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My Summer Undergraduate Research Experience Kinetics of Thyroid Hormone Receptor α Nuclear Import Xenopus laevis Background Thyroid gland secretes two similar hormones (T3 and T4) that play a pivotal role in development and maturation. The thyroid hormone receptor (TR) is a nuclear protein which, in response to T3, promotes or represses gene transcription. Therefore, the mechanism regulating the subcellular distribution of TR is an extremely important topic. Thyroid Gland Nuclear Pore Complex Molecules that are ~50kDa can passively diffuse through the NPC Energy-independent process Does not require NPC interaction Temperature-independent Molecules can be actively transported through the NPC Energy-dependent process Requires interaction with the NPC or import receptor Temperature-dependent Cytoplasm Central Transporter Nucleus http://www.scripps.edu/~stoffler/proj/NPC/npc.html QUESTION Why, if TR can enter the nucleus by an energyindependent process it has also evolved to follow a signal-mediated process. HYPOTHESIS: Signal-mediated import will occur more rapidly than passive diffusion. Xenopus laevis Surgically remove Xenopus oocytes. Manually microinject radioactively labeled TR into the cytoplasm. Vegetal Pole Inject 35S-TR Animal Pole (where nucleus resides) Block signal mediated transport of TR by keeping oocytes at 4 °C. Compare the nuclear localization rate of TR with its rate in oocytes incubated at 20 °C. 4°C 20 °C At various time points, manually dissect nuclei from “cytoplasms” (enucleated oocytes). Separate proteins using SDS-polyacrylamide gel electrophoresis. Detect radioactive TR by exposing the dried gel to X-ray film. Cytoplasm Nucleus Autoradiogram Experiment incubated for 2.5 hours. Oocytes incubated at 20° C have a noticeably greater amount of TR in the nucleus as compared to oocytes at 4° C. 35S-TRα TRα Nuclear Import Kinetics 0.45 0.4 %TR Nuclear 0.35 0.3 0.25 20°C 0.2 4°C 0.15 0.1 0.05 0 0 0.5 1 1.5 2 2.5 3 3.5 Time (hours) General trend of increased nuclear localization with time. Oocytes at 20°C consistently had a greater %Nuclear TR than 4°C. Benefits of Research Patience, Determination and Perseverance Surrounded daily by people with the same interests Perfect my public speaking skills. Drastically different experience than classes Analyzing novel data vs. regurgitation of facts. Hands on Experience- learn by doing. Get to know Professors in the Biology Department Dr Allison and I are now best friends. Lab meeting twice a week with Dr. Shakes in the summer. Scientific ethics seminars with Dr. Heideman. My Future Plans Next year I will attend medical school. Applied to schools with excellent research programs. Hope to combine my affinity for research and medicine. Continue molecular biology research pertaining to human disorders. Get involved with clinical research. Acknowledgements Dr Allison for her expert advice, time and incredible patience. Funding from NSF and NIH for L.A.A. Supported by HHMI Summer Research Fellowship, HHMI Minor Research Grant, Minor Research Grant from the College of William and Mary.