One aspect of my internship this summer is a project that I will present to the lab staff in August. I have been informed that the only requirement of this project/presentation is relevancy to a current or prior research conducted by the HPL (Human Performance Lab). Because both heat related studies are ongoing, I have decided that, while I continue to help out with them, I will start focusing my attention on a recently concluded HPL study looking at the relationship between cortisol and obesity and start developing a presentation based on this research. Since this study has already been concluded and all samples have been collected, it presents the perfect opportunity to do a little original research (under Joel’s guidance). I expect more specifics next week but until then, I can tell you that we are going to be looking at GR (glucocorticoid recceptors) in plasma samples from the cortisol/obesity study. GR was proposed as a logical candidate for follow up research because it is an important cortisol receptor. Right now, our main objective is to isolate and characterize our GR receptors. The appropriate ELISA kits have been ordered and we started our first receptor isolation dry run today using a western blot to isolate and quantify nuclear ER (estrogen receptors) from blood I was able to get from a team member. (People are remarkably generous with their blood here). Hopefully I will be able to get a couple of western blot dry runs in before the actual kits arrive so I will be proficient enough to do them without Joel’s supervision. I said we started the western blot today because Joel told me that one of the best ways to reduce background is to incubate membrane and blocker (5% milk in this case) for 24hrs, which means we will be able to develop the membrane on Monday or Tuesday. The gel itself ran beautifully! Since we have been given the freedom to fine tune the protocol, we decided to try two methods of cell lysis: freeze thaw (3x) and sonication in order to break the ER free from the nuclear membrane. I must say the freeze thaws were particularly fun because we used dry ice to “snap” freeze the the samples after each thaw. And what a difference! the sonication lanes were almost clear while the freeze thaw lanes were muddles with cell debris! The transfer to the nitrocellulose membrane very quickly (7 minutes) using a new transfer machine although the gel marker lanes still has some visible bands, which might have indicated that not all the proteins were transferred to the nitrocellulose. We then submerged the membrane in milk for blocking in order to prevent non-specific binding of our antibodies and prepared the primary and secondary antibodies for development after the weekend. We are hoping that if we get clean results, we can use a similar protocol in the coming weeks with the new kits to isolate and quantify GR.

Other than working on the western blot, this weeks highlights included two presentations on the the effects of preventing the dimerization of GR in mice and my first real lab meeting, where everyone shared progress reports on whatever they had been individually working on this week and we discussed a protocol issue with the heat tolerance study. Apparently the ASCM guidelines meant to screen for diabetes mandate no food intake for 8hrs prior to the measuring of fasting glucose levels, a key biomarker collected prior to the VO2 max test. The issue, as I understood it, was that subjects’ max scores might be significantly influenced by this rule. In the end it was decided with the input of one of the lab physicians that fasting glucose levels could be accurately measured after only 4 hrs of fasting and that stressing our subjects to the fainting point was not necessary.

Every new intern is required to complete 5 successful sticks before being allowed to make blood draws from real subjects. My hope is that I will have all 5 by the end of next week so I can be the resident phlebotomist. The draw today went surprisingly smoothly, despite Josh’s very elusive veins. (Josh is an HPL research associate who also needs to get his sticks in). Because donating blood to inexperienced interns is supposed to be voluntary, its up to me to find some donors for next week. Thus far, I’ve lined up two other research associates and the HPL’s resident medic, the other Zach, who is a Navy Corpsman recently transferred to our lab from the Marines.

about to get stuck

returning the favor...

Where to start!? Its hard to believe but already my first week in sweltering Bethesda Maryland is coming to a close. After spending the first couple of days filling out endless paperwork and working on a few powerpoint presentations for my supervisor Stacey, I am beginning to feel the dust settle. Yesterday, I was given my very own desk and the only mac in the lab (since apparently very few people know how to use them in the lab, they have been deemed worthless and relegated to the ranks of the interns). I have since spent a significant amount of my time reading about the different assays I will be performing in the coming weeks. I will admit that, at first, the stack of manuals and background literature appeared somewhat daunting but has since turned out to be very interesting and I can’t wait to get my hands on a kit and actually run an ELISA (enzyme linked immunosorbent assay). The Interleukin-6 and Creatine Kinase assays are of particular interest because both are key plasma biomarkers that will be measured in the caffein heat study at several time points exertion under heat stress. I have been told that at some point in the next two weeks, I will help out with a multiplex bead assay, which is supposed to filter out different cytokines from plasma. Neat!

I should mention that one of the coolest things at the lab is the “Heat Chamber” where subjects in both the Exertional Heat Illness (EHI) study and the Rhabdomyolysis study spend a good deal of time on treadmills sweating more than they thought possible. The Heat Chamber is essentially a fancy sauna that can very precisely regulate temperature, humidity, UV exposure, and wind. The idea behind making subjects walk or jog for extended periods of time in the chamber is to simulate the environment of a Middle Eastern desert and thus get a more accurate idea of how their bodies would react to such an environment. A precursor to the heat chamber in both protocols is a VO2 Max test or aerobic fitness test. On Tuesday, I was able to take a break from registration paperwork and actually observe a Max test. Basically, subjects are outfitted with a mask that measures how much oxygen they are breathing in compared with how much CO2 they are breathing out, EKG leads, and a catheter to take regular blood samples. The subject then proceeds to run to exhaustion. If the subjects actually Maxes out, as determined by several criteria including their “exhaustion” O2/CO2 breathing ratio, lactate and glucose levels, and heart rate, then he/she is cleared to continue on the the heat chamber (not the same day of course).

Tomorrow I am supposed to spend all morning learning how to run the heat chamber and troubleshoot equipment so that I will be able to actually help out when we have a subject come in on next Tuesday.Tomorrow afternoon is to be spent with one of the lab fellows, Joel, who is from Birmingham England and will be teaching me DNA extraction using the DNeasy assay and how to quantify whatever I can get using spectroscopy. Tomorrow will also by my first day with a camera (I have been told everything except people is fair game) so hopefully I’ll have some pictures by next week.

Zach Smith ’13 received a Koshland Integrated Natural Sciences Center summer stipend to do research at the Human Performance Laboratory at Uniformed Services University.

Learn more about his project>