Duke University | Howard Hughes Undergraduate Program

 "Enzymes are things invented by biologists that explain things which otherwise require harder thinking." – Jerry Lettvin

Everyday I slice and dice tissue, use innumerable pipette tips, apply chemicals to slides, and spend hours on amazingly expensive lab equipment. Inevitably, what I do costs money – money that is largely provided by your wallet! Like many biomedical research laboratories, we here at the Stripp Lab receive a lot of our funding from government agencies like the National Institute of Health. Those departments are financially supported by your gracious yet compulsory donations as taxpayers. So, thank you! Whether you’ve realized it or not, you play a huge role in keeping our research afloat, and you deserve to know what we’re doing. You wouldn’t just hand someone your money without knowing what that money’s going to be used for, especially during times of economic crisis. Let me enlighten you with what you are allowing me to do during my time as a Howard Hughes Research Fellow.

It’s quite likely that you or someone you know has or had some sort of lung disease, whether that’s asthma, emphysema, or a kind of chronic obstructive pulmonary disease. If you do, you know how debilitating and life-altering these conditions can be. A person with lung disease may need to be provided with an inhaler, home oxygen therapy, a cocktail of prescription medication, and receive periodic vaccinations. Some of these diseases can be genetically influenced, triggered by pollutants in the air you breath, or caused by smoking.

Lung disease can have devastating effects on the anatomy of the lung. Once injured, the epithelial cells that line bronchioles begin to die, leaving the airway open to infection. The cells in the terminal bronchioles near the alveoli, where gas exchange occurs, can also be destroyed by lung disease; this can make oxygen intake and carbon dioxide release difficult, resulting in struggled breathing among other serious problems.

What if these damaged airways, compromised by cell death, could be restored? That is one of the long-term aims of Dr. Stripp’s research. One solution may come in the form of stem cells.

Stem cells have received a lot of attention in the news media and scientific community over the last few decades. Stem cells are those cells that have not been assigned a specific duty in the cell (e.g. red blood cells have a job; their job is to transport oxygen to your tissues), can self-replicate, and can give rise to differentiated cells (specialized, often localized cells with a “job”). There are basically two different categories of stem cells: adult (those found in deve loped systems like bone marrow, intestine, lungs) and embryonic (those found in the inner cell mass of an early stage embryo). The latter is quite controversial, because many scientists hold that they have a greater ability to differentiate into a larger number of different cell types (pluripotent) compared to an adult stem cell (multipotent) and some religious and ethical groups do not approve of killing an embryo to harvest its stem cells. Regardless, if a stem cell can create other types of cells, why couldn’t one be made to regenerate lung tissue? That is one idea we are exploring.

Lung stem cells are also quite controversial in the biology world. Stem cells are often very active, replenishing depleted supplies of cells at great rates. Lung stem cells are different, because, in the adult, they are not extremely active. They are most active during the embryonic development of the lung, and then they begin to quiet as the necessity to build lung tissue decreases. Other less stem cell-like cells are able to upkeep the lung in the adult; these Clara cells protect the bronchiolar epithelium by secreting Clara cell secretory protein (CCSP) and a component of the lung surfactant and by detoxifying harmful inhaled substances. cells also multiply and differentiate into the ciliated cells that line the epithelium.

Okay, so Clara cells can regenerate the bronchiolar epithelium, but what happens when Clara cells are wiped out by pollution? This question was recently addressed, and it turns out that there exists a population of rare pollution-resistant Clara cells (vCE or variant CCSP-expressing) that inhabit specific locations of the lung. vCE cells can replenish the depleted Clara and ciliated cell populations, ablated by pollutants like naphthalene and ozone. However, if the lung injury sustained is severe enough, some of the vCE cells can be wiped out. If enough are ablated, the lung will be compromised.

What if more vCE cells could be present and assist in epithelial restoration of Clara and ciliated cells? That is what my research focuses on. By increasing the concentration of a protein called beta-catenin in adult mice, I may be able to reprogram Clara cells to the vCE status. An accumulation of a particular form of beta-catenin has been shown to activate the transcription factors that are normally turned on during embryonic development, when the population of vCE cells is relatively high.

I spend most of my time in the Stripp Lab preparing mice tissue, staining it for proteins like CCSP and beta-catenin forms, and analyzing the immunofluorescence under a microscope.

Take a look at one of the cool photos that I have taken of one of my samples!

This is an image of the conducting airway epithelium of a mouse bronchiole. The blue staining shows where cell nuclei are located. The red/orange between and around the epithelial cells shows one form of beta-catenin. The green shows the form of beta-catenin we have tried to genetically introduce in our transgenic mice. What are important to notice here are those epithelial cells that show only green, indicating an increase in the beta-catenin that has been shown to induce stem cell (vCE in this case) reprogramming.

