UConn Health Center: MMSB

Rotation Projects

Rotation Projects

Lab	Rotation Projects(s)
John Carson Room L3040 Phone (860) 679-2130 Email carson@neuron.uchc.e Lab Web Site http://barbaresecarson.uchc.edu/ Fall 2005 Spring 2006	The Carson lab is studying RNA trafficking in neural cells using single molecule imaging, systems analysis and computational modeling in the Virtual Cell. Rotation students in this lab have the opportunity to learn cell culture, protein chemistry, microinjection, confocal microscopy, fluorescence correlation spectroscopy and computational cell biology. Possible rotation projects in this lab: 1) characterizing dynamic molecular assemblies involved in RNA trafficking using photo-induced biotin transfer from hnRNP A2 to other molecular partners 2) defining conditional operators (EITHER OR, BOTH AND, ONLY IF) controlling molecular interactions in RNA trafficking using cross correlation FCS.
Asis Das Room L2012 Phone (860) 679-3405 Email adas@neuron.uchc.edu Fall 2005 Spring 2006	We are interested in the regulation of gene expression in bacteria with an emphasis on genetics and genomics. Rotation students are welcome in one of the following areas under investigation: 1. To elucidate the regulation of stress response in E. coli and Salmonella by the Gre factors, two conserved RNA polymerase binding proteins that are induced by several forms of stress. 2. To test the function of Gre factors and various two-component systems in the pathogenesis of model gram-negative organisms using C. elegans as a surrogate host. The basic approach for these studies is to create knock-out mutants through chromosomal ìrecombineeringî, investigate the patterns of gene expression using reporter gene fusions and molecular techniques, and deduce the phenotypic consequences of the mutants by a combination of physiological experiments and infections of worms as well as human cells in culture.
Henry Furneaux Room E5032 Phone (860) 679-2374 Email furneaux@nso.uchc.ed Fall 2005 Spring 2006	1.) Does mir-16 regulate the expression of vascular endothelial growth factor (VEGF ) mRNA ? Mir16 is a microRNA that has been found to be deleted in human tumors and is thought to play a role in the development of cancer. It is already known that mir-16 anneals to the AU-rich regulatory element in COX-2 mRNA and regulates its expression. We have mapped a similar regulatory element in the 3'UTR of VEGF mRNA, an important growth factor involved in angiogenesis. In this project, we will test the hypothesis that VEGF is regulated by mir-16 . 2.) The further design of RNA decoys that inhibit the action of microRNAs. We have synthesized RNA decoys that inhibit the action of the let-7 and mir16 microRNAs. In collaboration with Dharmacon , we are now in the process of designing decoys against all human microRNAs . Such inhibitors will provide an insight in to the mechanism of action of microRNAs and also provide new specific therapeutic reagents. 3.) Do somatic mutations in microRNA target sites contribute to tumorigenesis? From database mining we have observed that patients with androgen independent recurrant prostate cancer have mutations in a putative microRNA target site in androgen receptor mRNA. In this project we will test the hypothesis that these mutations result in the up regulation of androgen receptor expression and confer resistance to androgen ablation therapy. 4.) Does the fragile X protein play a role in the action of human microRNAs? Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein. Fragile X protein is known to bind to RNA. Studies in Drosophila have indicated that fragile X protein may play a role in the regulation of gene expression by microRNAs. In this project we will test that notion in human cells.
Jeffrey Hoch Room L3053 Phone (860) 679-3566 Email hoch@uchc.edu Lab Web Site http://structbio.uchc.edu/HochLab_files/index.html Fall 2005 Spring 2006	The role of water as a mediator of biochemical processes has been studied for more than a century, but many fundamental questions remain unanswered. Recent technological developments provide new opportunities to gain insight into long-standing questions, such as a molecular description of the hydrophobic effect and the influence of co-solutes on the properties of water. In light of the increasing awareness that the biological milieu is not a dilute solution, these questions are especially important for correlating biochemical studies in vitro with real biological processes. Potential rotation projects involve application of modern nuclear magnetic resonance (NMR), vapor pressure osmometry, and differential scanning calorimetry (DSC) to these problems. 1) More than 30 years ago, Irwin Kunz observed that the NMR spectra of frozen concentrated protein solutions exhibit an unexpected resonance from fluid water whose intensity is proportional to the amount of dissolved protein. Advances in the sensitivity of modern NMR spectrometers should now permit investigation of this phenomenon for dilute protein solutions. This could potentially provide an empirical method for quantifying the amount of solvent-exposed surface on a protein, and the extent to which it is modified by conformational change, molecular association, or co-solutes. 2) Recent developments in NMR pulse sequences have enabled the observation of the transfer of nuclear magnetization from the protons of water to protons on the surface of a protein. This has been used to investigate ìorderedî water molecules that interact for relatively long times with specific sites on protein surfaces. Preliminary observations in our lab indicate that nonspecific interactions involving water molecules that are not tightly associated with the protein can also be observed. The modulation of the effect by co-solutes provides a potential method for quantifying the influence of co-solutes on the magnitude of the hydrophobic effect. 3) We have constructed a novel high precision, high dynamic range vapor pressure osmometer (VPO) for biochemical studies. VPO is a means for directly measuring free energy changes in water, and with sufficiently high precision it should be possible to directly observe the effect of dissolved protein on the free energy of water. One project involves characterizing the achievable precision that can be attained based on recent engineering changes to the osmometer, and the influence of coatings applied to the sensor (which utilizes surface acoustic waves) as a potential means for improving sensitivity. Projects 1 and 2 are highly complimentary, and both also involve the use of DSC to determine protein stability.
