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Grace Wang, Weeks 2-3: New Techniques and Analyses

     During these past two weeks, I have been introduced to the various research projects my lab is working on. Luckily, the project is very similar to the primary literature I have read throughout the year, so I understood my postdoc's description of it. Our main project focuses on the research question: given that NF-κB signaling in muscle stem cells causes telomere shortening, what is the specific cause of the shortened telomeres? In the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway in muscle stem cells, spurred by growth factors and other stimuli, a phosphorylation cascade causes the transcription factor NF-κB to be released. Our lab has previously shown that NF-κB signaling causes telomere shortening, but we have repeated the experiment more meticulously to confirm these results and record clearer data. Elisia allowed me to prepare grids of telomere assays (muscle stem cells plated in wells) in which the telomeres were tagged with fluorescent probes. Since telomeres have the sequence 5'-TTAGGG-3', the fluorescent probes have the opposite sequence, 3'-AATCCC-5'. We later identified the telomeres by the fluorescence using imaging. I used an imaging software called ImageJ to analyze the lengths of the telomeres. 
Using ImageJ to analyze telomere length. Essentially, I would use the software to overlap images of the muscle stem cell nuclei and telomeres, each of which were stained. By using specific functions of the software, I could record the lengths of the telomeres in both the nuclei of injured and non-injured mice.
     
     As mentioned in my previous post, my lab is working in collaboration with the Veteran Affairs Medical Center and Penn's bioengineering department for a neurology project. Essentially, we are attempting to regenerate rat neurons in conjunction with skeletal muscle such that an injured rat's body can accept this transplant. Given that this project has many possible practical applications, we have lots of funding for it. This is why we use larger rats (with larger muscles!) instead of the tiny mice we usually use. The lab technician, or grad-student-to-be, David, taught me a technique called tissue fixing. I had to come to my lab early at 8:30 AM on Tuesday, June 12 to learn it, but it was well worth my time. Essentially, we place target muscles into cube-shaped molds and fill them with OCT (optimal cutting temperature) gel. We then freeze these gel cubes using dry ice and liquid nitrogen since they must be kept very cold (anywhere from -16 to -24 degrees Celsius!).
Fixing rat muscles into molds using OCT gel.
Freezing the molds using dry ice and liquid nitrogen to solidify the gel cubes
    
     I have received training to use the cryostat, a machine which facilitates the subsequent process of tissue sectioning. I watched David attach the fixed gel cubes to a black chamber on the back of the cryostat and use a lever to cut thin slices of the gel cubes. He then imprinted the thin slices onto a microscope slide. This technique enables us to analyze cross-sections of target muscles under a microscope. 
     While my second week was focused on learning and employing new techniques, I focused on analysis during my third. I have analyzed the areas of various skeletal muscle fibers for the neurology project in addition to telomere length analysis. Both can be tedious since they involve using the imaging software to trace muscle fibers and nuclei, but I'm grateful that I get to do a variety of work in the lab. Next week, our lab will most likely have results.
Using ImageJ to analyze the areas of muscle fibers in a cross-sectional image.
     
     Besides my lab work, I have found time to visit the nearby Franklin Institute with Srinidhi, which was really fun. It was so ethereal to see the Philadelphia skyline at night from the uppermost floors of the museum. I also thoroughly enjoyed eating birthday donuts with my lab, in addition to the cake I  shared with my family when I went home last weekend. Other than that, I will be pretty busy after work for the next few weeks because I have to study for the ACT. Luckily, since I have my PennCard ID now, I have access to the biomedical library, a quiet study space great for studying, Hopefully that will go well. While ACT work isn't very exciting, I'm nonetheless looking forward to spending the next month here in Philly. I'm especially excited to see the results from our experiments, maybe even as early as next week!
    

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