Remarkably, this one image has taken months to cultivate. I only took over when the lung tissue was embedded in wax. From that point on, I needed to spend about a week cutting the wax, putting the samples on microscope slides, staining with immunofluorescent antibodies, and analyzing with the microscope. Before all that work, the experiment needed to be set up, the mice injected with certain chemicals, their progeny screened, and the lungs dissected, fixed, and embedded in wax. All the work and number of people involved in the generation of just one image like the one above are testaments to the cooperation, patience, and ingenuity required in research.

I know that this information is a lot to take in! It was certainly difficult for me to wrap my head around the details during my first few days in the lab, but stay tuned to my blog updates and everything will start to become clearer!

- Tyler

"Q: If both a bear in Yosemite and one in Alaska fall into the water, which one dissolves faster?"
"A: The one in Alaska, because it's a polar bear." - Mariano Cecowski

Before I recount my interview with Brian, let me just fill you in on what I've been up to in the lab. I'm still perfecting the protocols that I'll be following throughout my research project; I'll fill you in on the details of that project in my next blog. Mean while, I'm getting much more comfortable with our microscope and PCR techniques. Linda, another Duke undergrad, has shown me how we humanely extract our lung tissue from transgenic mice. Next week, as I get more involved with my project, my pace will begin to pick up so stay tuned!

Before I started research in Dr. Stripp lab's, I did my own research on who I would be working with this summer. From reading his short bio on the Stripp Lab website, I could tell that technician Brian would be both brilliant and highly entertaining, an assumption that would be proven entirely correct. This is a portion from Brian's blurb from the Stripp Lab site:

"As a former teacher, my daily focus has changed from interacting with students to interacting with noxious respiratory toxicants. This is proof that one's life can, in fact, improve."

Clearly, Brian has some interesting opinions to share so I was very eager to ask him a few questions in the lab. 

What are your current research interests?

TGF-beta and SMAD / Basically, there are a lot of genes. We take away one at a time and see what the effect is on lung development and repair, especially after injury. We are simulating injury by cigarette smoke, pollutant exposure, and possible remodeling in situations like asthma.

What’s your educational background?

None. Just kidding. A Bachelor of Science in Biology from Slippery Rock University of Pennsylvania.

What were you doing before you came to work with Dr. Stripp?

I taught senior and junior high school - science, gifted and talented students, video, and stage productions. I didn’t enjoy it. The last year they gave me junior high students, who should live on an island until 9th grade. Kids should learn sports, games, hobbies, and getting along together for 7th and 8th grades. You should not teach them anything until 9th grade. I can teach them anything they should’ve learned in 2 years in 2 weeks.

What’s your favorite donut and why?

The éclair, because it’s BIG. (I'm not sure if they éclair can be categorized as a donut, but I'll give it to him.)

What’s your favorite part about you what you do in the lab?

Being able to see results directly, like by microscopy, unlike people who work with things that look like water before and after their experiments, and then they use a machine to tell them whether or not the final product is actually water.

What’s your least favorite part?

Molecular biology, because I can’t see it.

What’s your favorite area of science?

Histology (the study of tissues; plant and animal tissues, that is), micrsocopy

What’s your vision for a better tomorrow?

Wow! An improved economy that allows more science to be done. If the economy gets bad enough, they will shut down science.

What’s your favorite soda, movie, candy, and immunofluorescent color?

Coke, Matrix (only the first one!), Laffy Taffy, far red (you can’t see it but the camera says it’s there)

 

 Brian is a constant source of clever hilarity and science genius. He has and will continue to add so much to my research experience, so thanks Brian!

 

“A biologist in my lab gave birth to twins a few days ago! She decided to name one Mary Therese after her mom, and she named the other Control.”

I thought I’d try to start my blog posts from here on out with great, albeit slightly dorky, science jokes. I hope they keep you entertained, if only for a few nanoseconds.

In this post, I want to share my expectations for this Howard Hughes Research Fellowship that I embarked on about 10 days ago. I think it’s best that I describe the presuppositions I developed before my first day in Dr. Stripp’s lab, my first day in any research lab.

