Tag Archives: UGSRF

Frozen In Time: The Story of a Tree Frog and its Potential to Help Humans Freeze Their Organs, and Possibly Themselves

I know what you’re thinking. What in the world does a tree frog have to do with humans freezing their organs? And yes, before beginning my undergraduate research project, I too would likely be thinking this exact same thought. But nature allows animals to do a very funny thing: adapt according to their environment. And the gray tree frog that you are itching to read about in the following paragraphs has adapted in one of the most extreme ways found in the animal kingdom: it has the ability to freeze itself and survive.

Credit: John White, University of California, Berkeley

My 2017 summer research project, funded through the American Physiological Society, is focused on Cope’s gray tree frog, Hyla chrysoscelis. H. chrysoscelis has developed a dramatic strategy to combat the cold during the winter months, known as freeze tolerance. Freeze tolerance permits this tree frog to withstand the cold by allowing it to convert 50-65% of its total fluid body water into ice. During its time spent frozen, Cope’s gray tree frog experiences fundamental changes in its physiology and biochemistry, including the abandonment of:

  • Blood circulation
  • Breathing
  • Nerve conduction
  • Brain activity
  • A heartbeat

Yes, you read that right. It has no heartbeat while it is frozen.

However, as wintertime comes to a close and external temperatures rise, the thawing process begins, vital signs reactivate, and normal life resumes for this frog. My research project for the summer is aimed at understanding a specific cellular process involved in the whole body freezing of H. chrysoscelis, which may provide future scientists with the capacity to not only freeze human organs in the future, but also freeze our entire bodies.

H. chrysoscelis experiences a variety of stresses during freezing and thawing, most of them being osmotic stresses. This is because the majority of the fluid surrounding the frog’s cells freezes, leaving a higher concentration of solutes outside the cell than prior to freezing. To combat this disruption of fluid homeostasis, H. chrysoscelis has evolved a transmembrane protein known as aquaglyceroporin HC-3 that acts as a channel for water and solutes to flow through, which helps to re-establish a fluid homeostatic equilibrium. In non-science terms, this protein, HC-3, helps to keep the cells of this frog from shriveling up like raisins or bursting open like a balloon with too much air.

My hypothesis demands a week long experiment, involving a variety of techniques that enable me to begin with the blood of this frog, fresh from its brachial artery, and shrink all the way down to the molecular level to analyze HC-3, the protein that I suspect to be a vital player in the freezing process. The first part of my experiment requires culturing red blood cells from Hyla chrysoscelis over a period of 48 hours, which in non-science terms translates to: keep 90% of these cells alive over a period of two days. Sounds simple, right? Well, after the first two weeks of failed attempts in maintaining proper viability of the cells in culture, I was left devastated, demoralized, and with numerous flasks of cells contaminated with who-knows-what. Science, without mercy, and in just a period of two weeks, showed me just how difficult it would be to successfully complete my week long experiment by halting me after just the first two days.

As the summer has progressed, I have finally achieved viable cell cultures for the full 48 hours through troubleshooting and controlling for one variable at a time, leading me to the conclusion that a certain reagent I was adding to the media my cells were kept in had been contaminated earlier in the summer. Currently, I am now focusing on more demanding techniques, such as western blotting and immunocytochemistry, while still always encountering new obstacles to overcome and problems to solve. New to research, and even newer to researching full time, I have come to enjoy the independence and autonomy that I’ve found in the laboratory. Every day is a new opportunity, and a new challenge, demanding my full attention and effort to successfully complete my experiments for the day. Through the few successes and many failures of my summer of research thus far, I have gained a deeper appreciation for science and continue to aspire to engage in scientific research throughout the rest of my professional career.

References

  1. White, John. “Hyla chrysoscelis, Cope’s Gray tree frog.” University of California, Berkley Regents (2006).
Dante Pezzutti is a rising senior majoring in Pre-Medicine at the University of Dayton in Dayton, Ohio. He is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Carissa Krane’s laboratory at U.D. over the summer. Dante’s fellowship is funded through the American Physiological Society. After graduation, Dante plans to attend medical school to become a physician, and also aspires to engage in bench-to-bedside translational research as a medical doctor.
Research Project Narrated for My Parents

My summer research project, which will span much of the next year, revolves around Classical Ferroportin Disease. Firstly, before explaining the minutia of Classical Ferroportin Disease it is important to know what ferroportin is; ferroportin is an iron transport protein that is responsible for exporting iron out of cells. Classical Ferroportin Disease is a loss of function disease, meaning that a mutation has occurred in the protein that makes ferroportin less efficient [1]. Seeing as though ferroportin is exporting iron out of cells, patients who have Classical Ferroportin disease can get iron into their cells, but their cells struggle to get iron back out of the cells. This ferroportin malfunction results in a net accumulation of iron in cells above what is physiologically healthy; macrophages of the liver and spleen become particularly iron overloaded because they metabolize a lot of iron. The disease can present clinically as anemia because the body is unable to acquire the adequate iron it needs for red blood cell production since the iron is being sequestered in the liver and spleen macrophages with no way to be properly exported [2]. The disease can also cause diseases of the liver such as fibrosis and cirrhosis because iron overload of liver cells is detrimental to liver health and functioning.

