Student Research at Duke

I probably should’ve posted this earlier but I’ve been quite busy since coming home! I actually stayed an extra week to finish up some work on the project so Adrian could have some extra data to include in his paper. On my last day we all went to LocoPops to celebrate the end of my summer research. If you haven’t been to LocoPops, you should really go. I only wish Peggy could’ve been there but she’s training for a triathlon along Lake Superior.

I had a fantastic time at the poster session!!! I think it was one of the best experiences of my life. I really enjoyed explaining my project to others and surprisingly, I got to meet some people who were specifically interested in it. Dr. Di Giulio is a PI in the Nicholas School of Environment and he had a high school HH scholar who worked on how PCB acts through the AhR receptor to affect populations of killfish. This is the cool part; the student’s mother, Carole Lannon, is a pediatrician at the Cincinatti Children’s Hospital and she was working on a database for Hypoplastic Left Heart patients. She asked me to send her a copy of my poster pdf and I was really flattered. I was really impressed with the questions I got too! Dr. Truskey, a BME professor who was Racquel’s mentor, asked me if we thought there were more AhR receptors in the left ventricle than in the right. We had never really thought about it and now we’re thinking of doing westerns for AhR receptors.

I also got to meet Dean Scheirer who is the new Chief Prehealth Advisor and we talked about what career paths I’m looking into.

Anyway, I really enjoyed the poster session and I’m very thankful to everyone (Dean Nijhout, Alex, Suzanne, Ms. Wahl, Dr. Kirby) for giving me this opportunity. Mary (Hutson) asked me what I took away from this experience and I said that I learned that research can be fun. In my previous experiences, I definitely did not think so! I think I finally found what area of biology I’m interested in and I’m so glad. Having super nice lab mates is a plus too. I recently got a comment from a mother who had to terminate her pregnancy because she had a child with HLH who could not be saved. She was so encouraging and motivational and really made me realize how important research is. There are people waiting out there for us to keep on working.

 School starts in about a week so I need to start packing. I look forward to coming back to the Kirby lab in the fall!

Like most other college students, I’ve been asked numerous times what sort of career I picture for myself. In high school, I volunteered at a couple wet labs at the University of Michigan Medical School and officially worked for the first time at the University of Michigan School of Public Health in an epidemiology lab. Right from the start, I knew that I wanted a career in public health and epidemiology. I took Global Health (PubPol 154) my first semester here and absolutely loved it. This is a quote from an article done on the class: “Defining the typical student in Duke University’s first Global Health class is difficult: there were students studying medicine, nursing, public policy, biology and cultural anthropology; the class included undergraduates, graduates and professional students. It even included students who are also employees.” Basically, I thought it was fascinating to use an interdisciplinary approach in solving public health problems and apply the molecular aspects of infectious disease and chronic disease to the greater population. After all, context is everything; looking at health differences in populations truly emphasizes how much our phenotype is a combination of both genes and environment.

The best part about coming to Duke is knowing that the opportunities are endless so I love being able to keep my options open. That said, I have three main interests as of now (the first two from my Howard Hughes research): environmental health, pediatric cardiology, and international health.

The source of interest for my project in the Kirby lab comes from an epidemiological study that found close correlations between the presence of environmental toxins and abnormal heart development. It scared me to think that we can do the best to preserve our health yet suffer so much due to regulations that not everyone chooses to take seriously until a disaster or the general lack of conscience. In my Spanish 105 class last semester, we watched a documentary called “Maquilapolis,” which explored the negative effects of huge multinational assembly plants (maquiladoras) created in Mexico and Central America as a result of globalization. The children born to mothers working in these factories were often exposed to toxic chemicals while in the womb. While growing up, they would drink and bathe in the water with the run-off waste. Toxic spills in the US are usually due to mistakes and usually bring about heavy investigation, however, in places like Central America, things like this occur all the time and we somewhat devalue the lives of people living in these conditions by ignoring them.