Weeks before my return to Duke University, my mind had conjured fantastic images of what my summer in lab would be like. There would be mice weaving through test tube racks trying so desperately to escape the clutches of a mad scientist whose torturous plans for these sacrificial animals were only intensified by his maniacal laugh and gravity-defying hairstyle. For two months I would be plunged into the darkness of a windowless basement with the only respite of light coming from Tesla coils and GFP-engineered lab rats that had escaped from their cages. My day would be quiet as my fellow researchers would try so hard to achieve an unparalleled level of anti-sociality in order to better focus on the experiments at hand. Talking would be frowned upon, and evil crackling at a hypothesis proved correct would only permitted on nights when the dark sky was scarred by thunder and lightning. I, outfitted with enormous goggles and a white lab coat stained with glowing, green ooze from my past experiments, would derive pleasure from the crisp smack that latex gloves would make against my skin, growing paler every day from the unholy number of hours spent in darkness, as I pulled and released the cuff. My role would be to mix chemicals whose names were unpronounceable except for the expert organic chemist. Within days, I would find stem cells collecting at the bottom of my Erlenmeyer flask, and the Nobel Prize would be within my reach. Slowly, I would become a modern-day Victor Frankenstein or Dr. Jekyll, or maybe even a Dr. Evil commanding my henchmen to contact a chemical supply company for its latest batch of sharks with laser beams attached to their friggin’ heads.

All right, I guess that was a slightly romanticized version of my pre-lab expectations. Not surprisingly, things ended up quite differently. However, I really do like the satisfyingly crisp sound that my gloves make when I put them on like a crazed scientist. I’ve also learned that science often moves very slowly; it can takes months from experimental setup to data collection. Every single tissue that I view under the microscope has undergone months of work.

Now that I have a better sense of what biomedical research is like, I can better develop my expectations. Contrary to any stereotypes of social awkwardness in the research community, I have learned that even brilliant scientists are outgoing and eager to share their knowledge. I’ve even come to love and appreciate the science jokes that are told in the lab. Thankfully, the mice are well-contained and we are far from the darkness of a basement. I’m well on my way to learning the tools-of-the-trade – the techniques and protocols that have been perfected over the years by Dr. Stripp and his colleagues. I’m slowly honing my skills – my immunostaining, microscopy, tissue sectioning, and lung collection from mice are all getting better. I can expect that these techniques will only continue to improve over the next several weeks, and that I’ll be introduced to more as I progress through my own research project.

Everyday, my understanding of what we are studying – conceptually, molecularly, genetically, atomically – improves. A light bulb goes off every few hours as I reach an intellectual epiphany; also, to save energy, the light bulbs literally go off every few hours in different parts of the lab. From the roles played by different lung epithelial cells to the signaling pathways that we focus on, more concepts become clear as my brain turns on the defogger. I’m sure that, until even my last few hours in Dr. Stripp’s lab, I will continue to learn something new and useful.

One of the largest goals that I share with my mentors is that I can contribute something positive to their research. Two months may seem like a long time, but I can tell already that my project will move slowly. We hope that, during my time in the lab, I’ll even be able to contribute enough data and insight so that I will have co-authorship on Dr. Stripp’s next publication. It would be amazing to have my name in a research journal, even if I’m listed as the 9th author!

I think I found a niche that makes me comfortable and satisfied. Every morning, despite how difficult it is for me to wake up to the sound of an alarm at 8:00am, I look forward to coming into the lab and working with a great and enthusiastic team of researchers. I can honestly say that a future in research is a goal I hope to realize.

- Tyler

 

 The reprogramming of Clara cells to behave more like pulmonary epithelial stem cells and monitoring their proliferation through injury models. That’s what I’ll be doing with my summer at Duke University, and a few days ago that phrase would have made absolutely no sense to me.

Thanks to the Howard Hughes Research Fellows Program, an eight-week fellowship that throws rising Duke sophomores into the seemingly daunting research community, I’ll be learning as much science as I can possible house in my tiny, undergraduate brain. This program allows Duke students to get a feel for the scientific research process by pairing each sophomore with a mentor, whose interests can range from housefly mating habits to gene regulation in plants.

Before I get carried away with describing what I’ll be researching in the lab, let me tell you a little bit about myself. As far as where my academic interests lie, I’m somewhat of an enigma. I’m lucky enough to know what I’m passionate about though – science and foreign languages. There is just something intriguing about the idea of uncovering and creating knowledge, and then being able to break down barriers and share that understanding with a foreign language. I have recently declared a double major – the first being a BS in Biology with a concentration in Genetics, the second French and Italian Studies. That will certainly keep my busy over my next three years at Duke! In my time away from the labs and lecture halls, I enjoy playing tennis, ping-pong, and board games, running, riding rollercoasters, watching James Bond movies, reading, and trying foods from around the world.

Hailing from the Jersey Shore, I have spent most of my summers relaxing on the beach in between workings as a tutor of inorganic chemistry and mathematics for high school students in the area. After only a few days in Durham, I can tell that this summer is going be quite different!