It is known that Classical Ferroportin exhibits autosomal dominant inheritance; my summer research project is interested in shedding light upon how Classical Ferroportin Disease is inherited via an autosomal dominant route. There are two models to explain the autosomal dominance that classical ferroportin disease exhibits. One model is a haploinsufficiency model, the other is a dominant-negative effect model [1]. These terms may sound complex but they are fundamentally simple. The haploinsufficiency model can simply be thought of in terms of dosage. When you make a cake the recipe may call for five eggs, if you were to only put one egg into the cake, said cake would turn out abysmally. Haploinsufficiency is exactly the same way; you inherit one copy of ferroportin from each parent. You need two good copies to be a well baked cake and make enough functional ferroportin protein, but if you only get one good copy and one mutant copy then you will be an abysmal cake with not enough functional ferroportin to properly export iron. All in all, in the haploinsufficiency model you don’t have enough functional ferroportin to keep up with the export rates needed to be healthy. The second model, the dominant-negative effect, is a little more complex but still fundamentally simpler than the name implies. This model is best envisioned as an arts and crafts project. Your task is to make a sphere out of two objects and super glue. THE TWO OBJECTS MUST COME TOGETHER TO CREATE THE PRODUCT. In the working art studio you’d be provided with two hemispheres and super glue; you would with ease construct the sphere and happily go about your day. In the art studio run by people lacking infant geometry knowledge, you are provided with one hemisphere, one cylindrical object, and super glue. No matter how hard you try with the materials provided you cannot construct the desired spherical object. Out of frustration you glue the block to the sphere permanently and leave full of rage to throw the mushroom shaped object at a wall.  The complete sphere, representing a healthy person without ferroportin disease, can be made when you are provided with two good hemispheres, one healthy ferroportin gene from each parent. The second art model, where the person is forced to construct a mushroom shaped object, represents Classical Ferroportin Disease via a dominant-negative effect. The person was forced to glue the hemisphere, a healthy ferroportin gene product, to a cylinder, a mutant ferroportin gene product, creating a non-sphere and thus a ferroportin protein unable to export iron. They key distinction between the haploinsufficiency model and the dominant-negative model is that in the haploinsufficiency diseased state you simply do not have enough of the good product, whereas in the dominant negative model the bad product hinders the good product from working all together. Overall my research project revolves around trying to figure out which model Classical Ferroportin Disease follows and my tools to do so are Xenopus laevis oocytes, human mRNA injections, confocal fluorescent microscopy, and scanning electron microscopy. This project falls into a huge sector of molecular biology and physiology that is cellular transport and it will aid in the treatment and understanding of one type of a family of diseases that fall under the name of Hemochromatosis.

Realities of Research

Experimental design is key; hard work pays handsomely in the form of pretty graphs; don’t start to genotype at 3 PM’ and experiments will be repeated a frustrating amount of times until those variance bars look pretty.

The importance of well thought out experimental design became a frustrating reality about 5 weeks into my project. I had been tirelessly performing experiments for weeks in a way that could only be analyzed via a one-way ANOVA. An ANOVA is a commonly used statistical test used to analyze data; all statistical tests require an individualized experimental design in order to be analyzed via a specific test. One day at lunch my PI and I were discussing my progress and he looks at me worriedly and says “How have you been setting up your experiments and how have you been analyzing the data?” He then went on to inform me that my experimental design had a critical weak point and that a one-way ANOVA would not provide the insight needed to make a strong claim on my hypothesis. So the rest of the lunch was spent redesigning my experimental approach so that I could run a two-way ANOVA, yet just another statistical test. This taught me that experimental design is worth troubling your mind over for a long while because good data is meaningless unless it is masterfully collected with a specific statistical analysis in mind.

