Author Archives: Hannah Ahrendt

The Summer in Review

I can’t believe that the eight weeks of BSURF are already over.  Although on a day to day basis it sometimes seemed like I wasn’t learning much, looking back over the experience as a whole, I realized how much I really learned.  I think the most valuable knowledge that I can take away from this experience is knowing what a career in science really entails.  Throughout the summer I came to the conclusion that having my own lab is probably not a path I want to pursue, but I have come to enjoy the process of research, the challenges of analyzing data and asking questions to better understand biological processes.  There were definitely challenges along the way, but I learned that is just part of the research process.  Very few, if any, projects go exactly according to plan, and the interesting part of research is figuring out new experiments and approaches to best answer the research questions.  Of course I also learned a lot about computational biology, although I still have so much to learn.

The other part of this experience that I enjoyed the most were the faculty seminars that we had three mornings a week. Although I loved hearing about the different types of research that is happening on the Duke campus, it was most helpful for me to hear about the different paths that each professor took to reach where they are now.  As someone who came into this fellowship hoping to get a better idea of what my goals are for the future, it was beneficial to get an idea of opportunities for researchers, outside of academia.  I also got to hear about subjects that I previously hadn’t considered much, such as evolution, and this has opened my mind to possibly pursuing a different field of study.  Overall, BSURF was a wonderful program that opened my mind and gave me a wonderful introduction to research.  Last of all, thank you to Jason, Dr. G and Trinity College for making this experience possible!

How did we come to exist?

Throughout the past seven weeks, we have had the opportunity to hear from many faculty members about the fascinating research they are doing on campus.  The work I found to be the most interesting was that of Dr. Mohammed Noor.  Evolution was something that I grew up simply believing to be true, but I never really gave thought to how it occurred, beyond natural selection (or genetic engineering, in this day in age). I recently listened to a podcast on which an evolutionary biochemist spoke about a current theory of how eukaryotes evolved from prokaryotes.  The very simplified version of that story is that one prokaryote swallowed up another, then the inner cell developed into the mitochondria and this source of energy allowed to cell to evolve beyond the reaches of prokaryotic cells and thus eukaryotes were born. Essentially, all eukaryotic species developed from that one, singular event. This singular event took over a billion years to occur.  This theory blew my mind and made evolution a much more intriguing topic to me.  It seems unlikely that such a diverse array of species developed from that single event, but on the other hand it makes sense that such an improbable event would occur only once ever.

Anyway, I only bring up that podcast because it is what sparked my interest in evolutionary biology, the field in which Dr. Noor works. His research questions focus on the more recent evolutionary events, specifically the genetic evolution that allows new species to develop and persist.  However, I learned that defining a species is a very difficult process because there are often many subtle differences in populations that are debated as to whether qualify organisms to be a new species.  Interestingly, there are barrier traits that exist between gene pools, to deter species from interbreeding.  These barrier traits could cause sterile offspring or an absence of attraction.  Dr. Noor’s specific questions focus on the genetic changes that produce barrier traits and how the barrier traits are driven, in order to drive speciation.  I am very glad that I had the opportunity to hear Dr. Noor speak about his research and I look forward to taking his class next spring!

Progress Report

My work in the lab has had its ups and downs during the past six weeks; some days are very productive while others are not so much.  Working on a computational project in a lab that doesn’t do very much computational work has presented some difficulties.  For example, there have been times when I am stuck on how to use a software or how to format data to input into a new software, so I had the opportunity to step outside my comfort zone and approach people in other departments for help with these questions.

Although slower than I initially expected, I am making progress on my project. I am learning that research projects are rarely as straightforward as they seem when first described.  There have been setbacks along the way, from working with the wrong genome to looking for incorrect epigenetic marks.  However, my project has made more progress recently and I have learned so much about the vast range of computational tools available, and how to use them. One thing I have figured out this summer is that in computational biology it’s much harder to just go through the motions without knowing why you’re doing what you’re doing.  This is something that I have enjoyed about my project so far, because I the methods I’ve been using have pushed me to understand the data that I am working with and think about how I can manipulate that data in various ways to reach the next step of my project.

