Well today I got my poster (4×6 is much bigger than I had imagined) and it looks beautiful. Look for it at Haverford Summer Research Symposium Sept. 24. In other news, my presentation went very smoothly. I was presently surprised to have two Colonels in attendance! Afterwards, the lab had a little celebratory brunch which was delicious and another wonderful surprise.

What follows is a brief synopsis of my summer and a couple of acknowledgments.

Summer Research Experience Synopsis
Human Performance Lab
Uniformed Services University of the Health Sciences, Bethesda MD
Summer 2011

For the past 10 weeks, I have been interning in the Human Performance Lab at USUHS where I have had the privilege of training with world-class scientists working to understand the molecular mechanisms behind different health disparities and human performance injuries and illnesses. The experience, while challenging, gave me the opportunity to not only learn, but actively pursue important questions in the field of health disparities by putting what I had learned into practice.
I especially benefited from a close relationship with my supervisor who throughout my internship taught me how to perform countless new lab techniques (DNA extraction, enzyme-linked immunosorbet assays, western blotting, phlebotomy, and Chemiluminescence and UV analysis to name a few). While these new skills will surely prove valuable in the coming months and years, the opportunity to pursue original research during my 10 weeks was equally if not more exciting. Under the guidance of my supervisor, I designed and carried out my own research project examining the relationship between an important protein in the stress response cascade (the glucocorticoid receptor), BMI, and ethnicity. This project gave me the opportunity to learn first-hand how research is conducted from the design and implementation of the experiment to the analysis and presentation of data. It is worth mentioning that the opportunity to characterize a protein in a novel way and contribute something to the scientific community was particularly gratifying.
Looking back, I am grateful for the support of my supervisors and the KINSC steering committee who made it possible for me to ask important questions about the biochemistry underlying health disparities. I believe this experience of asking and then pursuing these questions was particularly valuable in my development as a researcher and something I fully expect to inform future research endeavors at Haverford and beyond.

Also special thanks to Jenny Punt for her support!

Last week at USUHS! Yes, Its hard to believe, but 10 weeks has flown by. After my supervisor made final edits to my poster yesterday, I submitted it to the Media office to be printed. [It is supposed to be ready on Friday, my last day, so fingers crossed everything comes though.] In addition to preparing a poster to bring back to Haverford to present at the Summer Research Symposium Sept. 24, I have been preparing a short presentation that I will be giving to everyone working in the Human Performance Lab on tomorrow. Basically all the summer interns present upon the conclusion of their internship.

Well the day has finally arrived. After a full day of running the sample (which turned out to have a few Caucasians mixed in) I have data. And not just any data! After running an ANOVA (analysis of variance) test with SPSS I can say that our results are significant and that they show Obese subjects expressed GR at a higher level than Overweight subjects and Overweight subjects expressed GR at a higher level than Normal subjects. The data is much cleaner than I anticipated because the samples were 3+ years old and in some cases had significant coagulation. Since the poster is basically prepared and I just need to play around with the data a little more, I will have time to try to find more correlations with GR levels. This afternoon, I correlated GR to ethnicity and found after an ANOVA test that ethnicity was not significantly related to GR expression. This was somewhat unexpected because African Americans tend to have higher levels of the ACTH hormone which is upstream of GR in the HPA feedback loop. If I have time, I would like to look at the relationship between GR and serum glucose. Other than that, I plan on spending my last week doing final edits on the poster and preparing to present it to the lab at our bi-weekly lab meeting next Thursday.

Yesterday, my long awaited ELISA kits finally arrived wrapped in ice packs leaving me with exactly two weeks to gather some data, analyze it, and past some data into my poster. At this point, we I am still reviewing/tweaking the protocol, but Joel and I have decided to give it a shot on monday. Fingers crossed!

Preparing for the GR ELISA aka labeling hundreds of tubes…

So this past week I was informed that the kits would arrive sometime next week leaving me with about 2 weeks to get some data and analyze it, which is not a lot of time at all but will have to do. In that light, I have spent the last several days doing everything I can so that when they actually do arrive, I can hit the ground running. I have organized all the sample into the appropriate subgroups [normal weight, overweight, and obese], prepared a workbench, and begun working on my poster. On Monday I will be adding PBS to all the samples and hopefully begin sonications. Other than that, I have mostly just spent my time helping out with data entry and screening subjects for the ongoing heat studies. Hopefully by the end of next week, I will have some data to talk about.