A lab reality surprise I encountered was how rewarding good data and graphs can be. I grew up playing baseball and soccer and I excelled at them, therefore I know the feeling of hitting a homerun and scoring a goal. They are full body highs encompassed with an adrenaline surge and confidence boost. I was surprised how close to those feelings I got when I got my first very usable data set and graphs. It’s very comparable to sports because you train in much the same methodical way for so long so that when you finally see results it feels amazing. Hard work does in fact pay handsomely with good data and graphs.

Another lab reality is planning your day saves your dinner date. I learned this the hard way multiple times when I started an experiment, genotyping, or prep way too late in the day.  It has become increasingly apparent that planning is your best friend in the lab. Fifteen minutes of thoughtful planning at the start of the day may save you hours each day. AKA don’t hold off on very pressing genotyping until 3 PM unless you plan to be in the lab until 9 PM.

Lastly, a frustrating lab reality is that some experiments will have to be repeated so many times you that start to worry you’ll be running the same experiment with the same experimental groups forever, or even worse you fear they will never give you interpretable data…. gasp. This is a frustrating reality that can never truly be realized until you have suffered first-hand through running the same experiment for weeks on end. It always gets better though; see baseball homerun feelings above.

Life of a Scientist

The most striking part of the day to day of a scientist is how social it is. I always assumed scientists were reclusive and withdrawn. I was wrong, outright wrong. The lab I work in is bustling with life. Intellectual exchange and idea bouncing occurs incessantly as we pipet about running assays. We come together at lunch and effortless fill the air with debate and jovial disputes. But the chit chat goes beyond academics, which I was most surprised about. Beyond bouncing ideas off each other and discussing relevant academic papers, a plethora of enticing conversations about extracurricular activities also buzz loud. We talk about music, the best coffee places, the trendiest foods, sports, and philosophy. Science is inescapably social in a beautiful way. Another thing I love about being a scientist is that your schedule is decently malleable as long as you get plan well. You are able to catch lunch with a friend or leave a little early one day if you make up for that time and work somewhere else; I am trying to get at the fact that science is not a strict 9-5 because as long as you get your work done no one is going to hound you about when you do it. This is a huge plus because it plays very well into an adequate work-life balance.

References

  1. Hal Drakesmith, Elizabeta Nemeth, Tomas Ganz, Ironing out Ferroportin, Cell Metabolism, Volume 22, Issue 5, 2015, Pages 777-787
  2. Mayr, Roman et al. “Ferroportin Disease: A Systematic Meta-Analysis of Clinical and Molecular Findings.” Journal of Hepatology5-3, 2010
John Bonamer is a senior majoring in Medical Sciences at the University of Cincinnati in Cincinnati, Ohio. He is a UGSRF fellow working in Dr. Bryan Mackenzie’s lab at the University of Cincinnati. John’s Fellowship is funded by the APS. After graduation John plans to pursue an MD/PhD with special interest in Urology.
My Summer Experience Observing Changes to Sarcomeres with Titin Regulation

Muscles are comprised of repeating units called sarcomeres, which can be seen as bands in myofibrils. Prior research has shown that the consistency of the lengths of these bands correlates to muscle strength – the more consistent the bands, the stronger the muscle. My project focuses on the relationship between the consistency of these sarcomeres and titin, the longest known protein which is proposed to maintain sarcomere length. Our lab developed an inducible skeletal muscle specific Bmal1 knockout mouse that demonstrates a to a longer titin isoform. By altering titin and comparing the variance of lengths of these sarcomeres to a control group, I hope to demonstrate a correlation between sarcomere length and the expression of titin. My project takes a histological approach to study changes in sarcomere length and A-band centrality in the skeletal muscle by staining for alpha actinin and myosin. These images are overlaid and peak fluorescence is measured. Knowledge from this experiment would contribute to knowledge about titin and could eventually lead to more targeted drugs or therapies. Results of my experiment thus far indicate that there is a trend toward a longer sarcomere length and no significant change in variance, but there are still other stains and trials I would like to do to strengthen my findings. If my hypothesis is supported then my project would support the assertion that titin has a structural role in the sarcomere.

The myosin stain allowed me to see if there were any differences in the location in the A-band in relation to the Z-line.

The alpha actinin stain labels the z-lines, allowing the sarcomere to be visualized and measured.

Realities of Research

Research in Dr. Esser’s lab was very different from what I had anticipated. Generally, people think of scientific research as solitary, but what I have found is there is a lot of collaboration in academic research. When we encountered an error in the RNA sequencing program of the model, and we reached out to other people at the university in order to help understand and correct this error. Additionally, we borrowed equipment such as the cryostat (a machine used to cut muscle at cold temperatures) and microscopes from other labs. Lab meetings were also a useful part of my experience because they allowed for brainstorming when I faced challenges. In addition to the RNA sequencing obstacle, there was also a problem with a control antibody stain because there was fluorescence in an unexpected region. After discussing the problem, we came up with the hypothesis that the secondary stain for one of the regions was attaching to the conjugated antibody of another region, which was why we were seeing the unexpected fluorescence. To correct this, I applied the antibodies in two steps instead of combining them. All of these obstacles were easier to overcome because I had people around to assist me, an invaluable asset that I did not expect.

