My Summer Undergraduate Research Experience

1. My Summer Undergraduate Research Experience

2. Kinetics of Thyroid Hormone Receptorα Nuclear Import Xenopus laevis

3. 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

4. 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

5. QUESTION • Why, if TR can enter the nucleus by an energy-independent process it has also evolved to follow a signal-mediated process. • HYPOTHESIS: • Signal-mediated import will occur more rapidly than passive diffusion.

6. Xenopus laevis • Surgically remove Xenopus oocytes.

7. Manually microinject radioactively labeled TR into the cytoplasm. Vegetal Pole Inject 35S-TR Animal Pole (where nucleus resides)

8. 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. 20 °C 4°C

9. 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

10. Autoradiogram 4° Nuc Experiment incubated for 2.5 hours. 20° Cyto 20° Nuc 4° Cyto Oocytes incubated at 20° C have a noticeably greater amount of TR in the nucleus as compared to oocytes at 4° C. 35S-TRα

11. General trend of increased nuclear localization with time. • Oocytes at 20°C consistently had a greater %Nuclear TR than 4°C.

12. 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.

13. 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.

14. 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.