Summer 2018: Researching ALS across Different Model Organisms

By Sophia Nelson

One of the world’s leading neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS), causes its destruction of the nervous system in largely unknown ways. Recent research has shown that a hexanucleotide repeat, GGGGCC in intron one of c9orf72, is the most frequent genetic cause of ALS, with the number of repeats in affected patients ranging anywhere from hundreds to thousands. While these repeat sequences are found in the non-coding region of the gene, they still appear to contribute to toxicity through repeat associated non-ATG translation, which can begin at any point without the presence of the start codon, ATG. This unconventional translation allows repeats to be translated into five different dipeptide repeat proteins (DPRs): GA, GP, GR, AP, and PR. One of these proteins, GA, forms paranuclear amyloidogenic aggregates which have been found in the brains of human ALS patients. However, the direct role of GA—or any DPR— in disease pathology and toxicity is not yet known, particularly because the toxicity of GA varies heavily based on the model system in use.

As a Velay Scholar this past summer, I got the chance to work with professors Robert Fairman and Roshan Jain to investigate the protein GA through a comparative study characterizing the aggregation and toxicity of GA in three model systems: worms (C. elegans), zebrafish (D. rerio), and fruit flies (D. melanogaster). The bulk of my work was in worms and flies, as my fish have not yet grown large enough for testing! I expressed GA in the neurons— ALS is known to attack motor neurons, so neuronal expression is important to study— in each of the two model systems and then performed behavioral and confocal imaging analysis for comparison. In the imaging studies, I was looking for multiple things: firstly, the large, paranuclear puncta that are hallmark of GA aggregation; and second, the localization pattern within each organism. Behaviorally, I was attempting to understand whether or not GA expression within neurons was toxic by comparing organism performance in simple behavioral assays both with and without GA. For worms, this behavior was thrashing; for flies, it was the ability of larvae to crawl. I learned so much throughout the summer! I dissected fruit flies (both larvae and adults) and removed and imaged their brains, which are barely the size of a poppyseed. I learned behavioral testing across all three organisms, as well as PCR genotyping, staining and imaging techniques, confocal microscopy, and the beginning stages of biochemical assays as well, such as lysate preparation.

The dissection of fruit fly larvae and brains showed that GA was heavily concentrated within their developing brains, particularly in the progenitor of the neural column and the neuronal ganglia that develop and associate with the eyes. The confocal images of my fly brains were likely the coolest result of my whole summer! In worms, GA puncta is found throughout the body; heavily concentrated in the brain and along both the ventral and dorsal cord. Behavioral assays indicated that the presence of GA had a negative effect on C. elegans thrashing. Larval crawling data was collected and differences between controls and GA positive larvae were found, but this data is still being followed up on to conclusively determine any effects. Biochemical results, obtained through SDD-AGE, will be gathered this spring. Overall, this study indicates that the form of GA aggregation is consistent across species despite slight differences in localization, and the presence of GA appears to have a negative effect on behavior, indicating it may have a role in disease toxicity and should be tested further.

Performing these experiments was an incredible experience! My mentors were both amazing and taught me so much. It was my first time working in a research lab, and as a biology major with minors in neuroscience minor and health studies, it has only confirmed my interests further. After graduation, I now plan on going to medical school and graduate school for an M.D.-Ph.D so that I can continue to perform research while also gain a clinical perspective and directly help patients. I am so excited to be able to apply the knowledge I gained this summer (and will continue to gain in my last year at Haverford) in my future career and work with a topic that has the potential to have a direct impact on many people’s lives.

Dissected and removed fruit fly larval brain neuronally expressing the protein GA. The middle section is the progenitor of the neural column.

Summer 2018: Mitigating ACEs at Vanderbilt Medical Center

“It’s easier to build strong children than to repair broken men.” – Frederick Douglass

Adverse childhood experiences (ACEs) come in many shapes and forms, including neglect, abuse, and household dysfunction. But how influential are they in a child’s health outcomes? Research has repeatedly shown that ACEs can significantly affect brain health enough to contribute to cognitive impairment, risky behavior in adult life, and long term risks of disease and mental illness. Therefore, we move onto the next question: How do we mitigate ACEs? That’s when I come in.