Day-to-Day of a Scientist

I look forward to coming in to the lab because I like the challenge of overcoming the unforeseen obstacles. Designing ways to solve problems is definitely my favorite part of working in a lab. The production of ideas about how an experiment or procedure should work and testing the methods is exciting. While these obstacles can be a nice challenge, they can also become tedious. The problem with the RNA sequencing was especially frustrating because it was out of our hands. Despite these complications, my research was overall very enjoyable. I am very appreciative of the APS for allowing me to pursue my interests this summer, and would encourage others to do a program because the experience was very rewarding.

Joseph Mijares, also known as Robby, is a junior at the University of Florida in Gainesville, FL. He is grateful to be a 2017 Undergraduate Summer Research Fellow in Dr. Karyn Esser’s Lab at the University of Florida.  Robby plans on applying to several MD and MD-PhD programs after his junior year, with hopes of attending after graduation.
Amniotic Membrane Supplementation in Rotator Cuff Reconstruction

Amniotic membranes have been of recent inquiry in the surgical world because of their composition of stem cells. These stem cells can differentiate into the desired type of cells in the body. In this study, amniotic membranes were placed on the insertion of the rotator cuff muscles after rotator cuff surgery to see how this affected recovery time. In order to determine this, the strength of the individual muscle fibers was determined using the single fiber isometric strength method. This method involves pulling out a single muscle fiber from a bundle of fibers obtained from the rat, tying it onto the apparatus and exposing it to high levels of calcium in order to allow for a contraction to occur. After determining the cross-sectional area and the peak isometric force, the specific force (the force per cross-sectional area) can be calculated in order to compare the relative strength of fibers from different samples. This study consisted of four different groups; uninjured control (no surgery performed), control repair (no amniotic membrane supplementation), injury only (no rotator cuff surgery performed), and experimental repair (surgery with amniotic membrane supplementation). There were six fibers obtained from each rat sample with 8 rats per group totaling 192 fibers. Once the specific force is determined for every group, comparisons were made to see if the amniotic membrane supplementation helped restore the specific force of the rats 4 weeks post intervention. This is applicable to human rotator cuff surgeries because it could help patients recovering from this surgery and decrease the recovery time. This would allow for these individuals to return to their normal activities more quickly. In addition, this method can be used in multiple different surgery sites to help improve recovery time.

Realities of Research

This project got frustrating at times because of the variable conditions of the fibers. There were some bundles that contained large, healthy fibers and some that contained extremely small and frail fibers (size comparison can be seen in images 1 and 2). While we did not know what groups these fibers were a part of because of the double-blind format, it was possible to infer which groups these fibers were from. Another interesting thing about this study is that it took three weeks to train for this technique. It started with learning how to tie the minuscule ties used to tie the fibers and then learning how to tie them on a practice machine. We practiced on separate machines because of the high probability we would break the real apparatus if we started on that. However, once data collection started, around 12-18 fibers were run each day and real progress was made. It was also interesting to see the variability of specific forces in a given bundle. This is something that we were not expecting, but was most definitely present. Luckily, the research question never changed, but there were many times that we would have to re-run fibers due slippage, ripping or any other issues that presented themselves.

The difference in cross sectional area for given fibers from different rats.

 

 

 

 

 

 

 

 

 

Day-to-Day Life of a Scientist

Life as a researcher was much more relaxed than I initially thought it would be. The arrival and departure time for each day was variable. While it was expected that you completed all of the work for the day, this could be done earlier or later in the day. This made it very low stress and everyone in the lab was extremely friendly and fun to be around. The best part of the research was definitely at the end of data collection when we were able to compare the specific forces and see if the amniotic membrane supplementation was effective. However, the worst part was definitely the struggles that I had with a specific rats sample that took me three days to get 6 good fibers run. I also had a great time in the collaborative lab meeting that was help in August with the entire research team. This gave an amazing opportunity to share results from multiple aspects of the project and see how all of the data fits together.