Larry Klobutcher Room L3012 Phone (860) 679-2816 Email klobutcher@nso2.uchc Fall 2005 Spring 2006	Research in the laboratory currently focuses on the analysis of phagocytosis using the ciliate Tetrahymena thermophila as the model. In higher organisms, phagocytosis plays a major role in immune defense, but also serves as the entry process for a number of pathogens. Excellent genetic tools available in Tetrahymena make this a strong system for learning more about this multi-step, but poorly understood, process. Rotation projects related to any aspect of phagocytosis will be considered, but projects in the following two areas are now available: #1. Generation and analysis of mutants in genes encoding phagosome proteins. We are carrying out an analysis of the Tetrahymena phagosome proteome by mass spectrometry, and have already identified more than phagosome 30 proteins. Many of these proteins are of unknown function. As such, there are numerous genes for which knockout mutations can be generated and their effects on phagocytosis evaluated. Projects in this area would provide practical experience in molecular biology methods, microscopy, and cell biological techniques. #2. Isolation of Phagocytosis Mutants Using Antisense Ribosome Technology. A second project in the laboratory involves carrying out a screen to identify novel mutants in phagocytosis. Phagocytosis is non-essential when Tetrahymena is grown on nutrient medium, but is required when cells are grown using bacteria as a nutrient source. This allows for a screening strategy that will be used in conjunction with a ?mutational? process unique to Tetrahymena, antisense ribosomes. This project would provide practical experience in genetics, molecular biology, and microscopy.
Juris Ozols Room L3018 Phone (860) 679-2211 Email ozols@nso2.uchc.edu Fall 2005 Spring 2006	Project: Identification and degradation of short-lived proteins in mammalian endoplasmic reticulum (ER) mebrane. The degradation of short-lived membrane proteins is poorly understood. The half-life of ER proteins is quite variable. The mean half-life of an ER protein is approximately 2 days, but the turnover rate of the short-lived membrane proteins is quite rapid, less than few hours. It is generally accepted that degradation of these proteins is localized to the ER. The protease(s) involved and the mechanisms by which short ñlived proteins are selected and degraded in the ER are unknown. How the short-lived proteins are distinguished from misfolded or improperly assembled proteins remains to be determined. The ubiquitin-proteasome pathway is believed to degrade cytosolic proteins and is implicated in the disposal of improperly assembled or misfolded membrane proteins, however, attempts to implicate proteasome system in the degradation of short-lived ER proteins has not been successful. As a model protein for these studies, we are using the stearoyl-CoA desaturase (SCD). SCD is an short-lived, intrinsic membrane protein of the ER. It is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids (MUFAs). Although, MUFAs such as oleate are readily available in mammalian diets, for reasons unknown, dietary oleate fails to enter the subcellular compartment where oleate is incorporated into phospholipids and cholesterol esters. Studies with abj/abj mice which have a natural mutation in the SCD gene and the ablation of SCD activity, results in decreased triglyceride, and cholesterol ester synthesis leading to increased fatty acid oxidation,and reduced body adiposity. Also mice lackind SCD activity display resistance towards diet-induced obesity. Students who whish to examine the mechanism of degradation of SCD in the ER of hepatocytes, or other cell line membranes are more than welcome. Projects on the SCD protease inhibitors in hepatic ER and their effects on triglyceride and cholesterol ester synthesis in mice or rats are also available.
Peter Setlow Room L3010 Phone (860) 679-2607 Email setlow@nso2.uchc.edu Fall 2005 Spring 2006	1) Analysis of the mechanism of the uptake of a major spore small molecule during spore formation; 2) isolation and characterization of a mutation that increases the rate of spontaneous spore germination; 3) overexpression in spores of an enzyme that degrades the sporeís peptidoglycan cortex in the first min of spore germination, and analysis of the effect of this overexpression on spore stability and germination; 4) analysis of the effects of temperature and fatty acid composition on the permeability of the sporeís inner membrane, the barrier to entry of most small molecules into the sporeís protoplast or core; and 5) overexpression and purification of the B. subtilis transglutaminase that appears to be involved in crosslinking of specific proteins in the sporeís coats.
Sandra Weller Room L2034 Phone (860) 679-2310 Email weller@nso2.uchc.edu Fall 2005 Spring 2006	The role of the cellular DNA damage response in the Herpes Simplex Virus Life Cycle. It is becoming clear that viruses have evolved elaborate interactions with the cellular repair, recombination and checkpoint machinery in order to create an environment conducive to their own replication. The host cellís DNA damage machinery is alert for perturbations in DNA which could lead to genetic instability. After infection, some of this machinery is inactivated by the virus in attempt to remove obstacles to productive infection; however, other components are utilized by the virus to promote viral DNA replication. In this project, aspects of this fascinating cat and mouse game will be examined. Cleavage and Packaging of Herpes Simplex Virus genomes. Capsid assembly and genome encapsidation are critical aspects in the life cycle of any virus. Our goal is to gain a better understanding of the processes by which head to tail concatemeric DNA molecules are taken up into preassembled capsids. We have recently provided the first evidence that HSV capsids contain disulfide bonds which may be important for viral assembly and encapsidation. This rotation project will involve the introduction of mutations into the conserved cysteine residues of capsid proteins to determine whether proper disulfide bond formation is important for during infection.