Monthly Archives: August 2019

The Circadian Rhythm’s Role in the Kidney
Emilio Roig
Junior, Microbiology & Cell Science
University of Florida
2019 STRIDE Fellow

My Research Project

According to the Centers for Disease Control and Prevention (CDC), one out of every three people in U.S. is affected by high blood pressure, which is also known as hypertension. Hypertension is a serious health concern because it significantly increases the risk of heart disease, stroke and kidney damage. In healthy individuals, blood pressure dips at night, allowing the heart to experience a period of time in which it is not under significant stress. However, some individuals have been diagnosed with what is termed as “non-dipping” hypertension in which blood pressure is constantly elevated, putting them at greater risk for cardiovascular disease. The fluctuation of blood pressure between night and day is regulated by our body’s circadian clock. The circadian clock is the body’s intrinsic time keeper, telling us when to wake up, when to eat and when to sleep. At the molecular level, every cell in the body also contains its own clock, including kidney cells. To better understand the circadian contribution to blood pressure, my research project for the summer of 2019 has been focused on studying the role of Per1, is one of the main circadian regulators in the kidney. The kidneys are responsible for filtering blood and are directly involved in the control of blood pressure. By removing the circadian rhythm gene Per1 from a specific region of the kidney, its contribution to blood pressure can be determined by comparing it to normal individuals that have the Per1 gene. Our goal for this project was to demonstrate why some individuals develop hypertension or fail don’t experience the normal drop in blood pressure at rest. Understanding the mechanism behind why some people develop “non-dipping” hypertension could potentially lead to better cures and therapies, thereby lowering the risk of cardiovascular disease.

Realities of Research

Even though this was not my first time working in a lab, it was the first time that I began working full time. Five days a week, my day began at 9 a.m. and would finish at 5p.m. However, sometimes I would find myself in deep thought about my project beyond those hours. I learned quickly that research is taking a step out into the unknown, meaning taking time to truly understand the complexities of the body’s physiology. Often,the results of my experiments were unexpected and generated more questions than answers. Other times the experiments would simply fail; the first Western Blot I ever attempted was an adventure. By spending a large majority of time in the lab, I have gained a new appreciation for researchers. Being a researcher takes persistence, creativity and an open mind.

Life as a Scientist

My sheer curiosity about the world is what originally drove me to become involved in research as soon as I began college. The American Physiological Society gave me the opportunity to develop as a scientist, immersing me in the vast complexities of scientific phenomena. Science can often be frustrating because things don’t always go as planned. But the moment new discoveries are made, every failure along the way becomes irrelevant. Persistence took on a new meaning for me the moment I had begun trying my own experiments, and that’s the beauty of science. When something finally is successful, it can open a whole world of possibilities.

Emilio Roig is a junior majoring in microbiology and cell science at the University of Florida (UF), located in the city of Gainesville. He is a Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Michelle Gumz’s lab at the UF College of Medicine. His summer of research was funded by the American Physiological Society and through a grant from the National Heart, Lung, and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Emilio plans to pursue a career in medicine so that he can fulfill his dream of preventing and curing disease.

Blood Flow and Other Bodily Functions: An Investigation of Vascular Function and Endurance Sports
Andrea Rico
Junior, Health Sciences
University of Texas at El Paso
2019 STRIDE Fellow

My Research Project

My research project was focused on measuring the vascular function and rate of blood flow in arteries of the upper and lower body extremities using flow- mediated dilation (FMD) and plethysmography. We investigated the differences in vascular function on endurance sports that are upper-body predominant, lower- body predominant and mixed combination. FMD is an advanced test that uses ultrasound to measure dilation changes in the diameter of arteries, such as those in the forearm. This is a method to assess the endothelial vascular function in humans. Plethysmography measures changes in volume of blood in different extremities like the upper- or lower-body extremities. These changes are measured with blood pressure cuffs attached to a machine known as the plethysmograph. This test can dictate the amount of blood flowing through the limb and time where peak blood flow happens. It is highly effective when it is used to find changes caused by blood flow. An endurance sport is any sport that has prolonged periods of physical stress. Swimming, for example, combines both cardio and light strength exercises mostly in the upper body, which trains the body to use oxygen more efficiently. Cycling combines both cardio and light strength exercises mostly in the lower body, increasing leg strength and endurance. American football involves a lot of resistance training in both upper and lower extremities. Comparing vascular function and structure in these three sports can help to determine specific changes with training modalities.

Realities of Research

This is my first time working in a lab and my first real research project, so it was pretty scary at first. However, as time passed, I started learning something new every day, including new techniques and skills. I slowly began to understand more about my project and its importance. It has been very exciting to be able to work on this project and being able to see the results.