Before starting this program, I knew that stem cells were something I wanted to explore. After a preliminary interview with Dr. Barry Stripp, an expert in pulmonary stem cell research, I was glued. His awesome lab group, of which I am now a part, boasts about ten members, each with a different and impressive scientific background. Each of us is working on some aspect of lung stem cell localization, proliferation (or rapid growth), and secretion, with the goal of being able to use each other’s data to gain a better understanding of the structure and function of pulmonary epithelial cells.

Over the past several decades stem cells have been found in various organs and tissues throughout the body, often in small populations. Some of the parts of the body in which stem cells have been found include: brain, bone marrow, teeth, heart, intestine, liver, and skin. Stem cells must fulfill certain criteria: relatively undifferentiated phenotype (meaning that they are not already specialized for a specific function; some cells in the lung are responsible for gas exchange, metabolism of pollutants, etc. but lung stem cells have no specific "job"), pluripotent differentiation potential (stem cells can self-replicate to give rise to specialized cells and tissues of all three human germ layers), low rate of steady-state proliferation (if there's no need for new, specialized cells, stem cells should remain quiet), and localization to a stem cell niche (stem cells tend to inhabit specific areas in the body known as niches).

Stem cells in the lung are crucial to cell regeneration. The air that you breathe isn't always as clean as you might suspect. Even in the smallest amounts, gaseous pollutants find a way into the lungs. In some rare cases, a person may be subjected to highly polluted environments. These chemicals can kill the cells that line the lungs and metabolize dangerous pollutants, leaving the tissue very vulnerable and breathing difficult. If the damage is severe enough, the stem cell populations of the lung (which are often immune to the damage of the pollutants) will kick into high-gear and begin to regenerate those cells that were ablated (destroyed). In the lab, we subject mice to different pollutants and at different strengths and then monitor the activity of the lung tissue. We can get an idea of which types of cells were destroyed and where, what cells are seemingly immune to the pollution, and what cells are helping to regenerate the cells that were destroyed.

Unlike many other stem cells, such as intestinal or blood stem cells, those that appear in the lung do not proliferate very much. A large part of the research that we are conducting is actually proving that these seemingly lazy (we like to refer to them as “quiescent”) pulmonary cells are actually stem cells. We do this by injuring the lungs of mice with pollutants such as naphthalene and ozone, then we use immunofluorescence staining (really cool markers that glow under very expensive microscopes) to detect which cells are proliferating, which cells have been ablated (destroyed by the pollutant), and where in the airways the cells are found. We can use lineage tracing to detect progeny cells and the parent cell so that we can determine which cell is a stem cell candidate. By figuring out where, how, and to what stimuli these stem cells proliferate, we hope to one day be able to develop pharmaceuticals that will encourage lung repair in humans with damaged lung tissue.

After only one week in the lab, I have learned more about pulmonary cellular biology than I could possibly have imagined. Every lab member I meet is eager to tell me more about what they are working on, suggest journal articles to read, and teach me the techniques that they rely on. In an effort to avoid boring you, I won’t go into the details, but trust me, I’ve learned a lot!

For the past few days, besides reading and listening to the science behind our research, I’ve been sectioning and plating lung tissue embedded in wax into slices that are 5 microns thick (that’s about two 10,000ths of an inch!), staining lung tissue from mice with antibodies that are bound to molecules that glow under certain wavelengths of light, and analyzing those images with awesomely powerful microscopes.

I’d write more, but I have to get back to staining my latest batch of lung tissue!

- Tyler

 My very first set of micropippetors! Kodak moment.

Post-doc Josh explaining his next experiment setup. He has been an invaluable resource this past week, teaching me how to use our microscope and how to stain and section our tissue samples.

Huaiyong, a post-doc in Dr. Stripp's lab, pipets...umm...stains...umm...okay, I'm not really sure what he's doing.

 Fellow Dukie Linda and technician Jeff trying to figure out how to program the robot (or what I refer to as the "undergraduate replacement machine").

 Me using one of our most sophisticated pieces of high-tech equipment for antigen retrieval - yes, that's a microwave.

My work station - a notepad to chronicle my days in the lab, my MacBook (which seems to be getting a lot of negative attention from the PC users), and I just really like Diet Coke.

The microtome. This week I spent four straight hours using this machine to section my samples. I was told that this room is secluded due to the screams of frustration and occasional episodes of delusions that some researchers face when plating their tissues. Luckily, I escaped unscathed. 

 My lab bay in Dr. Stripp's lab! 

 

The cell culture room! I have yet to spend much time in here.

This microscope has a powerful mercury lamp that emits different wavelengths of light to excite the electrons and emit immunofluorescence in the antibodies that are bound to the lung cells we are interested in studying. 

 

I just thought the mouse in the microscopy room was pretty cool.

 Yeah, Danger's my middle name.