Category Archives: Uncategorized

Regulation of System xCT (SLC7A11) by ABL Kinases in Human Lung Cancer

The ABL family of non-receptor tyrosine kinases, ABL1 and ABL2, are upregulated in non-small cell lung cancer and promote lung cancer metastasis. Recent work has shown that ABL kinases promote lung cancer brain metastasis and colonization. Global transcriptome analysis of targets controlled by ABL kinases in lung cancer cells revealed SLC7A11 as being differentially regulated in ABL inhibited cells. SLC7A11 encodes the plasma membrane antiporter system xCT which has been shown to be overexpressed in cancerous cells. System xCT exports glutamate and imports cystine, an intermediate molecule in the cellular oxidative stress response mechanism. Real-time polymerase chain reaction (RTPCR) verified a reduction in SLC7A11 mRNA levels following ABL pharmacologic inhibition or genetic knockdown. Continued work is being done to reveal a reduction in cellular protein levels through protein isolation and western blotting. FACS analysis will be deployed to determine whether membrane-localized levels of SLC7A11 decrease upon ABL inhibition. Additionally, a glutamate assay kit will be utilized to determine whether export of glutamate by xCT is reduced in ABL knockdown cells relative to control. The ultimate goal of this project is to understand if ABL kinases promote SLC7A11 expression in order to alter the brain microenvironment and promote tumor colonization.

Abstract and Progress

I’m happy to say in the past few weeks I’ve made some progress with my project. While the beginning was a pit rocky, after a few rounds of optimization and discovering we were using a faulty antibody results have started to come in and I couldn’t be more excited. What I’ve been told is that’s kind of how science goes and that a lot of results come in very quickly towards the end of a given amount of time. I’m excited to see what comes out of these last two weeks!

 

Ubiquitination is a cellular response to damaged proteins caused by oxidative stress. But the mechanisms and targets of different ubiquitin structures have not been well characterized. This research investigated the ubiquitinating enzymes and relative targets related to K48 ubiquitination, a signal for protein degradation, and how the efficiency of relevant molecular machineries, the proteasome and deubiquitinating enzymes, is impacted by different levels of oxidative stress. This was done by using mutant yeast strains with ubiquitinating enzyme knockouts, exposing them to oxidative stress, and analyzing their K48 ubiquitin content with a western blot. Ribosomal isolation and K48 ubiquitin antibody tagging was used to examine ubiquitin targets. Proteasome and deubiquitinating enzyme activity was analyzed using a substrate that fluoresces when processed and tracking light levels emitted by stressed cell lysates. Our data suggests the K48 ubiquitination system involves multiple ubiquitinating enzymes and genes and the greater impact of oxidative stress on the deubiquitinating enzymes than previously anticipated. Improving our understanding of K48 ubiquitination in stressed cells could allow us to better understand diseases such as Parkinson’s and Alzheimer’s that are caused by protein aggregates in stressed cells, and potentially provide new targets for treatment to make cells more resistant to stress.

 

A Day in the Heitman Lab!

I have loved every moment of of my research project this summer. Although its been challenging adjusting to new terminology, vocabulary, and other hands on task, I could not ask for a better opportunity. My days in the lab have been pretty consistent for the most part, with few variations from the norm.

First I arrive around 10:30 where I go straight to my notebook to make sure I have documented everything I have done from previous days. Once I have done this I have a talk with my mentor, Ci, to understand what my next steps are in the project. Once we have had our discussion and I am sure that I understand what we are doing and the reasons why I begin setting up and preparing to run the experiment (most days this would be a PCR overlap). I write in my notebook every reagent that I will be using in order to ensure that I do not make any mistakes. Once I have completed this task, I begin conducting the experiment. I transfer the correct amounts of each primer and DNA template based on my master mix into PCR tubes.