Jeffrey Kepple is a senior at Gonzaga University in Spokane, WA. He is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Chris Mendias’ lab at the University of Michigan in Ann Arbor, MI.  After graduating, he plans on pursuing an MD/PhD.
It’s Getting Harder and Harder to Breathe! ~Maroon 5

This summer I have been hard at work in the Student Development Complex, here at Michigan Tech. It is a closed room with no windows, kind of odd for an exercise nut, but some of the best things are happening there! We have research going on testing the human body to its limits. Specifically, my research is testing the limits of our respiratory system during exercise. I believe that respiratory muscles are important in performance and if we can understand the limiting factors of the respiratory then we can help people with asthma, chronic obstructive pulmonary disease (COPD), and team and endurance athletes. I am taking members from our cross-country ski team and putting them through two arm cycling trials to failure, i.e. biking for your arms. One trial their whole body is 100% fresh, the second their respiratory muscles have been fatigued ~20%. During the trials, I track the time and all their respiratory and metabolic functions and compare them between the trials. I have had six people go through the tests and have seen a surprising decrease in time to failure when respiratory muscles were fatigued. Additionally, their breathing rate was low and volume high during the normal condition and it flipped during the fatigued condition, showing that when the muscles are already fatigued they work even harder trying to sustain performance. The next step in my study is finding out how and why this happens.

Tom Bye performing equipment tests while designing his protocol, Michigan Technological University

Here in the Upper Peninsula of Michigan it can get hot, for us at least. Our lab is air conditioned, unlike most buildings up here, so we can have a consistent climate for testing and it is the place to be somedays. Research in the lab is a blast! When I have subjects in for testing we keep the mood light and keep them determined with some tunes, conversation, and cheering. There are also pros and cons to the lab, because I have ski team commitments to come accomplish, my mornings arriving in the lab for set-up are as early as 6:15 AM and nights can be the same, ending late, crunching numbers or safety checking our laboratory. I have been learning a lot over the summer about different programs and technology we use in the lab. I’ve been fortunate enough to use ultrasound, metabolic carts, NIRS devices, and EMG. Currently my task at hand is to learn SigmaPlot, a graphing program that is Excel on steroids; I’m getting good at it! The main tests that I am running on subjects are maximum aerobic capacity and maximum upper-body exercise tolerance tests. Using flow meters I have also been able to test their basic respiratory function, one subject’s lungs could take in over 8 liters of air! A normal person his size would normally take in 5-6 liters of air. Our results are looking like we expect when we plot them out, but statistically only some are significant. The next step is to keep trucking on and see if the results stay consistent!

Tom Bye pushing a subject to their max during pilot data collection, Michigan Technological University

Our lab isn’t just me and my research host, we are a team of many undergraduate, graduate, and doctoral students! It is amazing to see how we transfer knowledge and share ideas with each other. It is great that I can go to a doctoral student (a goal of mine) and get info on schooling and my research. I have been in the lab a while now and teach some of the undergraduate and graduate students how the equipment works with their research project protocols. In a few weeks, we will have a testing blitz for my project and get five participants through five days of testing each! My worst experience so far is troubleshooting our metabolic carts and lab computer… It was supposed to take an hour and ended being the whole day. My favorite part of lab also surprises me too, it is the intensity that all the team members bring to the lab, but when we have a BBQ we are all relaxing and playing yard games.

Thomas Bye is a senior studying Exercise Science at Michigan Technological University in Houghton, MI. He is a research fellow in Dr. Steven Elmer’s laboratory at Michigan Technological University in Houghton, MI. He is supported by the American Physiology Society Undergraduate Summer Research Fellowship. After graduating, he plans on becoming a physical therapist and being involved with research in sports. Later in life, he would like to be a professor to pass on what he has learned.
Does Light Pollution Cause Heart Disease in Mice?

Light levels at night have significantly increased since industrialization1. During this same period of time, there has also been an increase in the incidence of heart disease. Previous studies have shown that light pollution, which is experimentally called dim light at night (DLAN), disrupts circadian rhythms1-2 and increases the likelihood for developing obesity2-3, but its connection to cardiovascular disease is not known2, 4. My experiments this summer determined if DLAN increased atherosclerosis the mouse aorta. I also analyzed eating behavior to confirm previous studies that DLAN causes a higher proportion of eating to occur during the day than normal2. My research is part of a larger project that focuses on disruption of daily rhythms and heart disease. The project will hopefully give new insight into possible causes of the global increase in heart disease. A greater understanding can lead to prevention and treatment of heart disease.