ACEs can range from being parenting related, to environmental.

This summer, I’m working alongside Dr. Seth Scholer, a pediatrician at Vanderbilt University Medical Center Children’s Hospital. Dr. Scholer has spent over a decade conducting research regarding ACEs, and how to successfully assess and alleviate them through pediatric primary care. With funding from the state of Tennessee, my research this summer has mostly focused on a randomized control trial (RCT) in which we hope to demonstrate that a brief parenting intervention can reduce unhealthy parenting tactics, thus nurture brain health in the clinic’s patients.

The utilization of an ACEs Screening Tool can improve health outcomes of children by identifying and addressing ACEs early in life.

My personal research project this summer is definitely simpler than an RCT, but has its own challenges. All previous research utilizing ACEs screening tools have taken place in pediatric clinics associated with research institutions such as Vanderbilt. However, the next step from here is employing a screening tool state-wide, which requires additional research that addresses how to implement the screening tool in private medical practices.

Therefore, I have been implementing an ACEs Algorithm and screening tool at a private pediatric primary care clinic for my summer research project. The screening tool is a quick survey that measures a child’s household/environmental stressors, and the degree to which their parent(s) use healthy discipline strategies. The ACEs Algorithm helps health-care providers interpret their patients’ scores, and points out when children are at low-high risk of ACEs. This is the first research study of its kind, and it requires working hands-on with the doctors and nurses at the private clinic to maximize the efficiency and effectiveness of the screening tool. Overall, this project has been a great opportunity to work along the front lines of ACEs research.

Health care providers use this ACEs Algorithm to interpret a child’s parenting-related ACEs and environmental ACEs (or other childhood stressors), after their caregiver completes a short ACEs Screening Survey. I worked with Dr. Scholer on the development of this algorithm throughout the summer, and this image is our final result.

As a Psychology major with minors in Neuroscience and Health Studies, this research experience perfectly fits the little niche formed from the intersection of my three fields of study. A typical day for me involves lots of patient/provider interaction and data management, with some manuscript and literature review writing stuck in between. This has helped me build concrete clinical research skills that are hard to learn in a classroom. Furthermore, I’m ecstatic about my ability to work within a research topic that is having a direct impact on people’s lives.

Aller guten Dinge sind drei*: 22 Prepositions, 264 Sentences, and One Test

—Claire Dinh ’16
*All good things come in threes.

Wie sehr schnell die Zeit vergeht! In German, that means, “How very quickly time flies!”

I can’t believe that summer is already over and that my last year at Haverford has begun. I really do have to thank Dr. Chatterjee and Dr. Jamrozik for making my first experience as a lab intern absolutely wonderful. But while my internship is over, my research experience is not. This fall, I am continuing my work at the Center for Cognitive Neuroscience!

But before I move on to a discussion of what we have completed so far and what we still have to do, here is a brief reminder about what we are trying to study. Let’s talk science!

BRIEF REMINDER ABOUT WHAT WE ARE TRYING TO STUDY
In my last post, I wrote that Dr. Jamrozik and I plan to create a test that will gauge the abilities of those taking it to use different kinds of prepositions (spatial; temporal; and abstract, metaphorical). The individuals with whom we plan to work are patients who have lesions in the parietal-temporal regions of their brains. We ultimately would like to use the test to answer the following question: Do impairments of spatial, temporal, and abstract prepositions co-occur, or can these uses be independently impaired?

Now let’s move on to a recap of what we achieved over the summer.

WRITING THE TEST
Over the course of 12 weeks, we went through a total of 10 iterations of our test—and it still is a work in progress. You may be wondering why the process of writing our test has been so tedious. Well, we need to make sure that it will measure what we want it to measure. The validity of our test determines the validity of our results, which, in turn, will tell us if our hypotheses have been supported or not.

The first step in writing our test was identifying the 22 most commonly used English prepositions. We used two corpora (two large sets of naturally occurring speech and writing) to accomplish this. We then proceeded to write sentences that we eventually will turn into fill-in-the-blank sentences for our final multiple choice test.

So a sentence testing whether or not someone can produce a spatial use of the preposition on would look something like this.

The book is _____ the table.
(a) at
(b) on
(c) through

Correct answer: (b) on.