I am now officially past the five week mark and half way done with my internship! Time has definitely flown here at the Human Performance Lab and looking back over the last several weeks, I’m amazed at how much I’ve learned and how generous everyone at the lab has been with their time, expertise, and bodily fluids (see previous posts). As I’ve described in previous posts, most of my time thus far has been spent learning new techniques, helping out with the various protocols the lab is running, and working on my GR project. Despite now having generated any data at this point, I feel that I have been able to do a significant amount of background research, which has allowed me to write a comprehensive introduction to my project (minus my findings of course) and develop a pretty solid plan for when the ELISA kits arrive and I can actually start analyzing samples. As I had mentioned in a previous post, one concern had been that since the samples consisted of frozen buffy coat with lots of red blood cells mixed in, the hemoglobin that would be freed (thawing blood cells tends to hemolyze them) might interfere with the ELISA by binding to the GR antibodies. However, after a lengthy talk with manufacturer of the kit, I believe this shouldn’t be a problem. While I have yet to test this theory, I was assured that for whatever reason, the GR antibody complex would fluoresce at a unique wavelength making identification accurate possible.
In other news this week, I learned how to use the lab’s new human DNA isolation/quantification kit had the opportunity to test out some new sweat patches that the lab is trying to incorporate into one of the heat tolerance studies because they might offer a much less invasive way to measure caffein and chemokines than sticking subjects with a needle and taking blood. Basically, I just jogged for an hour in the heat chamber to ensure they had plenty of sweat to work with. Looking forward, Josh has assured me my kits are on the way and I will try to have my PI (primary investigator) take a look at my abstract and introduction drafts so they are ready all set when poster time comes around.

Who knew the military loved to party so much. This morning I walked into the lab only to be told I would be attending the Navy’s Hospital Corpsman Birthday! (I think they turned 131 or something) Anyways, there was lots of delicious cake and solemn ceremony and a good time was had by all.
In my last post, I mentioned that I had gotten a very weak signal for my western blot. After discussing it with my PI and a couple of post-docs, it was decided that I either didn’t have enough buffy coat (leukocytes), which make up less than 1% of blood by volume or I had hemolyzed the erythrocytes and made most of my white blood cells sink because they were now heavier. To overcome these challenges, I decided to try two different techniques. The first was to “snap” freeze fresh blood immediately after spinning in our centrifuge at 3000rmp for 15min and removing the plasma layer. The idea was that I could then use a scalpel and razor blade to scrap off the top layer of white blood cells leaving them relatively intact and free of any contamination from the erythrocytes. However, scraping this precious layer off proved much easier said than done because, despite being stored in a -80C freezer, it melted after about 15sec. The second technique yielded a much bigger and cleaner layer of white blood cells. In order to effectively separate the white blood cells from the red ones, I used a “Serum Separator Tube” which is essentially a vacutainer filled with some kind of goo that weighs less than red blood cells and more than plasma and white blood cells. The buffy coat layer was a little thicker than I expected (think slightly thicker than jello) and it remains to be seen whether it will yield a stronger signal than I got last weekend. However, while a strong signal would be very encouraging and show that the western is working properly, the samples I will be using for the GR ELISA have already been frozen and I don’t think it would be possible to transfer them to Serum Separator Tubes without hemolyzing them so I might have to come up with yet another technique that would allow me to extract the buffy coat relatively uncontaminated with red blood cells from frozen epi-tubes.
In other news, I finished my Lab Animal Medicine training so am now officially allowed to take sample from mice if I run out of human donors (I think I’ve stuck everybody at least once who works here) and I have begun to set up my own bench space where I will run the GR ELISA once it arrives and I have a reliable technique to extract buffy coat from the samples.

I titled this post practice/plan because this week I’ve basically spent all my time working on the western blots (dry runs for my GR assay) and starting to hone in on on the context I will be looking at GR in. I developed the first batch of blots on Monday and Tuesday using a new chemiluminescence technique that was pretty neat but yielded a very weak signal for both gels.

Incubating secondary antibodies for western

I plan to run at least two more practice gels, which will hopefully yield a stronger signal next week.
In other news, I’m finally starting to put together a plan for the rest of my time at HPL! Right now, its basically centered around a modest research project to correlate GR sensitivity/expression level to obesity and insulin resistance in African American (AA) subgroups. If I can find such a relationship, it would lend itself to a greater understanding of how HPA (hypothalamic-adrenal-pituary) dysregulation is related to insulin resistance and obesity since insulin is an antagonist to cortisol and both insulin resistance and obesity affect AA at higher rates than Caucasians (CA). This is essentially based on literature that says HPA dysregulation can result from chronic stress and AA experience disproportionate levels of chronic stress and insulin resistance. I am trying to see whether differential GR sensitivity can help link the two. In other words, can the hypothalamuses’ inability to respond to negative feedback inhibition be linked to insulin resistance through GR sensitivity. While GR sensitivity can be influenced by several things besides competing ligands such as insulin, I believe quantifying GR in different AA subgroups offers an excellent opportunity to discover a real relationship. Like I said in the last post, the GR kits are supposedly on the way so as soon as they arrive and I can prove to Joel my western technique is up to bar, things should start to happen. (Even though I will be using ELISAs to look at GR, westerns are similar enough in concept that Joel is letting me prove myself with them using estrogen receptors). If everything goes my way, I will be using a cohort of 100 AA, who will hopefully represent several subgroups (obese, normal weight, insulin resistant, chronically stressed..etc.)