Dr. Pendergast often reminds everyone in our lab that results will not always support the hypothesis. However, she also says that unexpected or negative results are still relevant and can lead to new experiments. The first couple of rounds of my experiment were mainly just working out kinks in the experimental protocol such as adjusting light levels to set up the DLAN conditions. I also learned new techniques including genotyping using PCR, mouse care, analyzing eating behavior, and aorta dissection and cleaning. Preliminary eating behavior data show that exposure to DLAN may lead to eating at the wrong time of day in mice. The atherosclerosis data obtained so far has shown high levels of atherosclerosis in male mice exposed to DLAN, and normal levels in female mice. Although the sample number is still low, the results do seem to suggest that exposure to DLAN increases atherosclerosis in male mice.

Since I am doing research with mice, mouse care is an everyday task in my research experience. Using mice for experiments is exciting because they provide data simply by being housed in their light-tight boxes with food and water. Getting results and sharing them with the lab is one of my favorite parts of research because it always leads to a discussion of what the results mean and the future directions of the experiment. However, the analysis of this data, such as watching hours of videos of eating behavior, is not the most exciting process. Being part of a research lab is interesting because of the daily discoveries about animal physiology, which lead to thoughtful discussions about how they relate to everyday life. Overall, research has been a valuable experience for me that has strengthened my time management, leadership, and problem-solving skills.

References

  1. Fonken, L. K.; Aubrecht, T. G.; Melendez-Fernandez, O. H.; Weil, Z. M.; Nelson, R. J. Dim light at night disrupts molecular circadian rhythms and increases body weight. Journal of biological rhythms 28(4), 262-71, 2013.
  2. Fonken, L. K.; Workman, J. L.; Walton, J. C.; Weil, Z. M.; Morris, J. S.; Haim, A.; Nelson, R. J. Light at night increases body mass by shifting the time of food intake. Proceedings of the National Academy of Sciences of the United States of America, 107(43), 18664-9, 2010.
  3. McFadden, E.; Jones, M. E.; Schoemaker, M. J.; Ashworth, A.; Swerdlow, A. J. The relationship between obesity and exposure to light at night: cross-sectional analyses of over 100,000 women in the Breakthrough Generations Study. American journal of epidemiology, 180(3), 245-50, 2014.
  4. Morris, C. J.; Purvis, T. E.; Hu, K.; Scheer, F. A. Circadian misalignment increases cardiovascular disease risk factors in humans. Proceedings of the National Academy of Sciences of the United States of America, 113(10), E1402-11, 2016.
Robert Wendroth is a senior at the University of Kentucky in Lexington, KY, majoring in Chemistry and Biology. He is working at the University of Kentucky in Dr. Julie Pendergast’s lab. He is a 2017 UGSRF fellow, which is funded by the American Physiological Society. After graduation, Robert plans to attend medical school and become a physician who also performs research.
Stressed Rats and a Stressed Undergraduate

Chronic stress leads to a greater likelihood of the development of many conditions including post-traumatic stress disorder (PTSD), anxiety, irritable bowel syndrome (IBS), functional dyspepsia and other gastrointestinal (GI) dysfunction. This indicates a likely rearrangement of neural pathways and regulation, although the mechanisms of how this happens are not yet known. As an APS Undergraduate Summer Research Fellow, I worked for ten weeks under my research mentor Dr. R. Alberto Travagli studying the neurochemical oxytocin’s role in stress adaptation. My project focused on the regulation of oxytocin signals between the brain and GI tract under conditions of chronic stress in rats. In other words, I studied whether oxytocin has a different effect on the brain and gut of rats after they have been stressed.

Following a 5-day stress treatment on each rat, oxytocin was microinjected in the dorsal vagal complex (i.e. the brain area that directly signals the GI tract). The response to these injections on gastric tone and motility in two areas of the stomach were then recorded and analyzed. The research is still ongoing, but we hope to answer a few questions: How does the regulation of oxytocin change after stress adaptation? Does oxytocin work through different neural pathways after homotypic stress (i.e. same stress each day) or heterotypic stress (i.e. different stress each day)? Since females have a greater likelihood of developing GI disorders, do sex/estrogen levels affect the regulation of oxytocin under stress? Although we are still collecting data, I am very excited to see the results when completed and honored to participate in this research!

What surprised you most about working in the lab?

Upon starting this project, I was surprised by how much skill is required to complete the tasks at hand. Although the technology we use to inject oxytocin and record the gastric response is quite advanced, it can easily be faulted by a human mistake. For example, if I did not suture the sensors tight enough to the stomach, the responses were difficult to read and interpret. There was a huge learning curve to carrying out the research day-to-day and then it was another challenge to ensure I was as consistent as possible between animals. Additionally, I was surprised by how much my project changed between the beginning and end of the summer. For example, early on we injected a new pathway-blocker out of curiosity, expecting it to have little to no effect on oxytocin injections. Surprisingly, however, in one treatment group it seems to be blocking the effects of oxytocin. After, we used that pathway-blocker for every animal and its effects may be crucial to our final conclusions.