Once I am sure that the correct reagents are in each tube I then place these tubes into a thermocycler (also known as a PCR machine). I program the machine based on the protocol of the polymerase that I am using. Once I confirm the time and temperature of each step, I start the machine to allow it to do its task of amplifying and constructing the DNA strands.

After the machine has finished amplifying the DNA strands, I take each PCR product and prepare it for gel electrophoresis. I do this by adding 5 micro-liters of 10x dye to each tube. Once this is finished I transfer each reagent into a separate well in the gel and run the gel in the electrophoresis machine.

Hopefully after doing this process I have successfully constructed and amplified the DNA strands that I need to continue my project. To confirm whether or not I have done this, I take a look at the gel using Ultraviolet light and see whether the position of the bands compared to the ladder matches my predicted outcome. If not, then I have to go back to the drawing board to see what went wrong. If so, then I will purify each PCR product that came out successfully and then continue on to the next step in the project. And that’s my day!

A Day in the Lab

Every day I spend in the lab is different. So it’s hard to choose a prototype of my day. The protocol for my lab work usually runs on a weekly basis and each day is an incremental step of completing a western blot in combination with optimizing previous western blot procedures and troubleshooting problems with antibodies and film resolution. I start my morning by doing prep-work, usually getting ice, labeling tubes, or turning on centrifuges, and thinking through what I have to get done for that day. On our lighter days of the western protocol, we conduct additional experiments that analyze K48 ubiquitination from different approaches. On these days, a lot of coordination is required to keep the timing correct on both experiments.

When people have a break in their work, around 12 PM, everyone in the lab goes for lunch together. Having lunch together is very important in my lab. They want to facilitate a culture that encourages us to just hang out outside of the lab in a more casual setting. It’s one of my favorite parts of the day. Besides the fact that by noon I’m starving and in need of my third coffee, I like to get a chance to talk to the members of my lab about their weekend and random topics without the immediate distraction of experiments in the background. The dynamics of the lab are great. Since it’s a relatively new lab, there are only 5 people in it: my PI, the lab manager, a postdoc, another summer undergraduate research assistant, and me. This has allowed us to get to know each other better due to such close contact. Some days we’ll take a break and go out for coffee and watch the world cup game that’s on. In the Silva lab, it’s not just about developing the ability to work with one another, but also developing relationships between lab members.

After lunch, we pick up where we left off on our experiments. I’m usually kept really busy while in the lab, running around mixing solutions and processing samples. More often than not, Latin music is being played in the lab while we work and sometimes big world cup games are streamed over the speaker. It’s a really good environment to work in and I’m really happy to be there.

Stage 4: A Quick Intermission

Although a life of research often leads to an extremely narrow field of expertise, reading and learning about new research and knowledge from other, sometimes completely unrelated, fields can be both exciting and intriguing. One such example is Ayana’s project on Cryptococcus neoformans, specifically on searching for links between the BZP4 gene and their virulence in humans.

C. neoformans is a very common yeast, often found clinging to the dirt and other plants and animals. While widespread, infections from the fungus is rare, as most people’s immune systems are capable of defending against the pathogen. However, occasionally, they opportunistically infect the lungs, which can then spread to the central nervous system where the fungi can cause meningitis or encephalitis- two very dangerous conditions. Therefore, Ayana’s lab is researching the pathology and potential factors that could help the development of some form of prevention or cure. Currently, her project revolves around the curious BZP4 gene within C. neoformans, as the gene has been previously known to fluctuate in gene expression levels in different conditions and upon knockout, the virulence of the fungi disappears.  Thus, she has decided to investigate its relationship to the virulence of the fungi by directly interfering with it function.

