Author Archives: Melissa Horowitz

8 Weeks Later…

In the weeks before the beginning of the program, I was really unsure of what was to come, since I had absolutely no research experience. I entered Duke with an extreme interest in researching genetics, though I’m majoring in BME/ECE, so my academic goals are a bit in contrast to my research goals. Although I spent a significant proportion of the summer growing increasingly worried about what I wanted to do with my life, I think I’ve begun to accept that it’s all right to still be uncertain. If there’s anything I’ve learned from the faculty seminars, it’s that scientists don’t always plan on being scientists, and people don’t always end up where they expect to end up. So, even if I’m still a bit worried and unsure of my future as I’m writing this last blog post, I’m definitely more open to the idea of approaching the remainder of my undergraduate career without the rest of my life supposedly set in stone.

Throughout this summer, I learned so much about the daily life of researchers, and I feel exponentially more comfortable in that type of academic environment. Even though I’m initially intimidated by everyone with a Ph.D., my mentors have been extremely supportive and I now know that I can rely on faculty for great advice in the future. Personally, I think I’ve grown a lot over the summer, as well. Other than the fact that I am becoming more skilled at handling plates without dropping them, I am significantly more capable of doing work independently than I was before this summer. Honestly, after having to run statistics of data from five different variables in R (a program I’ve never used before, with no programming experience other than MATLAB, and no college-level statistics class as of yet) with what initially seemed like a million different interactions I could examine, I’m a bit more confident in my ability to face challenges that come my way.

Even though I really enjoyed this exposure to research and my introduction to genetics research, I wouldn’t say I’ve had anymore clarity on what I should pursue in the future. While I still want to participate in undergraduate research and think research in genetics is a possibility for me in the years afterwards, I’m still in love with being an engineering major. For now, I think I’ll hold on to the dream of combining both my interests in genetics and engineering (maybe gene therapy?), but I really look forward to possibly continuing my involvement with the Donohue Lab, if my dense schedule permits. Thank you so much to everyone who made BSURF possible (including Dr. G and Jason!), because I was able to spend my summer involved in amazing research, learning more about Duke’s faculty and more about myself, and it has definitely made an impact on my life. I also enjoyed learning from all of the other fellows and what their experiences were from the summer (like when I recounted how I filled 2300 plates with agar, and Demi responded with “oh, I just have a plate pourer to do all that work for me.”)!

Biology and Engineering

Hearing about the research and life stores of various Duke faculty members was really enlightening; every speaker took a different path in science, yet all seemed to truly love where they ended up in their research. While some knew from a young age that they wanted to pursue research, it was reassuring to learn about the large number who had to explore a range of possibilities before arriving at their current profession.

One speaker I really enjoyed listening to was Dr. Charles Gersbach. As a biomedical engineering major, I found his story about making the decision between research and industry extremely relatable. Also, the way in which his research combines genomic research and engineering (two fields that really interest me) is fascinating, and the possibilities of using genome editing for gene therapy opens up a number of possibilities nearly unimaginable to me. DNA used to be considered this unchangeable aspect of our selves, and for a long time there was nothing that could be done about life-threatening mutations, even if it was a simple base change. CRISPR-mediated genome editing is truly thinking outside of the box by manipulating a bacterial mechanism in order to potentially treat diseases (such as muscular dystrophy), whose causes are locked within our own genome.

To me, Dr. Gersbach’s research really exemplifies the unique interdisciplinary type of research that occurs at Duke. I first heard about his research while doing an AP Biology project in high school, and I think that was the first time I was really taken aback by how far science has come and the possibilities that are awoken by research. Someday, I hope that I can also find a way to combine my interests in genetics and engineering in a way that not only contributes to scientific knowledge, but also makes me look forward to continuing my work every day.

Data, Data, and More Data

As the summer is getting nearer to its completion, so is my project, and fortunately with usable data! After four weeks of counting germination proportions in 1000+ petri dishes with around 20 seeds each, it’s a relief to know that each plate was actually accounted for and that the excel sheet is exactly the size it’s supposed to be. From what I’ve seen so far, there are some pretty interesting trends in the data even if not entirely what I expected (though the results will be more obvious once I finally figure out how to use R). Though by the time the poster session arrives, I’ll have graphs ready that can better illustrate the interaction between light quality and Flowering Locus C expression and their impact on germination!