I would love to work for the US Department of Health and Human Services, WHO, or the Natioinal Institute of Environmental Health Sciences (a branch of the NIH in the RTP) and look at public health policy, specifically dealing with environmental health in the US and other nations. I would also definitely want to have the medical expertise to treat diseases or conditions induced by environmental factors. I think it’s important that more MDs enter the world of policy since they can have great influence with their background. Working for these organizations and specializing in infectious disease or chronic disease epidemiology interests me, as well.

I am also very interested in doing research on congenital heart disease like I am now in the Kirby lab or doing pediatric cardiac surgery, since I’m familiar with the heart and shadowed some fantastic pediatric cardiologists and surgeons.

My Howard Hughes lab experience really helped me discover some of these interests and I hope to continue developing new interests with the help of new experiences in the next few years.

Right now, I’m working on optimizing our Westerns. Western blotting itself is not a difficult procedure, however, it’s difficult to cater to YOUR specific protein. The protocol can give you a set of directions to follow, but you must modify concentrations/times to best suit the nature of your protein or model from which it came.

Most Westerns tend to have nonspecific bands and some background because the antibody may recognize other proteins so it might look like this:

During optimization, our job is to reduce the number of nonspecific bands and background so that we can feel more confident about our target protein’s presence. A cleaner Western might look like this:

Adrian, the rockstar of the lab, did a great job targeting p53 levels in zebrafish so he has been able to help us out a lot. This is Adrian:

He just got his PhD so we’re all very happy for him. Anyway, Kyle and I have made progress in making these Westerns look better but we can still do a much better job. We’ve gotten E2F to work, but the cyclins are being very stubborn. It may just be that the antibody isn’t recognizing the zebrafish cyclins as well due to sequence differences. Tomorrow, I’ll be looking at Rb, or retinoblastoma, which is a suppressor.

When Rb is phosphorylated, E2F becomes an active transcription factor and cell cycle genes are expressed. We’re expecting to see lower levels of E2F in PCB fish (lighter bands on the westsern), however, Rb is a bit more complicated since it has two forms: phosphorylated and non-phosphorylated. Therefore, we might see two bands and must analyze it from there. Rb is not well-characterized in zebrafish at all. The closest protein we’ve been able to find is called Rb1, or retinoblastoma-associated protein, which is significantly smaller than the human Rb.

Kyle and I decided that we were looking at too many proteins at once. We can start doing that once we know that all of these antibodies work but right now, we’re going to focus on two proteins at a time and decide which ones have hope.

Aside from the zebrafish, the chick work is great! We might do Westerns on chick cell proteins as well…should be interesting.

Hey guys!! I’m pretty excited right now because in a few hours my lab is having a Fajita Fiesta on Harriett’s farm!! Anyway, I thought I should make an update about my research. It has been a very intense week in terms of work! I stayed at the lab pretty late most days but once you get into it, you just don’t feel like dropping everything and going home. The project has gotten significantly more interesting with the use of two animal models. The zebrafish is fantastic because it is transparent, breeds fast, and readily available. Nevertheless, it is SO small. We are using a strain of fish known as cmlc2s which have their hearts stained with Green Fluorescent Protein (GFP). They look so cool under the fluorescent microscope!!

We spent a good amount of time extracting the hearts for protein extraction. We have someone from Proteomics helping us with this part so it’s nice to have experts around. Protein work is probably the most tedious of genetic techniques. Although the concept of Western blots is simple (you buy an antibody that recognizes your protein and forms bands if it’s present), it’s actually very complicated and sensitive, especially with tiny proteins. Right now our problem is that we are getting a lot of background and too many non-specific bands, so the next few days will be a troubleshooting period for us. That’s why I’m really happy we have the chick model, as well. The chick cardiomyocytes have the potential to be a good model since we at least know how to culture them. Using a compound called BrdU, we can tell which cells are proliferating (the DNA of a dividing cell uptakes the BrdU and stains it green).