In Ayana’s first aim, she hopes to confirm the link between virulence and BZP4 in C. neoformans, by taking a BZP4-knockout strain and blasting it with the BZP4 gene to observe for a recovery in lethality. This is cool primarily because it involves the use of a gene gun, which is just a really awesome machine that exists apparently, transforming cells with DNA by blasting them with a gene-coated bullet- and it works! At some point in my life, I need to devise a transformation experiment that requires that machine just to see it in action. Furthermore, her work would further pinpoint a site of target for treatments of C. neoformans infections, which is a monumental success in the world of disease prevention and care. Additionally, it could demonstrate a stronger link between virulence and the BZP4 gene, as the gene’s interactions and expression post-transformation could be identical to pre-knockout levels which would indicate greater independence between the gene’s mere presence and virulence over the possibility of gene expression interactions and post-translational activity with nearby genes being the source of virulence.

In Ayana’s second aim, the goal is to determine if there a competitive advantage given by the BZP4 gene. To study this, she will be inoculating a BZP4+ strain of C. neoformans and a BZP4- strain in close proximity, and measuring their growth and interactions upon reaching one another. This will perhaps give some insight in the purpose of the BZP4 gene, as not much is known beyond the excitatory effects it has on melanin production, which at most can be suspected to boost tolerance to environmental oxidants such as UV radiation. This is also important because the results can be used to determine if virulent C. neoformans growth can be stymied by the introduction of nonvirulent BZP4- strains into important sites of infection. However, something I wondered in this component of the project but did not remember to ask was the activity of BZP4 in various environments. As antioxidants are so versatile and diverse, many function differently in different conditions, so I wonder if there are any environments where BZP4 activity is optimized, boosting the vitality of C. neoformans in that environment. Similarly, which environments is activity dampened in? This could perhaps explain what BZP4 is specialized for (or what the melanin is meant to do) and what pathway exactly that it manipulates in the body to cause so much damage. Studies like these fascinate me and remind me of just how essential research is to the field of medicine, even in straight biology settings. While not at all related to my research in Alzheimer’s disease, this project gave me some energy and exciting plans to bring to my work.

Weekly Updates

“A piece of my gel fell on the floor once and I didn’t notice, so renovations re-varnished the lab. Now it’s forever imprinted on the floor.”-Stuart Sundseth detailing one of many Western fails

“I accidentally threw my gel into the sink once.”-Stuart Sundseth

“I’ve torn a gel before”-Stuart Sundseth an hour before tearing another gel

“Have fun at the beach this weekend!”-Christine O’Connell
“Wow, you’re going to the beach again?”-Dang Nguyen
“Yeah, I left some potatoes in the cupboard”-Joan Wilson

“I feel…so dizzy…I think…I might fall over”-Hui Fang as she aggressively hoses the floor with liquid nitrogen

“And now I’ll just seal up this bad boy and throw him in the cold room for the-” *throws dead spider in box at Dang* “-night and let him blot since I can’t find the antibody.”-Stuart Sundseth
*graphic screaming*-Dang Nguyen

Sea Urchins, Baboons and Mice, Oh My!

Over the past week, myself and other members of the BSURF program had to give chalk talks; 8-minute talks about our project with nothing but a whiteboard and a marker. Even though I was nervous about giving my talk, I was looking forward to the talks. I had previously talked to some of the other students about their projects, but I wanted to know more about everyone’s project. Hearing about the diverse research opportunities gave me the opportunity to expand my knowledge on topics including Alzheimer’s and sea urchin embryos.

One talk that I found fascinating was entitled “Neural Circuitry for Vocalization in Mice”. The chalk talk was given by Alina Xiao, who is working in the Mooney Lab. Her project involves looking at a specific area in the brain to see if it is activated when male mice vocalize, whether it be for defense purposes or courtship. The project got my attention because humans vocalize all the time, yet I rarely think about how and why other animals vocalize. Seeing how mice use this trait compared to humans is interesting. I also enjoy learning about how how the activation of different pathways illicit different responses in the body. Besides being fascinated by the project, I was also impressed with the quality Alina’s talk. It was apparent that she had thoroughly rehearsed her talk and she enjoyed what she was doing. I cannot wait to see the results for her project at the end program.