Fortunately, there’s not much you can do to seeds that they won’t manage to survive or recover from. Nonetheless, I think I now have the lab record for the highest number of plates dropped during seeding (which involves placing the seeds onto the agar in each petri dish). I somehow even managed to fling a petri dish across the room using only parafilm (luckily, I had prepared 50 extra plates with agar, because I had zero confidence in my ability to not be clumsy). However, we had our last census this week, meaning that all of the plates were tossed in the autoclave bin to be subject to intense heat that kills cells so that the altered genotypes don’t mix with natural populations. After spending six weeks preparing the plates, seeding them, and counting germination proportions over and over and over again, it was a bit of a relief. I’ll definitely be much more satisfied by the complete graphs though, since I’ll know that all of the work yielded actual results!

Until then, there’s still a bit more work to be done!

Chalk Talks and Mutations

Though I’ve heard bits and pieces about a variety of people’s research projects throughout the past five weeks, I haven’t heard them described in as much detail as they were during the chalk talks. Therefore, I found it really interesting to hear the diversity of fields that our projects covered, from plants models to animal models and genetics to engineering, yet there were definitely some common threads present. This week has really shown me how one field can branch into so many questions, yet so many different fields can also converge into one question (if that makes any sense).

One chalk talk that I really enjoyed listening to was Demi’s, for two major reasons (but not the only reasons!):

  1. The genome is a really interesting subject, and the ability to study its self-repair mechanisms and the functions of the genes within it always amazes me.
  2. We learned about a lot of these concepts in BIO201 (mutagenesis, DNA mismatch repair, cytosine deamination, etc.) and it’s refreshing to learn about real research involving them (also without being tested on it).

Demi’s project involves a transition mutation in the gene CAN1 in yeast and how the strand bias of the mutation significantly increases after removing the DNA mismatch repair mechanism. The driving question is what exactly causes this bias?

I’ll be looking forward to see in the upcoming weeks as to whether her results support either hypothesis (a polymerase causing mutations during replication or a stronger consensus sequence for a cytosine deaminase) and if any broader generalizations can be drawn from the research!

Weekly Seed Activities

My daily activities in lab really depend on what point of the experiment we’re at. Since my project is such a giant experiment, it takes up most of my time during the day, though I do help work on another smaller experiment that’s also going on. I generally arrive in lab around 10 AM. The more intense days have me working continuously from 10 to 5, while on the easier days I have time to read papers and work on some smaller assignments (like blog posts or the upcoming chalk talk).

The first few weeks at lab were pretty intense, since we all had to work together to prepare the experiment due to the sheer quantity of plates (2,304) needed. In the mornings, I would cover an entire lab bench in petri dishes (it turns out your standard lab bench can fit exactly 450 3 millimeter petri dishes on it). Then we needed to prepare the agar, which can fortunately be done on a larger scale using the autoclave machine down the hall in the Biological Sciences building. This way, we can heat up around 12 bottles of agar at once in about an hour. After filling up all the plates, they needed to be chilled overnight so that no mold or fungus grew in the agar. Making all of the plates basically took up the entire first week.

The second week, we had to put 20 Arabidopsis thaliana seeds in each plate. The maternal plants for this project were grown before the program started, but a bunch of plants of each genotype were grown, and then placed in their respective simulated environments. Each of the plants had to be wrapped in a plastic tube in order to prevent cross-pollination. Then, at maturity, the seeds of each plant were collected into tubes, which I then placed into their respective plates. Since the seeds are extremely small (not much more than a speck), I had to use a thin probe and place them one by one on the agar. Then, trays with filters were filled with 36 plates in order to simulate a certain light quality environment. After being filled, we had to take them to temperature-controlled chambers that are either down the hall in BioSci, or in the phytotron (a rather dark and scary place filled with dozens of tall, humming, machines) in French Science.

I was able to start doing germination censuses for the project in week 3. This involves taking the trays out of their chamber, examining the plates under a microscope, and counting the number of seeds that have germinated (which is when you can see the radicle poking out of the seed coat). If all of the seeds are fully germinated, then they can be thrown in a bin to be autoclaved (which kills all of the cells so that they don’t cross-pollinate with natural genotypes!). Then the trays go back in the chambers. The whole process is usually finished by lunch, especially since we have 3-4 people helping out at once. The rest of my day is spent getting caught up on papers or taking care of other plants down in the phytotron. By next week’s census, I should have enough data to begin analyzing the results!