If we can show that there are less PCB-dosed cardiomyocytes than normal cardiomyocytes, it will tell us that the chick might be a good model for hypoplastic left heart as well. Still, it’s important that we don’t get too caught up in this while collecting data. To prevent bias during cell counting, we randomize the wells so that we don’t know if we’re looking at PCB cells or normal cells. I think it’s important that all researchers make sure they take these sorts of measures because it prevents them from affirming their hypotheses for comfort.

Hopefully, we will get some results after modifying the Western protocols for zebrafish protein. It would be great to have since Peggy (Dr. Kirby) is applying for an in-house grant next week and we can show our need for a 2-D gel. The title of this post is actually something my developmental psychology professor, Gary Feng, said.

I’ll post some pictures from the fiesta in the next post! take care

Many people put scientists and healthcare professionals on a pedestal. Why wouldn’t the public regard the people who alleviate human suffering to a higher standard? Hearing about scientific scandals such as the Poehlman scandal and the South Korean stem cell scandal scares the public; moreover, it reflects how the scientific community is just a microcosm of greater capitalist society: money is power and people in power will go to great lengths to keep it. What shocked me most about Poehlman was that he had been fabricating data for almost 10 years. This wasn’t a one-time thing; it wasn’t due to increased stress for ONE grant…a chunk of his career was a lie. Poehlman just wanted to stay in the cycle of publishing papers/obtaining grants, however, he failed to understand that life exists beyond this cycle. Every paper he published and all the findings he presented at conferences shaped peoples’ lives. Scientists such as Poehlman forget that the ultimate aim of their research is not to write a 10 page paper for an obscure journal that only scientists in that field to read. Furthermore, I love whistle-blowers. Nothing would’ve happened to this pseudo-scientist if it weren’t for DeNino, a young guy with guts and a conscience.

“And the Band Played on,” the movie about discovering AIDs, showed the negative impact of politics on science. EVERYTHING is about politics and it makes me sick! I would love to work for the NIH, WHO, or CDC in the future but these political battles are a daily part of working in those places. I loved listening to Colleen Denny, a research associate at the NIH Dept of Bioethics. Although she mentioned the balance between government involvement and research, I think it’s imperative the government get involved positively in order to solve public health issues. I cannot imagine the frustration of scientists and doctors like Don Francis who was doing his best to get more funding for AIDs research. I was just very angry after seeing the movie. Had we better controlled AIDs when it began, we wouldn’t be in this awful situation right now. With the current healthcare crisis in the US and abroad, I just hope people of our generation can ameliorate the situation. I would hate to leave so many problems for future generations to solve.

I hope everyone’s having a great week! It is WAY too hot outside. I step outside and I can’t breathe. Thank you, Michigan, for preparing me so well for this weather. What makes this unbearable condition worse is the occasional smoker. Second hand smoke and I do not get along. Of course there are people (the smokers) who think that people like me do not respect their choice. I’m sorry, kiddo, but it is NOT your choice to spread second hand smoke to innocent citizens, especially those poor babies and children who must deal with their irresponsible parents.  Duke Med just prohibited smoking in DUMC however I still see people smoking in the area. I just don’t understand how people working in healthcare or planning on doing so can do that to their bodies. They’re supposed to set an example. My 5th grade class had a really good DARE program so reality hit me a little hard. Lesson of the day: This reminds me of the Bodies exhibit I just went to on Sunday. It was phenomenal!!!! To see the human body and the organ systems as they are was just incredible. In one room, they had many of the normal organs and then the cancerous organs right next to it. Thanks to all the HHers doing cancer research because it does not look pretty and I’m pretty sure it feels a lot worse than it looks. In one display case, there were normal lungs and right beside it there were blackened lungs with cancer and emphysema. Then right beside that there was a glass box for people to throw away their cigarettes and there were a good number of boxes in there. Anyway, my favorite display had to be the one with the brain and then all the nerves extending from it. Then there was the display with JUST skin. yep SKIN. It looked like a rubber suit. Just seeing the body intact was very cool. Go see it at Southpoint if you haven’t already! So onto lab-related work…We’ve made great progress on the project. But first, I should tell everyone exactly what my question is.