Life as a Scientist

Working in a lab and being able to work with individuals who share the same passion has truly being an extraordinary experience. One of the greatest things that I personally have witnessed is seeing how all lab members collaborate with one another and help each other out. It has truly been an unforgettable experience to get to know everyone and share endless memories with one another. I love being part of a lab!

Andrea Rico is a junior at the University of Texas at El Paso majoring in health sciences. She is a 2019 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Gurovich’s lab. Andrea’s fellowship is funded by the American Physiological Society and through a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Andrea hopes to pursue a PhD in occupational therapy and work at a local hospital or practice.

2019 Summer of Science – ABC, PCOS, NAFLD the Summer Science Alphabet
Jessica Myer
Sophomore, Health Science
University of Missouri
2019 STRIDE Fellow

My Research Project

Infographic produced by the National Polycystic Ovarian Syndrome Association containing statistics about PCOS and its symptoms.

This summer I had the opportunity to be an American Physiological Society (APS) Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow and work alongside Dr. Stanley Andrisse in the endocrinology laboratories at Howard University and Georgetown University. Our labs study the mechanisms of polycystic ovarian syndrome (PCOS), non–alcoholic fatty liver disease (NAFLD) and insulin resistance. PCOS is the leading cause of infertility among women and affects many more women than statistics suggest. As a consequence of premature use of hormonal birth control, a large population of women may be unaware that they have symptoms of PCOS. In order for our mouse model to exhibit the symptoms of PCOS, we gave them low-dose testosterone and monitored them. NAFLD is a continuum of liver inflammation that inhibits the liver’s ability to process lipids normally, which causes fat accumulation. We induced NAFLD in our mouse model by feeding a high-fat diet for 30 days before tissue extraction. We were specifically looking at the mechanisms behind the lipid accumulation in hopes of discovering how therapies for the reversal of consequences are associated with insulin resistance, NAFLD and PCOS. The better understanding of the processes will be beneficial to combating obesity and the sister diagnoses that come along with it.

Realities of Research

Example of a protein assay, which is completed to determine the concentration of proteins in each sample.

There have been many parts of research that surprised me or were not as I expected. The biggest shock to me was how long it would take to complete one process. For example, running a Western Blot —the main technique I have been doing—takes an entire day for each step. Western blots are used to detect specific proteins in samples. The entire cycle for one blot takes a week, but thankfully I was able to work with four blots at a time. I was surprised at how relaxed the lab environment was, as there was a lot of down time while tests are being run, but there is always something to work on. In the lab, I learned many techniques that were used to discover protein concentrations, RNA concentrations, protein presence and so much more. As expected, the experiments had their ups and downs. We had some great weeks of data and some days where I would take an image and not get any significant results. Overall, I would say that we made great progress this summer. Most of our results have been as expected; although, when we cross a road bump, there are many tweaks we can make. We can increase the amount of sample in our Western Blots, increase the time we block the blots between antibodies, increase wash time or increase the concentration of antibodies. If none of those steps resolve the problem, we go back to published research to see what other scientists have done and how we might be able to learn from them. We never had to start over due to error, but we did complete an extraction during my last few weeks of research which was the beginning of the sampling process.  I thought it was so cool to see exactly where the samples come from and how they are obtained. The research question has not changed. In fact, it has become more focused as we gained more data for the control and knock out samples. Our research is ongoing and I am excited to see what the future holds.

Life as a Scientist

The day-to-day life of a scientist is very rewarding. It is exciting to go into work and be able to see changes and progress that are being made. I was surprised by the laid-back environment and the independence of it all. Once I was fully trained on a technique, I was able to run it on my own and also how to correct errors. I was impressed with how much I was able to multitask in the lab. One of the best parts of working in a lab was being able to see the data come together as publishable images and also images that I took was a great experience. The biggest adjustment for me was getting up so early, since I worked in the lab—across the city—starting at 7a.m. Although this seemed so early at the beginning of the summer, it turned out to be perfect time. I was able to manage well my schedule and had the late afternoons and nights to explore the wonderful city of Washington D.C. I accomplished so much in the lab as well as had a wonderful tourist experience. The worst part of this summer was ending my summer research experience and leading back to school! I loved being in the lab and working with Tina and Bobby and the other lab assistants. Tina is about to start her third year of medical school at Howard University and Bobby went to international medical school and is applying for his Master’s in Public Health.

References

PCOS Challenge Inc. (Ed.). (n.d.). What is PCOS? Retrieved from https://www.pcoschallenge.org/what-is-pcos/

Stewart, C. (2016, November 14). Pierce BCA Protein Assay Kit For Quantitative Total Protein. Retrieved from https://www.biocompare.com/Product-Reviews/239559-Pierce-BCA-Protein-Assay-Kit-for-quantitative-total-protein/

Jessie Myer is a sophomore majoring in health science at the University of Missouri in Columbia, Mo. She is a 2019 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Stanley Andrisse’s lab at the Howard University College of Medicine and Georgetown University Medical Center in Washington, D.C. Jessie’s fellowship is funded by the American Physiological Society and through a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Jessie plans to attend medical school and become a pediatric cardiologist.

