Download Student Presentation to the W&M Board of Visitors

My Summer Undergraduate
Research Experience
Kinetics of Thyroid
Hormone Receptor α
Nuclear Import
Xenopus laevis
Background
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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
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Molecules that are ~50kDa can passively diffuse through the
NPC
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Energy-independent process
Does not require NPC interaction
Temperature-independent
Molecules can be actively transported through the NPC
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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
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QUESTION
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Why, if TR can enter the nucleus by an energyindependent process it has also evolved to follow a
signal-mediated process.
HYPOTHESIS:
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Signal-mediated import will occur more rapidly than
passive diffusion.
Xenopus laevis
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Surgically remove
Xenopus oocytes.
Manually microinject
radioactively labeled TR
into the cytoplasm.
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Vegetal
Pole
Inject 35S-TR
Animal Pole (where nucleus resides)
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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
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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
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Patience, Determination and Perseverance
Surrounded daily by people with the same interests
Perfect my public speaking skills.
Drastically different experience than classes
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Analyzing novel data vs. regurgitation of facts.
Hands on Experience- learn by doing.
Get to know Professors in the Biology Department
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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
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Next year I will attend medical school.
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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.
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Acknowledgements
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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.