generalHow does the toxin PCB affect heart development?

a little more specificHow does PCB induce abnormal heart development?

a little more specificHow does PCB cause underdevelopment of the left ventricle leading to a congenital heart defect known as hypoplastic left heart?

what I ask myselfWhat cell cycle proteins are up-regulated or down-regulated when the heart is exposed to PCB during the early developmental stages?

Peggy (Dr. Kirby) recommended that we use chick cardiomyocytes because it will make our case stronger if we can show similar effects of PCB on heart development in two different animal models. Plus, chicks have four-chambered hearts so we can specifically look at the left ventricle.chick embryos How do we obtain the left ventricles of chick embryos? MICROSURGERY!(someone else extracting the embryo…doesn’t require a microscope at later stages)Harriett and Peggy taught me and Kyle (the grad student I’m working with) how to take out the embryo from the egg (just normal eggs from a farm) and then how to take out the heart and THEN how to cut the left ventricle out using micro-scissors. This entire procedure is done under the microscope and it is FASCINATING. Taking the embryo out was tough because sometimes I’d pop it and there would be blood everywhere. Plus, it would plunge into the yolk so I had a hard time finding it. Cutting the heart into 4 chambers was tough as well but it should be easier next time because Harriett ordered finer tools for us. Overall, microsurgery was one of the most difficult things I’ve ever had to do but now I know what techniques to use and what to look for. The coolest part was that the heart would STILL be beating after extracting it from the embryo. Seeing that under the microscope blew me away. I just checked those cells today and you can see them beating against the bottom of the petri dish. wow.Before I end this, I have to tell the elk jerky story.Ann Marie, a grad student in the lab, tricked Kelly, another grad student in the lab, into eating elk jerky!!! Ewww. Kelly just thought it was beef jerky but she immediately knew it wasn’t when the meat tasted like “a mix of beef and chicken.” Kelly said she would get back at Ann Marie for making her eat “Bambi’s uncle.” And that is why I had a great day even though I couldn’t eat lunch until 3 pm (it was a very busy day).                                                        