The Environment and Genetics of Germination

When discussing the development of any type of organism, the topics of both genetics and the environment are strongly considered, which has contributed to the debate known as “nature vs. nurture.” At the Donohue Lab, both heredity and the environment are studied as contributors to the germination patterns of Arabidopsis thaliana seeds, but the two factors aren’t as black and white as some “nature vs. nurture” lectures may make them seem. While a human development class may ask the question “Is the parents’ genetics or the environment of the offspring a higher indicator of gene expression?” the Donohue Lab is currently addressing the question “Is the maternal environment or offspring environment a higher indicator of gene expression and germination patterns?” In this research, genetics and the environment are considered together while their interconnection is being further explored.

It was originally expected that the seed’s environment would be a stronger predictor of its germination patterns that the maternal environment. After all, it’s nearly impossible for the mother plant to know what conditions the seed will face when it germinates due to the varying length of dormancy. What if the maternal plant lived in a dense canopy, but a fire comes and wipes out most of the seed’s possible competition? Unexpectedly (as researched by lab members Lindsay Leverett and Gaby Auge in a paper that’s currently in press), the maternal environment seemed to have a much more significant impact on the proportion of seeds that germinated. Furthermore, when the maternal plants lived (while the seeds were maturing pre-dispersal) under a simulated canopy, meaning greater competition when the seeds dispersed and germinated, the seeds were more likely to germinate. Which is really strange time to germinate, as you’d might expect that the seed will be less likely to survive when there were more plants around competing for the same resources.

The maternal environments used in the previous study were simulated with filters. A clear filter allowed white light to pass through, mimicking little to no competition. A green filter both lowered the irradiance of light that passed through and altered the red to far red light ratio, mimicking a dense canopy. My project introduces a neutral filter, which lowers the irradiance of light but does not change the light ratio, which could help explain the results of the previous experiment. This will answer the first question of the project: is light acting as a source of information (light ratio) or a source of energy (level of irradiance)?

The second objective is to determine what role FLC (FLOWERING LOCUS C) plays in the germination patterns. Four genotypes will be used: Ler (nonfunctional FLC), LFC (high FLC expression), 145, and 146 (both of which have high FLC expression that has been knocked down through RNA interference).

Therefore, this project has a lot of different variables that will be analyzed to determine their impact on germination. We’ve already prepared 2304 plates of agar (!) and put 20 seeds in each according to genotype, maternal light treatment, germinant light treatment, and temperatures. We’ve also seeded them, so the plates now look like this:

Seed Pic

We’ve also done the first week germination census, so some data has started to come in! After another week, there will hopefully be enough data to begin analyzing the results. All in all, the goal of this project is to explore the variety of information sources that can have an impact on germination and how genetics and the environment play a role together.

The Circle of Life: Plant Edition (as studied by Dr. Gabriela Auge)

(My primary mentor, Dr. Kathleen Donohue, has been away from the lab for the past week and a half, so I interviewed my secondary mentor, Dr. Gabriela Auge, instead.)

Whenever I travel to Duke, the four hours of flights make it seem that my home in south Florida is so far away. However, my initial journey to Duke wasn’t even comparable to that of my secondary mentor, Dr. Gabriela (or Gaby) Auge. She spent a significant portion of her life in Argentina, having completed both her undergraduate and graduate education there.

At the Donohue Lab, Gaby works with the seeds of the \Arabidopsis thaliana plant and is currently researching the effect of light quality on their germination patterns. Though the seeds of the Arabidopsis thaliana plant don’t look like much in their dormant stage (in fact, they’re so small that they aren’t much more than a speck, and we need to use a thin probe to place them on the agar), Gaby passionately described their tremendous potential. A seed barely larger than a period can grow several inches tall with dozens of flowers in only a few weeks. Though much of her career has been focused on seeds, she didn’t originally expect them to be such an integral part of her life. She began by studying biotechnology at the National University of Quilmes in Argentina—at which point she knew she wanted to be involved in research—and then started working towards her PhD in 2002 at the University of Buenos Aires. It was during her first post-doc in the School of Agronomy at the University of Buenos Aires that she first began working with seeds, which eventually brought her to Duke University and the Donohue Lab.