Using CRISPR to Explain the ART of Artemisinin Analogs
Suhayl Khan
Senior, Health Science
Benedictine University
2019 STRIDE Fellow

My Research Project

Diagram showing how the CRISPR-Cas9 editing tool works.

Artemisinin is a drug derived from the Artemisia annua plant. It is known for its anti-malarial properties, but has also been found to have anti-cancer properties. The active portion of artemisinin is an oxygen-oxygen bond called an endoperoxide. When in contact with free iron in a cell, this endoperoxide breaks and creates oxygen radicals which are extremely reactive. These oxygen radicals then proceed to react with cellular components such as membranes and proteins which eventually leads to cell death. Previously, it had been found that DMR1 and HSM2—two analogs of artemisinin— are particularly effective in inducing cell death in cancer cell lines but not in normal cell lines. This summer, my lab and I worked on figuring out why this is so.

 

It has been found that cancer cells contain a higher iron concentration than normal cells. This higher iron concentration is due to higher concentrations of transferrin receptors—the receptor that transports iron into the cell— in cancer cells when compared to normal cells. We believe that the specificity of our artemisinin analogs to cancer cells is due to the higher concentration of iron in cancer cells. To test this, we planned to use Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR, a gene editing technique that can remove the transferrin receptor gene in lung cancer cells. Then, we would test our analogs on these transfected cells to determine if a lower iron concentration would show the analogs as ineffective. However, we were unable to test our analogs on the transfected cells because the transfected cells died three days after successful transfection. This proved to us that transferrin receptor is required for cell growth and the proliferation of cancer cells, and that cancer cells cannot survive with low iron concentrations. In the future, we plan on using CRISPR to overexpress the transferrin receptor gene in normal lung cells and testing our analogs on these cells to see if the specificity of our artemisinin analogs is indeed due to iron concentration within the cell.

Realities of Research

Cell culture flasks and media in a laminar airflow hood.

Doing research this summer has been very enlightening. In all honesty, before starting research, I imagined it to be a bit boring. I couldn’t see myself really enjoying sitting at a bench and waiting for experiments to run and cells to grow. Surprisingly, when doing research on a subject that you enjoy, it all becomes very exciting. I have learned so much about cell culture techniques and how to maintain a lab this summer. I found myself waiting in anticipation for an experiment to finish because I was so curious to know the results.  I couldn’t wait for cells to grow to large, usable percentages because I wanted to get the next experiment running. Admittedly, it was always disappointing when certain experiments didn’t go as planned or when a lengthy experiment needed to be done multiple times due to errors in previous runs. However, I have learned that even when experiments yield unexpected results, those results still contribute to the research we are conducting. It is not uncommon for an experiment to produce strange results that only make sense after hours of thinking “How could this have happened?” Fortunately, all data that we obtained this summer—expected and unexpected—contributed to my original hypothesis

Life as a Scientist

My day-to-day life as a scientist consisted of waking up early, getting to lab and checking on the cells. Every Monday, Wednesday and Friday the cells have to be fed. If they have grown exponentially, they needed to be split into a new flask. The cell media must be warm, so I had to turn the water bath on and place tubes of media in the bath well before I needed them. I checked the cells under a microscope and estimated the amount of cell growth of each individual flask. If a flask had less than 80% cell growth, the media needed to be discarded and replaced. If a flask had cell growth of 80% or above, then the cells needed to be removed from the current flask and placed into a new one to give them more room to grow. After feeding and splitting was completed, I met with my research mentor and discussed what needed to be done for the rest of the day. The biggest surprise about being a scientist was realizing how little I know about my field of research. Going into research, I believed that I had decent knowledge of physiology and biochemistry. Despite this, I spent every day learning something new and interesting about these fields. My favorite part about research is that there always seems to be more to do. Because of this, there was never a moment where I was bored with nothing to do. That being said, my least favorite part was that there were certain days where an experiment was particularly long and I found myself either overwhelmed with the amount of work to be done or exhausted by the amount of work I completed. Fortunately, working as part of a lab team took a huge amount of stress and burden off of my shoulders. It was very nice to have people to talk to and help me out whenever I need help with a task. Overall, life as a scientist is very rewarding and I have learned so much since I started research this summer.

Suhayl Khan is a senior majoring in health science at Benedictine University in Lisle, Ill. He is a 2019 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Jayashree Sarathy’s lab at Benedictine University. Suhayl’s fellowship is funded by the American Physiological Society and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Suhayl plans to pursue a Master of Healthcare Administration or Master of Public Health.