Having an awesome PI has made this experience very enjoyable so far. Meet Dr. Margaret Kirby, Professor of Pediatrics and Cell BiologyrnrnDr. Kirby is internationally reknown for her research in heart development. She has done breakthrough research on many crucial features required for normal heart development (arterial pole, neural crest cells,etc). Not only does she focus on these aspects, but she also looks at the BIG picture. Our project for example has its roots in epidemiology since it is looking at the effects of a toxic spill in Baltimore. She looks at her research and asks important questions such as, “How is the environment shaping this? How can this finding be directly applied to problems in society?” Dr. Kirby also teaches pediatric cardiology fellows and grad students at Duke. She is so down-to-earth and accessible. She cares a lot about everyone in the lab and makes the lab a fun place. I sat down with Dr. Kirby for a half hour yesterday and got to ask her some questions about women in science, her greatest discovery, her hobbies, and more.rnrnrnWhat got you interested in science and biology in particular?In 10th grade, my father gave me a microscope and said “I know you’re going to be a scientist.” In college, I started out as an English major but quickly switched to Biology. I didn’t know what to do with that, so I just decided to go to medical school. In medical school, I realized that research and new knowledge was what I was really interested in so I switched into a PhD program(there was no MD/PhD program at the time at my school.)rnrnrnrnYou must have been one of the only women in the field at the beginning of your career. Did you feel like there were many obstacles you had to overcome as a woman?rnIn my med school class, there were 110 people. I was one of six women. Now 50% or more than 50% of med students are women. Some say that this has made medicine “kinder and gentler.” There is still a fair amount of prejudice but we’ve come a long way. I remember the course director of my neuroscience class in med school made remarks such as “women shouldn’t be in medicine.” This came at the same time as when the Equal Rights Amendment was on the table and a senator said that all women should be “barefoot and pregnant.” There are still obstacles out there. Although more women are in the field, there are very few women in the higher ranks.rnrnMale PIs greatly outnumber female PIs here and elsewhere. What would you say to young women who are interested in research and medicine but scared about balancing professional and family life?It’s hard…no question. I’ve had many women come in and out of my lab trying to balance family and professional life. I myself had two young kids and a husband when I was getting into the field. My husband took care of the kids. It’s important to have a partner who is responsive to your needs and requirements, especially during the big steps such as tenure or a promotion. Many peoplernusually go back and forth saying “It’s your turn, now it’s my turn, etc” and it’s usually women who end up taking the hit because they just can’t stand to see their babies going around in dirty diapers. You just have to work really hard at both and you need a lot of support. You can’t do it all by yourself.rnrnrnrnWhat was your greatest discovery? How did you come to find that out?Well, what I am known for is my discovery that neural crest cells are necessary for normal heart development. In France, this group created quail/chick chimeras to observe neural crest cells in the vascular system of the neck. I used the same model to study the heart. I remember I had two high school technicians at the time because I couldn’t afford too much (they were great technicians!). They put these slides in front of me and I immediately realizedrnsomething was really wrong about their hearts. I was expecting functionalchanges, not such obvious structural changes.rnrnYou recently wrote a textbook called “Cardiac Development” which was published by the Oxford University Press.  Did you enjoy the experience?It was one of the hardest things I’ve ever had to do. It was a sustained effort. I would write it at 4 o’clock in the morning because scientists just don’t have extra time for things like this with all the grant writing, etc. It was a labor of love. This was actually in association with a medical illustrator. We had a lot of pictures so I just wanted to make sure her message got out there. That was the impetus.rnrnWhat impact are you hoping this book will have?Heart development was so mysterious in my time (it still is!).It was poorly taught and just a big mess. It’s been an uphill battle though…it’s taken me 15-20 years to feel comfortable. I thought this book might help people in the future so they don’t have to go through that. In previous heart development books, many people would be working out but they’d each write their own part (what they specialize in). This is the WHOLE story. I’m hoping it’ll help pediatric cardiology fellows, physicians, grad students, and researchers.rnrnYou are quite the photographer…How did this become a hobby of yours?It started in junior high when my neighbor taught me a lot about photography. I then came back to it in 1998 when I took a course at the New York Institute of Photography. It’s just a really fun thing to do.Here are some of Dr. Kirby’s fantastic photos! rnrnWhat else do you do in your free time?I LOVE the outdoors. That’s my only regret about working in the lab…not being outside more.rnrnWhat is your ideal day at work?hmmm…work in the lab in the morning, have a session with a pediatric cardiologist, work on some manuscripts/reviews, journal club;I LOVE journal club and of course, local meetings here in the lab.rnrnrnrnWhat do you see as the biggest challenge in biomedical research?We’re really good at solving technical problems. We’re bad at understanding the ramifications of what we’re doing. For example, in the 40s and 50s when open-heart surgery was possible, we were doing it on children with all sorts of defects. Now that we know that many of these defects are genetic, the picture is totally different. Now the children that we fixed back then can have children and pass on those genes. We’ve subverted evolution and bred genes into the population. We need to be responsible socially. That is our biggest challenge as a species. Global warming falls in that same category. We’re not seeing the BIG picture. If we’re gonna repair hearts, we have to learn how to repair genes. We’re able to develop things technologically…we’re just missing that other side. It’s a humanistic perspective.” 