Though Gaby’s first post-doc was focused mainly on the effect of temperature on germination and dormancy, she is now expanding the frontier a bit more by considering the lifecycle as a whole. For example, the Donohue Lab contributed to the publication of a paper a few years ago (Chiang et al. 2009) that showed how FLOWERING LOCUS C (or FLC), a gene that repressed flowering in A. thaliana, also plays a role in germination. Therefore, germination and flowering are two steps in the life cycle that are not necessarily two separate units. Though Gaby has made a lot of discoveries at the Donohue Lab here at Duke, she already has a secure position in Argentina (between the level of a post-doc and assistant professor) where she can work on her own project and possibly have her own lab in the near future!

Gaby’s education and scientific career hasn’t all been about performing experiments, though. Her first job was as a TA in Plant Physiology (a course in which she was also a lecturer), then Molecular Physiology, as well as other courses. She wasn’t fond of being a TA at first, since it was a bit of a difficult transition from learning to having to teach classmates that graduated after her. However, once she found the way that she taught best, she began to enjoy it. Though she hasn’t taught any courses at Duke in particular, she does mentor a lot of undergraduate students (including me!) at the Donohue Lab (once she even mentored eight undergrads simultaneously). Through the process of teaching and mentorship, she realized the she really enjoys sharing what she knows with students, especially those that are eager to learn.

After deciding that research was the path for her at the beginning of her undergraduate career, it isn’t surprising that Gaby’s favorite part of being a scientist is the research itself. She enjoys learning in and of itself, and how every day in the lab can be a surprise (on a related note, she described how she once lost several days of work by dropping plants that were cross-pollinated by hand. Next time I lose only a few hours rather than days of work, I’ll have to try to just laugh at my mistake and move on like her). While she said that writing and publishing papers are the most extrinsically rewarding aspect of research, Gaby prefers the experiments themselves (though she just got a paper approved, yay!).

Overall, I really learned a lot from my discussion with Gaby. Though she absolutely knew she wanted to be involved in research when she was at my point in life (whereas I’m ~90% sure I want to pursue research), she didn’t get thoroughly involved with seeds and their lifecycle until she began her PhD work. She seems to have found her calling though, so now I’m a bit more reassured that I have time to decide what I’d like to do with my life. And once I do figure out a future path for myself, I hope to find as much joy in it as Gaby does in hers.

From Theory to Practice

Having completed a year at Duke filled with science classes including biology labs and papers on genetics, I thought I had a decent grasp on what research involved. After a week of preparing an especially large experiment (studying germination in Arabidopsis thaliana) in the Donohue Lab, it’s quite clear that a few pages of a paper cannot fully convey the level of dedication needed for researchers to perform experiments. What may be a single sentence in the methods section of a paper may involve months of planning and work. My first day in lab, I was told that over 2300 plates of agar needed to be prepared for the upcoming experiment. At the time, I was oblivious to the hours of mixing, microwaving, pouring, and bubble-popping that such a number entailed. However, it will surely be exciting to witness the progression of the plates throughout the experiment and the results that they bear, as well as actually participate in the process myself.

Overall, I hope to discover what research is like for myself. Although I hear about it every day from my professors, other students, and papers during the school year, I’ve already realized this week that nothing can really compare to your own experiences. Since I’m rather (read: greatly, ridiculously, hopelessly, etc.) undecided about my future and weather I want to pursue research (genomics, bioinformatics, or something else?) or engineering (biomedical or electrical?) or most preferably some combination of the two, I hope that this experience will help give me some sort of indication. As I’ve already begun to see from the speakers this week—and hope to continue to see in the remainder of the program—scientific research isn’t so cleanly cut along divisions. It can involve multiple disciplines and require an array of skills that necessitates cooperation among scientists and those from other departments, which I hope to continue to hear about in the future.

While the research projects themselves are certainly a big part of this program, I expect that the weeks to come will offer even more insight into the world of research. Whether listening to seminars from Duke faculty, discussing projects both formally and informally with other students, forming connections with our mentors and hearing how they got to where they are today, or just learning general skills (both in and out of the lab), I’m sure that this summer will be a remarkable experience day in and day out.