For each preposition, we drafted four sentences per each use, for a total of 264 sentences (22 prepositions x 3 uses/preposition x 4 sentences/use = 264 sentences).

We did our best to write sentences that included preposition meanings that had been repeated across sources (we took note of such occurrences when conducting our lit review). We wanted to ensure that our sentences were as reflective as possible of everyday speech.

There were a number of other factors we had to consider when writing our sentences. Here are a few.

  • Word order: For the most part, our sentences had the following structure: article + noun + verb + preposition + article + prepositional object (e.g. The cat is in the box). Sometimes, though, it made sense for us to also include a direct object (e.g. The boy threw the ball over the fence) or omit an article (e.g. Monday is before Tuesday).
  • Frequency of verbs: We limited the number of verbs that we used, as they should not be so obscure that our patients will not understand them. We also were consistent in the frequency of our use of the verb “to be” across the different preposition uses.
  • Types of nouns: Prepositional phrases name a relationship between a figure and a ground (e.g. in the sentence, The cat is in the box, the figure is the cat and the ground is the box). In our sentences, we wanted to make sure that our figures were count nouns (i.e. nouns that can be counted), which means we never used mass nouns like coffee or happiness as figures (though coffee does make a lot of us happy). We also wanted to make sure that our grounds were reflective of the preposition use being tested. For example, a sentence demonstrating an abstract use of a preposition would have a ground that is an abstract entity or concept, as demonstrated by the sentence, The café is popular within the community, where the noun community is an abstract ground.

After writing our 264 sentences, we began to norm them. We asked healthy participants to rate how natural each sentence sounded to them, and for each set of sentences for each use of a preposition (e.g. we have four sentences demonstrating a spatial use of the preposition in), we asked our participants to select the two sentences that they believed best represented common uses of that preposition. Ultimately, we would like to cut down the number of sentences we have by about half, so that our patients will not be too overwhelmed by the test.

And that is where I am today! But before I leave you with a Tschüss! for the next several weeks or months, I would like to send a special shout-out to Misha and Melissa, my fellow undergrad interns at the CCN. I really enjoyed spending time with them during my internship and at the other places we visited in the city!

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From left to right: Cody, Misha, Melissa, and me enjoying music at Festival Pier.

Aller guten Dinge sind drei*: German, Preposition Metaphors, and the Brain

—Claire Dinh ’16
*All good things come in threes.

WILLKOMMEN!
In German, that means, “Welcome!”

As this is a science blog, you may be surprised to learn that I am a German major. That I have been able to explore all of my different academic passions at Haverford is one reason why I have found my liberal arts education so rewarding. I learned German at Haverford, while taking courses like Chem225 and Bio200, which deepened my love for scientific inquiry as a means to better understanding ourselves and the world around us.

I have taken my knowledge of these different disciplines with me to Penn’s Center for Cognitive Neuroscience, where I am spending my summer in the lab of Anjan Chatterjee, MD (Class of 1980). Under the direct guidance of Anja Jamrozik, PhD, I am studying the neural bases of preposition metaphors. I am taking an interdisciplinary approach to my project here (which is super cool!).

I hope you will join me as I embark on this new adventure! But before we get started, I will address a question you probably have—

WHY GERMAN?
As a child, I spent countless hours bonding with my dad over soccer. In 2006, the German national team first caught my eye. I knew that they were something special, and it was only a matter of when they would become world champions (and eight years later, they finally did!). The more interviews I watched of my favorite players, the more curious I became about that beautiful language they were speaking.

So I made learning German one of my goals at Haverford, which proved a real challenge at times. I had to wrap my head around the fact that German nouns have genders, but at least I could fall back on memorizing them. Prepositions were different. Their meanings changed depending on context. For the notorious two-way prepositions like “auf,” the presence of motion in a sentence dictated what the article and adjective ending preceding the prepositional object were. Yet it was these very complexities of the German language that also fascinated me. I had to rethink my way of experiencing the world, and I loved it. I therefore decided to declare German as my major.

Before I move onto my discussion of my research project, however, I should mention that learning the German language is only one aspect of German studies. In the past three years, my love for German culture has grown immensely; and I have Professors Ulrich Schönherr, Imke Brust, Brook Henkel, and Kathleen Wright, as well as the Gotthardt family in Berlin, to thank for that.