Now that’s something to think about. Dr. Kirby’s last point reminded me of discussions about bioethics in my Biotechnology and New Genetics seminar first semester. Good times. Tomorrow, I will be running the anticipated Western to see which cell cycle proteins are up-regulated or down-regulated in the PCB fish (I’m sorry for smushing all you little embryos in that tube…I needed your protein!)…I ordered $1,200 worth of antibodies from Santa Cruz Biotech just for this so it better work!!

Hello everyone! I’m glad I have this opportunity to lock in the Howard Hughes experience. First of all, it feels GREAT to be back at Duke. You know that warm, fuzzy feeling you get when you arrive home? I got THAT feeling. Nevertheless, I love home back in icy Ann Arbor, Michigan. In fact, that’s where this summer research experience really started.                                                     Living in a college town is AWESOME. Not only do you get to hang out on a great campus and see academia/city life merge, but you also have endless opportunities. To complement the research I would be doing on a congenital heart defect in the Kirby lab, I shadowed some of the top doctors and surgeons in the field at the University of Michigan Congenital Heart Center. It was there that I got to see the phenotype.  I was so impressed by Dr. Jennifer Hirsch, a pediatric cardiac surgeon at the center. What amazed me most was that she’d have Eminem or Fergie playing in the background as she inserted a piece of gore-tex to repair an ASD or VSD (septal defects). Now that’s talent. It was with Dr. Caren Goldberg when I got to see patients with….*drumroll* HYPOPLASTIC LEFT HEART SYNDROME, the defect I am studying in the Kirby lab, a cell and developmental biology lab with a focus on the early stages of heart development.    So what exactly IS hypoplastic left heart syndrome? It is the most lethal congenital heart defect, resulting in severe underdevelopment in structures of the left side of the heart (in diagram), namely the left ventricle which pumps blood to the entire body via the aorta. With reduced action of the left ventricle, there is mixing of blood and more strain on the right side of the heart to complete the important activities of the left side. Without intervention, babies will die within hours or days. Even after early intervention, patients must undergo further surgeries to reduce the strain on the right side of the heart. For example, i saw a few patients who were about to undergo the Fontan procedure in which the SVC and IVC (vena cavas) are connected to the pulmonary artery so that blood can just directly go to the lungs without passing through the right ventricle. This heart condition can be detected even at the fetal stage by an echocardiogram (notice the tiny left ventricle compared to the normal-sized right ventricle) :  What makes our research on hypoplastic left heart unique is that we’re studying it in an epidemiological context. A long-term study showed that PCB spills in the Baltimore area led to increased rates of hypoplastic left heart. We are studying how the PCB toxin interacts with different cell cycle proteins to inhibit cell proliferation in the ventricle, leading to its underdevelopment. Some of these cell cycle proteins we’re targeting may sound familiar, especially to those of you who took Bio 118…our current list includes p53, Rb, E2F, cyclin D1, cyclin E, and cyclin B. Most importantly, the zebrafish is being used to model hypoplastic left heart and has proven to be very effective. control fish (blue-stained heart)     PCB-exposed fish (notice the “stringy” heart and swelling of the pericardium, the sac around the heart). Working on this project has been a lot of fun so far…take a look!! Pictures courtesy of the wonderful Alaina Pleatman.   Looking up which antibodies would best recognize zebrafish cell cycle proteins…rnrnMartha, Laura, mewith Dr. Kirbywith the chick eggs, some lab members do microsurgery to extract the embryosrnrnrnrnrnrnI’m glad I finally found an area of research I’m really interested in. Don’t get me wrong, I do like finding out about new genes and obscure proteins but sometimes it’s refreshing to just look at the BIG picture, as well.  What I love most is that my project encompasses many of my interests such as epidemiology, pediatric cardiology, environmental science, and cell biology. Next post, I will introduce Dr. Margaret Kirby, my PI and Professor of Pediatrics and Cell Biology. Until then, take care!