-pathogen interactions in
seagrass “wasting disease”
Tim Sherman, Ph.D.
Department of Biological Sciences
We are examining factors affecting susceptibility of two North American seagrass species to the “wasting disease” caused by the protist Labyrinthula. We have taken two approaches in that work. First is a “biochemical” approach to the study of the infection process. The focus of that work is on characterization of factors that regulate the synthesis of phenolics, biomolecules that have been suggested to influence susceptibility of the young seagrass leaves to attack by the protist. We have taken a second approach that utilizes transmission electron, light, and fluorescence microscopy supplemented with immunological and cytochemical techniques to examine the infection process at high spatial resolution. These two approaches have produced insights into the process by which seagrasses are infected and respond to that infection. It is clear from our work that the plant responds to the attack, but it is unclear as to whether the response is one of general stress, one of specific to the pathogen, or (more likely) some combination of the two. Currently we are expanding upon our previous work to look into host response to pathogen attack, as well as other sorts of stresses, at the level of gene expression via analysis of plant and pathogen proteomes.
Characterization of functions and roles of
carbon-starvation-inducible
proteins that regulate stress response pathways in Salmonella enterica
serovar Typhimurium
Michael Spector, Ph. D.
Department of Biomedical Sciences
Salmonella enterica serovars are Gram-negative enteric bacteria capable of sensing, responding to and surviving the numerous environmental stresses that are typically encountered throughout its life cycle. One such stress is the starvation for an available/utilizable carbon-energy (C) source, e.g., glucose. This bacterium responds to C-starvation by generating a starvation-stress response (SSR). The SSR results in a smaller hardier cell that is capable of resisting the long-term effects of C-starvation as well as exposure to other environmental stresses, i.e., extremes in temperature, pH or osmolarity and exposure to reactive oxygen or nitrogen intermediates and antimicrobial (poly)peptides. In addition, Salmonella serovars are able to mount more focused resistance responses that afford resistance to one or just a few stresses, e.g., heat-shock response. Our lab is focused on the identification of genes/proteins that function during and are required for the SSR as well as other stress responses in this bacterium. These include:
1. The sigma (transcription) factors encoded by the rpoS and rpoE genes, sigma S and sigma E, respectively, have both been found to be required in order to generate an optimal SSR and many other stress responses in these bacteria. Sigma factors direct RNA polymerases to specific promoter sites on the chromosome and therefore direct the transcription of specific sets of genes. Thus, identifying and characterizing genes under the control of one or both these sigma factors is an aim of our lab.
2. In addition, many stress responses are specifically controlled by two-component protein systems composed of a membrane situated sensor-(histidine) kinase and a cytoplasmic transcriptional regulator protein (the target of phosphorylation by the sensor-(histidine) kinase in response to the presence or absence of an environmental signal). One way to control the function of these two-component systems is to dephosphorylate the sensor-(histidine) kinase before it can transfer a phosphate group to the transcriptional regulator protein. This can be accomplished by a phosphohistidine phosphatase. One such protein that preliminary evidence indicates is C-starvation-inducible and sigma E-regulated is the SixA phosphohistidine phosphatase. Preliminary evidence has suggested that SixA is needed for maximal survival during long-term C-starvation. Thus, further characterizing the function and role of SixA in the SSR and other stress responses along with identifying and characterizing potential pathways controlled by SixA activity is an aim of our lab.
Another mechanism used to control cellular physiology and stress responses in bacteria is through managing levels of certain intracytoplasmic signal molecules such as cyclic AMP (cAMP), guanosine tetraphosphate (ppGpp) and cyclic diguanylate (c-diGMP). The role of cAMP and ppGpp levels in the regulation of stress responses is well characterized, but the same cannot be said for c-diGMP levels. The levels of all these signal molecules can be controlled at both the level of synthesis and degradation. We have identified a gene cdgR encoding a c-diGMP phosphodiesterase (degrades c-diGMP to 2 GMP molecules) that preliminary results indicate is C-starvation-inducible and regulated by both sigma S and sigma E. This suggests that c-diGMP levels may play a role in the SSR. Thus, another aim of our lab is to identify and characterize genes/proteins/pathways that are influenced by c-diGMP levels and specifically in terms of their potential functions in the SSR and other stress responses.
Structure-function
analysis of squirrel monkey chorionic gonadotropin
Jonathan Scammell, Ph.D.
Department of Pharmacology
In higher mammals during early pregnancy the glycopeptide chorionic gonadotropin (CG) is secreted by the placenta. In squirrel monkeys and other New World primates, CG is also expressed in the pituitary gland and appears to substitute for luteinizing hormone in regulation of normal reproduction. Furthermore, based on prediction programs the C-terminus of CG in the squirrel monkey pituitary lacks some sequences that undergo O-linked glycosylation, a post-translational modification that can influence in vivo hormone stability. Rather, it is predicted based on sequence that New World monkey CG has gained a unique N-linked glycosylation site. A number of interesting questions arise from these findings. Among them, how is LH expression suppressed and CG expression turned on in the pituitary gland of New World primates? Has monkey CG lost O-linked glycosylation and gained N-linked glycosylation sites as prediction programs suggest? Is glycosylation of monkey CG the same in placenta and pituitary? What are the functional consequences of changes in glycosylation of monkey CG? These questions will be answered during the course of the NSF-REU.
The
Role of Nonpolar Interactions in the Mechanism of Action of Antifreeze Proteins
Andrzej Wierzbicki, Ph.D.
Department of Chemistry
Antifreeze proteins (AFPs) are highly specialized proteins which can protect select cold-surviving organisms from freezing by a non-colligative mechanism of the freezing point depression. This protective mechanism relies on the subtle functional design of these proteins which masterfully utilizes both polar and nonpolar residues to minimize the free energy at the water/ice interfacial region. Our objective is to study the structure-function relationships in the mechanism of action of the small a-helical proteins (Type I AFPs) in order to elucidate the importance of the contribution of nonpolar interactions to molecular recognition and binding. We will use quantum mechanical calculations and molecular dynamics simulations to study the various molecular interactions, both hydrophobic and hydrophilic, that influence the recognition and binding of AFPs at water/ice interfaces. The broader molecular mechanism by which AFPs inhibit the growth of ice and interact with phospholipid membranes will be also studied to elucidate how the AFPs may protect the living organisms from freezing. Based on our computational studies, we will design and experimentally test, using nanoliter osmometry, circular dichroism spectroscopy and nuclear magnetic resonance, functional AFP analogues to investigate the importance of nonpolar interactions to molecular recognition and binding at the water/ice interfaces. Since these studies deal with general aspects of functional protein design, protein folding and solubility, and interfacial adsorption, their impact extends beyond the field of antifreeze proteins.