Overall, everyone gave an amazing chalk talk. Even though we have four more weeks until we present our results (we hit the halfway point!!), I am excited to see how everyone’s projects turn out.

The Different Faces of Science

This week, I heard about the amazing research that my fellow BSURFers are conducting while listening to their riveting chalk talks. I learned about neurobiology, plant biology and all the different topics in between. Though all of the presentations were phenomenal, the one that stood out to me was Michelle’s talk. Her project this summer is on knocking out the gene Endo16 in Sea Urchins and whether or not that would affect gut development.

Endo16 is a gene in sea urchins that is fundamental in gut development. Her question is whether knocking out Endo16 will result in reduced gut development in sea urchins. To do this, she will design guide RNAs and inject them along with the Cas9 complex into sea urchin embryos with the goal of causing an indel in that particular gene. With a large deletion or insertion, a frameshift mutation may occur, causing a premature stop codon, and rendering the gene useless thus “knocking it out”. She will then monitor the embryos with a knocked out Endo16 gene to determine if gut development is able to carry on as it normally would or if the lack of the gene prevents the formation of a gut. Determining the function of this gene will aid in understanding how embryonic development works in sea urchins and could lead to information about its interactions with other genes. Her project will expand the Genomic Regulatory Network and she can continue to test other genes with unknown functions.

I enjoyed Michelle’s presentation because I saw the way it related to my own research project. The use of a CRISPR-Cas9 system to knockout a gene and test its function is a real-life application for the technique I am studying. While using CRISPR for medical treatment is what my lab focuses on, hearing about the many other ways it can be used for research makes me appreciate my project even more. I loved hearing about the different methods and aims for her project because I understood everything she discussed. I’ve had to design my own guides and I can relate to the various steps in her procedures. I am amazed by the various ways this genomic tool can be used, as determining the function of a gene is important for understanding its part in genetic diseases. By doing a gene knockout and finding its function, we grow one step closer to unraveling the mysteries of biology.

Everyone gave an amazing chalk talk

This week, I had the opportunity to listen to everyone give a chalk talk about their project.  It was nice to understand details about their project,  such as Alzheimer’s, and DMD instead of thinking, okay he works in an x,y,z lab.  Additionally, I enjoyed how everyone was able to present such complex topics in a short amount of time in a way that was easy to follow.

As someone who attended the NC Zoo school, I had the opportunity to go into the zoo on a daily basis.  If time allowed me to, I enjoyed going to the African side of the NC Zoo to watch the chimpanzees and baboons.  When Christine presented the idea that both the highest ranking (alpha) and lowest ranking baboon experiences the same amount of stress, but it might be due to a different kind of stress I was all for it.  Over the summer she is planning to answer the question, “Do alpha baboons and low ranking baboons experience different stressors?”.  She is planning to look at energetic stressors, so she will analyze a thyroid hormone(1), T3, in a wide range of baboon fecal samples. Another thing that caught my attention about the presentation, was the fact that there was a significant decrease in stress from the alpha to the second rank baboon (beta).  Finally, it was great to learn the behind the scenes of this research project.  She talked about how they have an area in Kenya and scientists in Kenya watching the baboon’s behavior (looking for changes in rank), collecting the samples, and shipping them back to Duke for analysis.

This is an amazing project and I can not wait to see the outcome of this project and other projects on July 27, during the poster presentation.

Next week, I will provide a glimpse into my daily routine (A day in the life).

1-thyroid hormones allow you to indirectly measure energetic stress because it is based on changes in metabolism.

So Baboons are like really cool!!

I really enjoyed listening to everyone’s talk this week! I learned a lot about neural circuitry, oxidative stress, and even the work that goes into gRNA design! One project that really stood out to me was Christine’s, titled: Identifying differences in Stress between Alpha and Low-Ranking male Baboons. Specifically, Christine is looking at whether or not the alpha and the low-ranking male baboons experience different sources of stress. I think what I really like about this project is the fact that it incorporates both a psychological and a biological perspective in addressing this question. Furthermore, I can really see the direct application this study will have on humans in terms of understanding stress and factors that cause it.