WHY RESEARCH ON PREPOSITION METAPHORS AND THE BRAIN?
When the topic of studying abroad came up in Intermediate German, Professor Schönherr reminded us that such an experience allows students to not only learn about another culture but also reflect on their own. I soon asked myself if I could apply his same comment to language. After all, as speaking and writing in German became more natural to me, my native language of English began to sound and look very odd. Why was it that in Professor Ashly Bennett’s class on the novel, we used phrases like “marry off” or “break up” to describe the characters’ relationships? Why was it that during finals week, when I asked my friends how everything was “coming along,” a common answer was that they were “pulling through?”

Never before had it occurred to me to think about how my professors, friends, and I used language to communicate with one another. We used preposition metaphors like “coming along” all the time. We immediately knew to interpret them abstractly rather than take them literally.

That all said, I am absolutely thrilled to be at the Center for Cognitive Neuroscience this summer. By studying preposition metaphors on a neural level, I hope to demonstrate that they are not just adornments to our language, and that such abstract language is more than just “useful” to us. It may actually be fundamental to how we communicate with one another as well as how we understand the world to be.

WHAT DOES “THE NEURAL BASES OF PREPOSITION METAPHORS” EVEN MEAN?
Now that you know more about my background and approach to this project, let’s talk science! I am investigating whether or not the brain uses the same areas to process spatial uses of prepositions (e.g. the cat is in the box) and abstract, metaphorical uses of prepositions (e.g. he is in love). There are also temporal uses of prepositions (e.g. it happened in one second) that are important to keep in mind.

I will be working closely with patients who have parietal-temporal lesions on their brains. If there are any patients who can correctly produce only abstract, metaphorical uses of prepositions but not spatial uses of prepositions (and vice versa), then we can conclude that a dissociation in the brain exists between the neural bases of these two kinds of prepositions.

In my first several weeks here, I read a number of studies and other scholarly works on the different ways in which we employ commonly used prepositions like “over” and “under.” I started with “Network analyses of prepositional meaning: Mirroring whose mind— the linguist’s or the language user’s?” by Sandra and Rice (1995) , and I recently finished reading “Well-Hidden Regularities: Abstract Uses of in and on Retain an Aspect of Their Spatial Meaning” by Jamrozik and Gentner (2015). This background information will help Dr. Jamrozik and me as we prepare tests for patients that will gauge their ability to use either kind of preposition. The findings from our tests will be applicable to our understanding of the uses of prepositions in the real world. For example, Coventry and colleagues have shown that we tend to use “above” and “below” when talking about where things are located, whereas we tend to use “over” and “under” when talking about how things relate to each other and fulfill the purposes we attribute to them (e.g. “the Viking is under/below the shield”; 2001). It therefore will be important for us to take into account such distinctions between prepositions when we prepare our tests.

Figure 10 from Coventry and colleagues' "The Interplay between Geometry and Function in the Comprehension of Over, Under, Above, and Below" (Journal of Memory and Language, 2001).

Figure 10 from “The Interplay between Geometry and Function in the Comprehension of Over, Under, Above, and Below” (Coventry et al., 2001).

My summer work so far has been quite a challenge, as I do feel I am learning an entirely new language. It is filled with terms and concepts like Coventry and Garrod’s (2005) “functional geometric framework” I never would have imagined could exist! There is still a lot of information for me to digest— and I cannot wait to be at the level where I can ask Dr. Jamrozik the kinds of questions that will really probe my understanding of the material before me.

That said, learning about the evolution of the different theories on our uses of prepositions certainly has been an adventure. This intersection between cognitive psychology and linguistics is unchartered territory for me. I have had a lot of fun building the foundational knowledge I need to really tackle this research question head-on.

One last thing before I leave you with an Auf Wiedersehen! for the next several weeks—

The story of the Class of 1970 is truly inspiring, as I learned on Alumni Weekend. I also learned from them that you should not have any fear in doing what you love. Loving what you do is doing what is right by you. Go where you think you are meant to be in life; your Haverford education will help you to get there.

Haverford College Class of 1970