I am very grateful to the American Physiological Society for providing me this opportunity because it has made me realize how challenging a career as a basic research scientist is! This summer has exposed me to how exhausting, long, and physically demanding lab research can be. But, I love the big-picture parts of research; designing the experiments, analyzing the results, and adjusting when results are not as predicted. It is amazing to work on research that could be part of a bigger solution (i.e. understanding of anxiety/IBS/colonic pain), especially when you can collaborate with other researchers and pool data to come to even more conclusions within each study. However, I will admit that lab research is grueling work and, like the rats, I was a little stressed at times! I look forward to next year’s summer project so that I can experience translational or clinical research and gain a more holistic view of the research world.

Julia Zimmerman is a sophomore majoring in Neuroscience at Swarthmore College in Swarthmore, PA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. R. Alberto Travagli’s lab at Pennsylvania State University College of Medicine in Hershey, PA, funded by the APS. After graduation, Julia intends to pursue work as an MD-MPH, or MD-PhD, bridging basic research into the clinical world.
Understanding Muscle Force During Cyclic Movements: Does Titin Play a Role?

During cyclic every day movements, such as running, jumping, and walking, our muscles go through cycles of shortening and stretching. While there has been extensive research on muscle function for the last 50 years, there is no current muscle model that can accurately predict natural movements. For example, when active muscle is stretched, it produces more force than expected based on current theories of muscle contraction. Likewise, when active muscle shortens, it produces less force than predicted by current theories. For years, scientists have been measuring properties of muscles under highly controlled conditions. The classic force-velocity relationship shows that force generated by a muscle is inversely related to the velocity of the shortening. However, this relationship changes during natural, more life-like movements. Recent work suggests that for a given velocity, muscle force is higher during cyclic contractions than the traditional force-velocity relationship. My research investigates the role of the elastic protein titin in the force-velocity relationship measured under different conditions. Using a mouse model with a mutation in titin, I conducted in vitro muscle experiments to compare the force-velocity relationship in cyclic and controlled (isotonic) conditions. Hopefully, my results will shed light on titin’s role as a spring in active muscle. If titin truly does store energy like a spring, this could account for the extra force and lack of force in the stretch-shortening cycles. This research will allow us to better understand movement on a whole organism scale, which can prove quite useful in prosthetic design and bioengineering, for example.

Much like the active muscle, doing research in a lab goes through cycles, except instead of stretch-shortening cycles, it is periods of challenge and reward. Some days, you go into lab, collect great data, and leave feeling utterly fulfilled. However, other days, you go into lab and it seems as though you spent your entire day trouble-shooting. Mainly though, our experiments worked and we were able to collect useable data. We have yet to fully analyze our results, but preliminary results seem to support our expectations.

In general, I have found my lab group experience to be very similar to my experience with playing college soccer. Both activities involve a group of people working toward a common goal. While in soccer, your team is working together to win, in the lab, there are many scientists working together to uncover a truth. Collecting and analyzing data is a collaborative effort and, to me, that was the best part of summer research. Working as part of a lab team allows you the opportunity to constantly learn and build off of others. It teaches you to adapt, be open to new ideas, and to use your time efficiently. The worst part of day-to-day life in the lab, is that sometimes data collection does not go as planned and you need to figure out what went wrong.  However, this aspect doesn’t seem so bad when you have your lab team to help brainstorm.

Overall, my time in the lab has been an incredible experience. It has helped me grow as both an individual and as a scientist and has stimulated my interest in future research opportunities. It is an experience I would highly recommend to other undergraduate students!

Lindsay Piwinski attends Pitzer College in Claremont, CA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research with Drs. Jenna Monroy (Pitzer College) and Kiisa Nishikawa (Northern Arizona University, Flagstaff, AZ). She hopes to attend graduate school in the future and continue pursuing research.
Neural Networks of Hypertension

Chronic high blood pressure, also known as hypertension, affects one in every three adults in the United States and nearly 1 billion people worldwide. It has been shown that the hormone, Angiotensin II (ANG-II), acts within the brain to stimulate the sympathetic nervous system, leading to hypertension. A region in the brain that is particularly sensitive to ANG-II and is involved in hypertension is the subfornical organ (SFO). The SFO is connected by neural projections to the paraventricular nucleus of the hypothalamus (PVN), and this pathway has been suggested to be important in cardiovascular regulation. The hypothesis of my study is that endoplasmic reticulum (ER) stress in the SFO-PVN projecting neurons is a cause of ANG-II hypertension. To test my hypothesis, the study will be split into two portions. In the first set of experiments, we will use staining of brain tissue (immunohistochemistry) to evaluate whether infusion of ANG-II causes ER stress in SFO neurons that send projections to the PVN.  If we find ER stress in these neurons, we will use genetic approaches (CAV2-Cre-GFP and AAV2-Flex-GRP78) to inhibit ER stress and determine whether this also prevented ANG-II hypertension from developing.