From my understanding, Christine’s hypothesis is that the alpha and the low-ranking male baboons do experience different forms of stress: with the alpha experiencing energetic stress and the low-ranking baboons experiencing psychosocial stress. When comparing the two baboons, she expects the alpha male to have lower levels of triiodothyronine because energetic stress suppresses this hormone. Last spring, I took an introductory neuroscience class, and we learned about the hormonal response to stress, and I think that it is awesome that Christine is applying her knowledge of this response to stress to understand how it is affected across different types of stress (psychosocial, energetic, etc.)

I think Christine was very clear in her explanation of her project, which I find to be really important in presenting a project because if you can’t convey your message in easy-to-understand words, what’s the point? She delivered her presentation in such a way that someone who is not in her field would understand it perfectly.

Interview with Dr. Ann Marie Pendergast

Last week, I had the opportunity to speak with my PI, Dr. Pendergast, about her path in science and the work that she currently does. She developed an interest in research, and particularly in Chemistry and Molecular Biology, during her undergraduate studies at the University of Michigan at Ann Arbor. She received her PhD in Biochemistry from the University of California at Los Angeles. Dr. Pendergast remained at UCLA for her postgraduate work in Molecular Cancer Biology. It was under the mentorship of Dr. Owen Witte that she developed an interest in normal and oncogenic tyrosine kinases, which eventually led to her focus on the Abelson family of tyrosine kinases. Dr. Witte discovered the tyrosine kinase ability of the ABL protein, as well as the role of the BCR-ABL fusion protein in leukemias. The Pendergast Lab researches the role of ABL in a wider range of cell signaling pathways. Dr. Witte was himself mentored by the great biologist Dr. David Baltimore, who received the 1975 Nobel prize in Physiology or Medicine for his work on tumor viruses.

The Pendergast Lab consists largely of graduate students. I asked Dr. Pendergast what she values most in students looking to join her lab. She emphasized the importance of understanding what scientific questions to ask. What distinguishes the best scientists, she said, is their ability to formulate important and appropriately ambitious questions, and not to lose sight of how each experiment is connected to these bigger picture questions. Although Dr. Pendergast enjoys advising her students and guiding their work to an extent, she understands the importance of giving them the freedom to be creative and design projects without restrictive instructions. Ultimately, she wants her students to gain the ability to formulate and answer scientific questions independently.

I asked Dr. Pendergast for her advice on pursuing a career in medicine versus one in medical research. She suggested that pursuing an MD-PhD gives you a greater range of opportunities than solely an MD (or solely a PhD, for that matter). For one, being qualified in both fields gives you far greater career security, particularly in difficult research funding climates. Additionally, physician-scientists have an edge in that they understand the clinical aspects of medicine to a far greater extent than pure researchers, and the scientific aspects of medicine to a far greater extent than pure doctors. The clinical side gives them an understanding of what scientific questions are most important (in that they can have the greatest impact impact on healthcare), while the scientific side enables them to actually address these questions.

Dr. Pendergast also suggested that researchers have a more exciting job than physicians. While doctors perform the same techniques again and again, researchers ask new questions and explore the scientific unknowns. A similar point was made to me by one of the lab’s MD-PhD students. Having experienced both the hospital and lab environments, he argued that researchers, to a greater extent than doctors, make use of the prefrontal cortex which gives humans our enhanced ability for complex thought.

Interview with Dr. Hammer

As mentioned in my previous post, this summer I have been given the opportunity to work in the Hammer lab within the Department of Immunology. The lab is run by Dr. Gianna Hammer and I had the pleasure to interview her this past week. Before I started the fellowship, I had the opportunity to read about her research and her amazing accomplishments, which include being named a Pew Scholar in 2015. I remember being excited to work with such a talented individual, which is why I was excited to have the opportunity talk to her about her research endeavors.