Chalk Talk Reflection

This week all of the fellows had an opportunity to share about our projects.  It was awesome to see the diversity of research projects everyone is working on.  Yillin’s project especially piqued my interest. The broad idea of her research is to understand if mutations of the Shank2 gene plays a role in bipolar disorder. Another gene in the Shank family, Shank3, has previously been found to play a key role in bipolar disorder, as well as multiple other neurological disorders.  However, little work has been done to look at the role of Shank2. This project is particularly interesting because of its clear application to human health and the understanding of bipolar disorder. It is also slightly related to my work, although really only in the fact that both of our research involves illnesses related to the brain, mine with addiction and Yillin’s with bipolar disorder.   It was also intriguing to see how her project is almost the reverse of mine. She started with wet lab procedures, running PCR on human samples, and then will transition to computational analysis after,and I started with computational techniques and now am beginning to shift to more wet lab methods.  Being in a lab that looks at the relationship between epigenetics and transcriptional mechanisms makes me skeptical that bipolar disorder is simply caused by a genetic mutation.  Epigenetics play a very important role in changing gene expression, so I think it’s possible that an epigenetic change is also involved in causing bipolar disorder. The implications of this project are fascinating, partially because of the obvious possibilities that this project holds for potential bipolar treatments, but also because this knowledge might alter the public’s view of certain mental disorders and reduce some of the stigma that exists.  I look forward to hearing about what Yillin finds with regards to the role of Shank2 in bipolar disorder.

A day in the West Lab

My project uses computational biology methods, so basically I spend most of my time on my laptop while I am in lab.  My time is divided between reading papers, coding, troubleshooting and googling how to use certain softwares.  The papers I read are usually related to either background information for my project, such as previous experiments using viruses for cell-type specific gene expression, or about computational biology methods, in order to determine which softwares would work best to complete specific parts of my project.  I have to spend a substantial amount of time learning how to code for specific programs.  For example, when I was figuring out how to use the program bedtools, to analyze a large data set, I had to learn how to do command line coding.  This proved to be much more of a challenge than I expected, because I also had to learn how to properly install the program and then troubleshoot figure out why the files were not working with the program (they were in a slightly incorrect format).

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I also spent a large amount of time on the UCSC genome browser, searching for ATAC-seq peaks unique to a certain cell type.

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Now that I have identified putative enhancer regions from the published data, my time in the lab will likely shift to more wet lab based experiments, which I am looking forward to (even though I just got the hang of command line coding).

Understanding Enhancers and Gene Expression

Glossary:

Promoter: A short sequence of DNA located just before the transcription start site for a gene, which is responsible for initiating the transcription of a gene, generally by recruiting RNA polymerase, the molecule responsible for DNA transcription.

Transcription factor: A protein that binds to specific sequences of DNA to control the rate of transcription, either decreasing or increasing it.

Enhancer: Similar to promoters, enhancers are involved in triggering the process of transcription.  Enhancers are located within 100 kilobases up and downstream of the gene promoter, and are generally binding sites for transcription factors.

PV interneurons: One of three types of inhibitory, GABAergic interneurons (the others being VIP and SST), which can be identified by the expression of parvalbumin.

UCSC genome browser: A website curated by the University of California, Santa Cruz, that allows access to the genome sequence data of various vertebrates and invertebrates, including human, mouse, worm and fruitfly.  Various annotations are available to provide visualization of various genomic factors along with the genes.

Adeno-associated virus: A common vector for genes that can hold up to 5 kilobases of genetic information.

Assay for Transposase-Accessible Chromatin with high throughput Sequencing (ATAC-seq): A technique that uses transposase Tn5 to measure areas of chromatin with low nucleosome density, or in other words, areas of accessible chromatin where transcription factors could bind.

ATAC Seq fig

The overall goal of my project this summer is to attempt to create a virus that can trigger cell type specific expression of genes. Previous methods to create such a tool have tried to use promoter sequences to achieve cell type specific gene expression, however these tools were not able to generate gene expression strictly in the targeted cell type.  So, my project is to identify a sequence motif that is common to PV- specific enhancers, which can be included in the virus, along with a specific gene, to trigger cell type specific expression of that gene, instead of promoters. Although there is still a chance that this technology will fail, it is possible that enhancers will provide more cell specific gene expression. Promoters are more commonly regulated among cell types, whereas enhancers are likely more involved in cell type specific gene expression, based on differential epigenetic signatures.

The initial steps of this project involved identifying potential enhancer regions that are specific to PV cells. This process began by finding genes that are differentially expressed between PV and VIP neurons, as well as PV and excitatory neurons, using the data published by Mo. Once these genes had been identified, I used the UCSC genome browser, with the ATAC-seq data, also published by Mo, for the three cell types, to look for peaks that appear only in the PV cells, within 100 kb of differentially expressed genes.  This part was slightly tedious because I had to search for the peaks by hand. Once the peaks had been identified, I converted the peak data to sequence data and entered it for motif analysis in the MEME-ChIP software, which identified novel and known transcription factor motifs.  Once these motifs have been identified, they will be tested in vitro to ensure that the sequences are truly promoters, by the production of eRNA.  Once that has been confirmed, we will begin to build the viruses and test their effectiveness as a tool to trigger cell specific gene expression.

