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Blog Post #3
Blog Post #3
Content Reflection
Content Reflection
The Western blot procedure was unsuccessful in detecting amounts of p-Akt in the cells. Additionally, the Akt Western blot was unable to detect protein levels for the Insulin Control and 150 uM Carbaryl Treatment groups. I think these limitations originated from the small scale of my project; because I had to culture cells in such small culture dishes due to the expenses of my project, there were fewer cells per sample, and thus less amounts of proteins that could be detected.
However, I was able to draw conclusions based on the Akt blot and data from the Baseline Control and 350 uM Carbaryl Treatment groups. Through my research, I found that levels of regular Akt were significantly decreased after exposure to carbaryl compared to cells that were not exposed to carbaryl or insulin.
Thus, I was able to find that, when exposed to cells, carbaryl treatment correlates with a decrease in the production of essential proteins in the insulin signaling pathway. As a result, it negatively impacts the efficiency of the insulin signaling pathway.
While more specific research is required before officially concluding the restriction of carbaryl usage, my research suggests that insulin signaling can be negatively affected by carbaryl exposure, indicating that there may be a need for caution surrounding the use of carbaryl in areas of high mammal contact.
Final Product Reflection
Final Product Reflection
This was my first experience writing a full-length research paper independently, and I faced many challenges in staying self-motivated and gathering enough justification for every protocol, assumption, and conclusion I drew. However, I was able to learn a lot about how to find past research and protocols specific enough to support my resources and time constraints; skills that will undoubtedly help me as I continue pursuing scientific research in college (hopefully!).
The final presentation and oral defense, as nerve-wracking as it was building up to it, went smoother than I had expected; it gave me the confidence to realize that after conducting research, you essentially become the person who knows the most about it, and that helped me enjoy scientific presenting more.
I decided to continue with scientific presentations by presenting my research at the Colorado Wyoming Junior Academy of Science annual symposium. After a presentation and Q&A session, I won second place in my session and qualified for the American Junior Academy of Science Annual Conference!
Research Process Reflection
Research Process Reflection
Rather contrary to the phrase "it's about the journey, not the destination," I learned that it’s a lot easier for me to reach goals when I focus on the final results. Especially when I was in the middle of my project, making the time to go into the lab every day and sometimes on weekends to check on my cells, and passaging them several times a week, was getting a little frustrating. However, I was able to stay motivated by remembering that I wanted to finish the project, and also that I was gaining valuable experience in lab techniques; if I was feeling bored while culturing cells, I took that as a sign that I was becoming comfortable in the lab and getting experience that would really help me in the future. I hope to carry these lessons forward by reminding myself how to stay motivated in these kinds of independent projects.
Acknowledgements
Acknowledgements
I’d like to thank one of my most influential guiding mentors, Mr. Andrew Neumann. He is a PhD candidate at the University of Colorado Anschutz Medical Campus in the Prekeris Lab, and has helped me not only by helping me through roadblocks in this project and allowing me to use the lab space, but also providing me with guidance throughout high school. After first shadowing in his lab as a sophomore, I eventually had the opportunity to run the assay for my independent research project as a senior. I’m incredibly grateful for his mentorship and the opportunities he has given me throughout this process.
I would also like to thank my biotechnology teacher, Mrs. Petri, for allowing me to use the BSC and resources of her lab, as well as for the support she has provided along the way. Taking the biotechnology pathway courses has been one of the most influential experiences of my high school career; as my teacher for the past three years, Mrs. Petri has been a major part of fostering my passion for science and research. I truly appreciate the time, support, and confidence she has invested in me over the years.
Finally, I would like to thank the AP Research Program, led by Mrs. Dobos and Mr. McBride for the help they’ve provided throughout this year. From guiding me through the lengthy process of deciding on a feasible research project to providing feedback to me and my peers on our presentations, they were integral to keeping me on track and motivated throughout this process.
And of course, thank you to everyone who has read this blog and supported me throughout my research!
Final Project
Final Project
Abstract
In the U.S., approximately 4 million pounds of carbaryl-based pesticides are used annually in agriculture, with an additional million pounds used in residential settings. Despite widespread exposure, the effects of carbaryl on metabolic health remain poorly understood. While other pesticides have been studied extensively, limited research examines if carbaryl interferes with insulin signaling and potentially contributes to conditions like Type II diabetes. Previous studies suggest carbaryl may disrupt insulin receptors, but lack definitive conclusions. This study investigated whether carbaryl exposure alters the insulin signaling pathway in NIH-3T3 L1 cells. The final step of this pathway involves phosphorylation of the protein Akt. It was hypothesized that carbaryl exposure would significantly reduce phosphorylated Akt (p-Akt) levels compared to untreated cells. Fibroblast cells were seeded in 6-well plates and differentiated into adipocytes to express insulin receptors. Two control groups and two experimental groups were established. One control received no treatment, while the second received 100 µM insulin for 20 minutes. Experimental groups were exposed to either 150 nM or 350 nM carbaryl for 3 hours prior to insulin stimulation. Samples were collected using trypsin, snap-frozen, and analyzed using Western blotting for Akt and p-Akt with tubulin normalization. The p-Akt blot failed to detect phosphorylation. However, normalized results showed reduced total Akt levels in carbaryl-treated samples compared to the baseline control. These findings suggest carbaryl may affect earlier stages of signaling by altering protein expression. Future studies should use larger culture platforms to increase cell yield and protein recovery for more reliable quantification.
Keywords: Carbaryl, Carbamate pesticides, 3T3-L1 cells, cell culture, Adipocyte differentiation, Insulin signaling pathway, Western blot
Click this link to read my final paper!
Click this link to read my final paper!
Future Steps
Future Steps
This project was my first, but hopefully not last, experience with cell culture. This fall, I will go on to pursue Biomedical Engineering at the University of Southern California and potentially take the International Health, Development, & Social Justice Interdisciplinary minor. I hope to continue lab research, and I know I will continue to carry with me the technical and personal skills I developed throughout this experience!
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