Dr. Hammer went to Eastern Washington University for undergrad and received her Ph.D. from UC Berkley. She, at first, was interested in microbiology but switched her focus to immunology after taking a class in the subject. She liked how immunology was relevant to the discoveries going on. During her academic studies, people were starting to realize that the bacteria in the body had critical functions. The science is what lead her to the intestines. Throughout her research, she kept questioning all the results she obtained. The questions that she asked in her research led her to investigate the role of the immune system in the intestines.

During our interview, I asked Dr. Hammer what her most memorable experience has been thus far. The experience happened to be when she wrote her first manuscript while in graduate school. She wanted her PI to check over every section of the paper as completed them but they refused to read it until it was complete. When her PI finally read the paper, he had many critiques, but this process ended up being transformative. Dr. Hammer was able to learn the most compelling ways to present research to an audience and how to precisely explain data/results. The tools gained while writing this paper have stuck with her throughout her career.

One of my goals, as mentioned in my first blog post, is to learn what to do after failure. I asked Dr. Hammer “How do you overcome/embrace failure in the research field”. She responded by saying “Failure comes in many forms”. Failure could be an experiment that did not go the way you expected or being rejected for a grant. She said that disappointment comes but its important to keep an open mind. Sometimes the failed experiment allows you to focus on a different aspect of a topic. Dr. Hammer also said to talk to the science with other people because sometimes they will notice something that you didn’t.

Since I am new to research, I asked Dr. Hammer if she had any advice for students like me. The first thing she told me was not to be afraid to diversify. Try out different aspects and types of research. She also said to pursue research because it’s what you love. Research does not always produce rewards immediately, it can take years before the benefits appear. If you do not love what you are doing, then this work can become miserable.

Interviewing anyone who has had success in a field that you’re interested in is always such an eye-opening experience. I am beyond thankful for the chance to talk to Dr. Hammer about her experiences in research.

Dr. Gianna Hammer (Source: Department of Immunology, Duke School of Medicine)

Interview with Connor

My mentor–Connor — is from North Carolina and is about to finish his final year at Duke. He plans on majoring in biology with a minor in neuroscience. According to him, his interest in these two fields began when he first took an introductory neuroscience class during the fall of his freshman year. At this time, he was in the Pratt School of Engineering pursuing a major in environmental engineering. While he did like engineering, he knew that he also had an interest in the intersections of biology and neuroscience. After taking a medical leave for six months, he returned back to Duke and transferred to the Trinity College of Arts and Sciences, where he sought out to study this intersection. He credits his medical leave as a major factor in deciding to pursue a career in medicine.

While Connor is pre-med, he plans to take a gap year to potentially work in the research field as a lab manager. Other possible plans he has thought to pursue are temporary careers in technical work, medical scribing, and in general, pursuing activities to strengthen clinical experience. When I asked Connor what piqued his interest in getting involved in research, he expressed his desire to examine the quantitative aspects of biology and neuroscience– engaging in quantitative research experiments to answer and even create questions. He didn’t want to just read about research experiments in the textbook- he wanted to see and to even conduct research that was related to neuroscience and biology.   So, Connor emailed professors and interviewed with my current PI, Dr. Calakos, and was interested in the way she conducted the interview–she asked questions that would challenge his scientific mind and cause him to think in a different way. Once he was comfortable working in the lab, he then started taking on independent projects.

During our interview, Connor also elaborated a bit on his first research experience, any lab disasters the has had so far in his research experience, as well as any accomplishments he is proud of. His first academic research experience was during the pre-orientation Project Search. During the program, students learn valuable lab techniques by doing the labs from Duke’s molecular biology class. Similar to BSURF, students listen to faculty talks and participate in Intro to Science Seminars. So far, Connor has not had any major lab disasters, but he says that he sometimes falls into autopilot mode when doing tedious benchwork, especially during a western blot analysis and pipetting samples.  Finally, in terms of accomplishments, Connor is most proud of his ability to take on independent projects in a lab as an undergraduate and working on things that no one else in the lab is doing, as well as “pioneering the way” for certain experimental protocols.