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The flow of the motif finding software, which is based on statistical methods, and then compares the motifs found to known transcription factor binding sites.

Interview with Dr. Anne West: The Importance of Following Your Passions

According to Dr. Anne West:

“There are three things to know about me:

– My compliment sandwich

-My perfectly teased hair

-My laugh”

Dr. West began her path to neurobiology research at Cornell University, where she started in the same place as most, as an undeclared freshman taking a broad range of classes.  She knew her main interest was science, so she started with the general courses in that subject: chemistry, biology and psychology.  As she learned more about psychology she found the underlying biochemical processes of the brain to be much more intriguing than the study of psychology. While taking courses in neurobiology, particularly those about how it relates to behavior and diseases, Dr. West found her passion for neurobiology and more specifically the neurochemical basis for decision making.

One lesson Dr. West has learned is the importance of incidental events, especially those that may occur when attending meetings. At a meeting about women in science, Dr. West learned about the Medical Scientist Training Program, which is a six-year program to earn both a PhD and an MD.  She applied to both MSTP and graduate school programs. In the end, she decided to attend Harvard’s MSTP because it would give her a chance to maintain broader horizons than if she were to attend graduate school, where she would have to choose a research question right away. Dr. West enjoyed the first two years of medical school, but when she entered the lab years of the MSTP, she fell in love with research. While studying protein trafficking in neurons, Dr. West went to a meeting where the concept of viral vectors was presented, which Dr. West was able to apply to solve a problem relating to her own research question, helping her to successfully complete her thesis.  After finishing her PhD, Dr. West returned to medical school and felt like an outsider, being older and an expert in something irrelevant.  She realized that medicine was not the path for her, so after completing her MD, she continued to a post doctoral program and then received a faculty position at Duke in 2005.   The greatest lesson Dr. West learned throughout the MSTP, is that it is much more important to follow your passions, because talent does not guarantee success, it has to be applied in the right setting.

Her research at Duke initially focused on the molecular mechanisms of neural plasticity, and now her lab’s view has broadened to use mouse models and develop a more holistic view of the process from gene regulation to neural development.  To Dr. West, the fun part of research is getting to follow your own passions and interests, but it is complemented by the challenge that sometimes people will be critical of your work, and you have to learn to enjoy the process of fighting for your research.  Dr. West is always aware of the possibility that her research may never lead to a great discovery. However, it is the possibility of that discovery, as well as a desire to understand the fundamental biochemical processes, that sustain her passion for research.

The Learning Curve

I walked into the West Lab on my first day, not really sure what to expect.  Generally, when people think of scientific labs, I imagine they think of people in white coats conducting experiments on cells or mice and mixing chemicals to make groundbreaking discoveries.  Or at least this is what I pictured growing up.

This past week I had my first entrée into the world of bioinformatics.  I have always thought the field sounded interesting and heard from others how important computational skills would be in research in the future, which is what made me intrigued in taking on the computational biology project proposed to me by Dr. West.  However, I wasn’t exactly sure what bioinformaticists do, other than analyzing large data sets and opening doors to whole genome research.  So, at first when my secondary mentor David described the first step of my project to me, I was a little disappointed because searching through spreadsheets for genes that are differentially expressed in multiple cell types didn’t seem like a very exciting project.  However, my perspective changed throughout the week as I progressed past the first step of the project.  I have come to realize that the work I am doing now will allow the wet lab experiments done later to be more effective, due to the background knowledge generated by my computational project.  I never pictured lab work as sitting at a computer, comparing maps of genomes, but I am now coming to realizing that I have so much more to learn about working in the field of science.

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I have learned that this is what computational biology looks like. This is an example of a section of a genome map that I am analyzing for my project.

I am hoping that throughout the summer, my understanding of lab research will continue to expand, as I hear about the variety of work being done within the West Lab, within the neurobiology department, as well as by the other BSURF participants.  As I realized this week, there is so much that I do not know about having a career in science, so I am hoping that this summer I will begin to understand what it means to be a scientist, the motivations of other students doing research and hopefully what I learn will help guide me in what I want to do after college. I am looking forward to all that I will learn this summer, from my work in the West Lab as well as from interactions with people inside and outside of the lab.