What did you learn working in the lab?

Working in Dr. Young’s lab has been enlightening, exciting, and frustrating at times. There are many skills that take a great deal of practice to master that are required for studies like this one. For example, in order to perform Immunohistochemistry to identify markers of ER stress in the brains—a technique I learned this summer—I first had to learn to use a cryostat to precisely slice and collect the brains onto glass slides. I sliced dozens of practice brains before I mastered the skill and even now after weeks of practice, I still face challenges and make mistakes while performing this task. I also learned skills like suturing, small procedures, injections, bench work, and many others.

I very much enjoy the lab environment. It is great to have such a close-knit team that genuinely wants to help each other succeed. I wasn’t anticipating the level of team work involved in running a research lab, but that is truly the foundation. I would say that the friendships are the best part of working in a lab. The worst part of working in the lab is the waiting. For example, I surgically placed ANG-II osmotic pumps in the mice and then had to wait two weeks before taking the next step in my study. During that time, I practiced on the cryostat a lot, but there was still significant down time that I filled with tasks unrelated to my project such as helping other lab members with their work, cleaning, etc.

Melanie Judice is a senior majoring in Biology at the George Washington University in Washington, D.C.. She is an Undergraduate Summer Research Fellow (UGSRF) working in Dr. Colin Young’s lab at the George Washington University. Mary’s fellowship is funded by the American Physiological Society. After graduation, Melanie plans to continue research in the Young lab while pursuing entry into the medical field.
Platelet and Ovarian Cancer Cell Interactions

The Larson Lab focused on two main goals this summer. Firstly, we were interested in knowing if exosomes (or small vesicles that bud off of many kinds of cells) secreted from ovarian cancer cells interacted with platelets and altered the way in which the platelets behaved. We isolated platelets from blood samples and exosomes from cell culture dishes using differential centrifugation (multiple centrifugation rounds done at increasing speeds). These were allowed to interact with each other either on a tube or on fibrinogen to analyze both platelet activation and platelet spreading.  For our second research project, we tested isolated platelets from Native American and Caucasian subjects to see if there were differences in platelet activation levels.  Washed platelets from the subjects were exposed to 4 different chemicals (found abundantly in the body) at different concentrations to examine any differences in platelet activity. These experiments are both pertinent to biology: 1) exosomes are poorly understood, but have the capacity of becoming a biomarker for cancer patients, and 2) by determining differences in platelet activity between populations, drug treatments could be more specific to patients.

Realities of Research

Working in a laboratory is as exciting and tedious as it sounds. The first two weeks were the most challenging, since my team and             I were all new to the lab, and had to habituate to the fast learning pace. Things became easier as we kept practicing, learning, and perfecting our techniques. My lab partners and I learned how to isolate exosomes, prepare and run a western blot, draw blood, wash platelets, and reinforced our practice in confocal microscope usage and cell culture. Many aspects of research work startled me, such as how long testing 7 blood samples can take, or how efficient a group of 4 individuals can be if there is communication. What surprised me the most however, was the amount of trial and error one has to go through to get consistent and reliable results. We are still analyzing data, so I cannot state whether the results are significant yet, but the data acquired looks promising. Thanks to the Augustana Nursing department and Sanford Research, we were able to make our project work.

This summer I got a glimpse of what the life of a scientist is: it is unpredictable, rewarding (but frustrating at times!), and tedious. I am glad our team consisted of 4 members, and I am certainly grateful we got along well; it made work far easier. Even though everyone did a little bit of everything, we all “specialized” in a particular task. Sarah kept records of the cell cultures, Katelyn imaged platelets, Kirby ran the flow cytometer, and I took care of the platelet test runs with the 4 chemicals. The worst part was the statistical analysis. It is tedious, it is messy, and it is not as…interesting. The overall experience however enhanced my technical and reasoning skills, and taught me more about how to behave, guide, and make suggestions as a leader.

Tania Rodezno attends Augustana University in Sioux Falls, SD. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Mark Larson’s lab at Augustana University. After graduating, she plans to attend graduate school and seek a PhD in either immunology or microbiology.