Week 2- The revelation of my project

Week 2 is complete and as promised in the previous blog post, here are some details for the upcoming project .

To begin, I will give you some background.  In cells, things are always in motion, whether it is RNA leaving the nucleus or proteins transferring from the endoplasmic reticulum (ER) to the Golgi apparatus (Golgi), or vise versa.  For this particular project, we are interested in the forward trafficking, i.e. from the ER to the Golgi.  This forward trafficking pathway is mediated by the Coat protein complex II (COPII).  COPII has five major components:Sar1, Sec13, Sec23, Sec24, and Sec31.  Additionally, posttranslational modification plays an important role in the regulation of the COPII complex. Recently, our lab and others have found that O-linked β-N-acetylglucosamine (O-GlcNAc), a single sugar modification added to serine and threonine residues of intracellular proteins, decorates many COPII components.

Now for the reveal of my project!  I will look at O-GlcNAcylation of Sec24D, a cargo-binding subunit of the COPII complex, because in humans, Sec24D mutations cause a subtype of osteogenesis imperfecta (also known as brittle bone disease), a collagen trafficking disorder. I will do this by answering the question, “What role does Sec24D O-GlcNAcylation play in collagen secretion?”

Here is how I hope to answer the question. I will first create a Sec24D knockout cell line using CRISPR-Cas9 genome editing. Then I will express unglycosylatable Sec24D mutants in the Sec24D knockout cells. Finally, I will determine the role of Sec24D O-GlcNAcylation in collagen secretion by using immunofluorescence microscopy and measuring collagen trafficking to the Golgi.

Here is what I think will happen after this project is complete.  I believe that mutations in Sec24D O-GlcNAc sites will cause collagen secretion to decrease. By doing so, I hope to enhance our understanding of the impacts of O-GlcNAcylation on the function of Sec24D in collagen secretion and allow for future questions on the impacts of O-GlcNAc on other COPII proteins.

Also, check out this paper similar to our project but on Sec23 from our lab.  It is filled with additional background information, and methods that I will be using.

This has been a very exciting two weeks, and I am looking forward to the next six weeks!

On next weeks blog we will have a celebrity interview, so please check it out.

What Am I Doing?

Week 2 of working in a lab is over and so far, it’s been a wonderful experience. Everyday I’m learning something new, which is always exciting.

As mentioned before, I am working in the Department of Immunology in Hammer Lab. This lab specializes in looking at the role of the immune system in the intestines. I will be joining onto a research project that involves dendritic cells.

In the intestines, you will find T-cells and B-cells, but you will also find mononuclear phagocytes (cells that engulf other cells) including macrophages (Mϕs) and dendritic cells (DCs). Both DCs and Mϕs are need for induction of active immunity in the intestines. Macrophages secrete cytokines. Even though both cells perform phagocytosis, Mϕs are better at it and frequently engulf bacteria/remove dead molecules in the intestines. Dendritic cells prime naïve T-cells and can prime T-reg cells. Unlike Mϕs, DCs can migrate between the intestines and lymph nodes. Both dendritic cells and macrophages share some surface markers. For example, both cells have MHC II (Major Histocompatibility Complex, Class II) protein markers. Each of these mononuclear phagocytes also have their own set of markers to differentiate the two cells. Mϕs have the markers CD14 and CD64. DCs have CD24 and CD26.

While performing flow cytometry (using a laser to count/sort cells based on programmable differences), a population of DCs that were CD14+ was discovered in the colon. CD14 is usually a marker found on Mϕs. The lab is currently trying to learn more about this population of DCs. The specific question that I will be working to answer this summer is “How does the ability of CD14+ DCs to do phagocytosis compare to not only that of the Mϕs and the ability of the other 3 populations of DCs?”. We hypothesize that the CD14+ DCs will be able to do phagocytosis just as well as the Mϕs.

I am excited to continue working on this project and to see the results we get back.

So what am I doing this Summer?

This summer, my mentor and I will be examining the role of intracellular structures-specifically, the biomarkers of these structures–in neurological movement disorders.

One of the main techniques that we will be using for this is western blotting. Western blotting is an analytical technique used to detect specific proteins. First, the proteins are denatured by the addition of sodium dodecyl sulfate, and then the samples undergo gel electrophoresis. Then, the excess primary antibodies are washed off, and finally, secondary antibodies are added to react with the primary antibodies. The secondary antibodies allow for protein visualization. Another technique we will be using is centrifugation. As the name implies, centrifugation is the process of spinning a mixture so its contents will be separated.

For this project, there is a large focus on isolating biomarkers. In cell biology, biomarkers can be used to understand susceptibility of disease or to even characterize the progression of a disease. For the purposes of our study, my mentor and I will be analyzing proteomic biomarkers, which can detect a variety of biological changes in the body.

I truly enjoy being a part of this project, because of the clinical applications that the results of this project may have, and its potential to begin a conversation in this particular field about new diagnostic measures and pharmaceutical treatments.

Opportunities to Come!

As I embark on this new research journey with the BSURF program, I am filled with excitement and eagerness to learn more about the process and importance of research as a whole. With acceptance into this program I feel as though comes the acceptance of a responsibility to take advantage of this opportunity as a means of learning and exploring the world in a different way than most.

Prior to this program I participated in another research opportunity at the North Carolina Research facility in Kannapolis, NC. As I young high school student, my involvement in the lab was limited, therefore limiting my experience altogether. I felt as though I was just an extra body for them to use to get their own projects done sooner. This feeling changed immediately  once I was accepted into the BSURF program and was assigned my very own project. Being apart of this program is giving me the opportunity for me to possibly make impactful change, and I want to make sure I take advantage of every moment of it.

I hope that being a part of this experience gives me the opportunity to contribute to science in some magnitude, even if it only contributes to a small part of an entirely new project. I want to learn the process of research and its importance to those who do it for a living. I hope this experience opens up doors in the future for me to further explore my interest in the sciences as I progress to higher level education.

BSURF Reflection

If I had two words to describe this summer, they would be: pipetting and grateful. While this may sound cheesy, this summer has made me realize that, no matter how much pipetting you give me, I will still enjoy working in the lab (which I think is a good sign). Learning what the everyday life of a researcher is like has solidified my goal of wanting to go into research as a career. I also was able to learn practical lab skills which I will continue to use throughout my research path.
And now to the three-cheese-blend of a second word to describe my summer: grateful. I am, of course, grateful to BSURF for providing me the opportunity to explore research this summer, and to Jason and Dr. G for fostering my curiosity about research at Duke with the faculty talks and seminars. I am also grateful to each person (and dog) in my lab for being so welcoming and friendly, and providing an environment that made me want to come back every day and to continue to do research. Thank you to everyone else that made this summer so enjoyable and fulfilling (looking at you, BSURFers)!

Looking Ahead

All summer we’ve had the opportunity to meet and learn from distinguished faculty which was super cool. One of my favorite talks I think this summer was from Dr. Christopher Kontos. Dr. Kontos is the director of Duke’s MD-PHD program. I really enjoyed this talk because before the program I was really contemplating striving for a MD-PHD. Listening to him talk really opened my perspective about everything. Especially because I work in a lab where people have all different types of academic backgrounds I think I’ve learned a lot! I really appreciated this talk because we got to ask lots of questions and learn more about the program. This has allowed me to explore my future even more which I was thankful for as BSURF comes to an end.