Author Archives: Sofia Guerrero

It’s time to reflect. Looking back at my first blog post, I originally emphasized the traits that would make me a more capable future scientist: independence, confidence, collaboration, and skepticism. I believe that, while there is plenty more room for improvement in each area, I managed to develop each of these traits throughout my summer.

I became more independent by mastering protocols, learning to be more organized/efficient, and taking on more and more work as the weeks progressed. I became more confident in my abilities through reading papers, analyzing data, and putting my own ideas out there. I was able to collaborate with my amazing lab and even with those outside of it, learning both from and with them. Finally, through the amazing mentorship of both my PI, Dr. Silva, and my mentor, Vanessa, I was able to think through aspects of my project, ask questions, and overall become a more critical student.

Regardless of what the future holds for my career, BSURF has confirmed that I enjoy the research process. As I move forward, I expect that research will continue to be in my life in some way or another, whether it be to solve a problem or to learn more about our world. More immediately however, I’m excited to continue my work with the Silva Lab this fall. Thank you to all who contributed to this wonderful experience!

Sofia Guerrero

Mentors: Vanessa Simões, Gustavo Silva, Ph.D.

Department of Biology

Oxidative stress, where cells accumulate reactive oxygen species (ROS), is among the most prominent types of harmful environment, damaging cellular biomolecules, fostering cell death, and contributing to neurodegeneration. Cells experience many endogenous and exogenous sources of ROS; however, mitochondria are one of the largest contributors. Functional mitochondria are important for neural and muscle tissue, which require large amounts of energy for proper function. Moreover, defective mitochondria are associated with several human diseases, including Alzheimer’s, Parkinson’s, and muscular dystrophy. Despite this, the mechanisms impacting mitochondrial function in response to oxidative stress are not fully understood. This study aims to elucidate the key role of a ubiquitin enzyme Rad6 in the regulation of yeast mitochondrial function. Through biochemical and fluorescent imaging methods, we demonstrated that deletion of RAD6 increases the number and function of mitochondria. These mitochondria are required for cellular growth as rad6Δ cells become more susceptible to mitochondria targeting drugs. Through further investigation, we will test whether Rad6 regulates the quantity and activity of mitochondria as an adaptive response to the harmful effects of oxidative stress.

This week I wanted to take the time to shout out the work being done by George in the Mooney Lab! George’s talk stood out to me for two different reasons. The first is the fact that he gets to perform bird surgery this summer (wow) and the second is the potential impact on the field of neurobiology.

The fact that George performs surgery on zebra finches is mind-boggling to me. For context, I work with single-celled budding yeast (nowhere near a whole bird). When I need yeast for an experiment, I take a colony and inoculate a tube. When I need to dispose of a yeast culture, I spray some bleach. When I want to tag a protein, I can use PCR and an antibiotic plasmid. Due to the ease of growing them as well as their highly conserved metabolic pathways, yeast are wonderful model organisms for understanding molecular biology. In turn, zebra finches are a wonderful model organism for the Mooney Lab, which works primarily to understand neural mechanisms behind language. That being said, performing surgery on a living bird and then having to “sack” that bird is in a whole other league to spraying bleach in a flask. It is crazy how different our days in the lab look!

Now, onto the project. To summarize, George’s project is to test whether a new technology, dubbed Tech X, is functional in the dopaminergic neuron cells of zebra finches. The specific mechanisms of Tech X are unknown to me (for proprietary reasons of course), but what George divulged was that Tech X binds to specific RNA and fluoresces using green fluorescent protein (GFP). What’s so cool about this is that (if it works) Tech X will allow neurobiologists to make specific neurons fluoresce and therefore study them! Another part of George’s talk that I found interesting was that he’ll be targeting dopaminergic neuron cells. Dopaminergic cells, as the name suggests, make the neurotransmitter dopamine! For the Mooney Lab, dopamine is important because of its role in the language pathways of zebra finches. Beyond language however, dopamine’s most famous role is in that of reward. Drugs, from caffeine to cocaine, act in the mesolimbic pathway to essentially prolong the time dopamine is in the synapse of the neurons in the nucleus accumbens. Hopefully, the success of Tech X in making dopaminergic neurons fluoresce will reach beyond language and into other important avenues of neuroscience!

This goes without saying, but the brain is an incredibly complex organ to study. Developing technologies like Tech X help neurobiologists further understand how cellular interactions form complex networks that enable us to think, regulate our bodies’ metabolism, and perceive the world. Neurobiology is so so cool (at least I think so) so I really enjoyed hearing the many neurobiology talks this week!

As in most labs, each day at the Silva Lab holds something different. There are routine tasks that must get done, like prepping cells, making media, and autoclaving flasks. There are frustrating tasks that must be dealt with, like troubleshooting protocols, testing new antibodies, or working with very old computers. And finally, there are exciting tasks that you hope for, like analyzing the interesting results from your latest experiment and planning what you should do next.

I’ll generally plan out my entire week ahead of time based on my progress/results from the previous one. Recently, my weeks have begun to look a little different as I’m learning some very cool and exciting protocols, including Myc tagging proteins through PCR and staining cells through microscopy! Despite these new additions to my week, one experiment continues to be a part of it. As my lab is focused on the cellular response to oxidative stress, the most common experiment I perform involves stressing out yeast cells!

The standard oxidation experiment involves several stages. First, I must inoculate all of the necessary strains of yeast cells in my desired media, allowing them to grow overnight. Next, I’ll dilute them in new flasks. I do this in order for the cells to be at the correct growth phase, specifically log phase, by the time I will be doing the experiment the following day. Next, I will subject the cells to oxidative stress by exposing them to low concentrations of hydrogen peroxide. Once treatment has been completed, I’ll collect cell pellets from each sample. The next step is to lyse (break) these cells open in order to extract their lysate. After that, I perform a Bradford protein assay which is a neat way of measuring the concentration of proteins in your lysate samples. This assay allows me to normalize the concentration of proteins across my samples for more accurate results later on. Treating, lysing, and Bradford generally take about a day. Thus, the following day is usually when I’ll run a western blot, which is a method used to detect and quantify the presence of specific proteins in a sample. This is done through antibodies. Once I run the gels, transfer the proteins from the gels to my membranes, incubate the membranes in the antibodies for the proteins I’m interested in, incubate in secondary antibody, and develop via film, I will go over results with my mentor and hatch a plan for what I should repeat, change, or test next!  

Image from: https://www.arborassays.com/products/by-research-area/oxidative-stress/

In between cell lysis or antibody incubation, I’ll make sure my lab notebook is up to date and (of course) read the literature. Each week I’ll have papers to read for BSURF’s journal club, for my lab’s journal club, and for my project. While oxidation experiments are yielding interesting results, I am most looking forward to mastering the new protocols and analyzing the data that comes from them!

Dr. Gustavo Silva has always enjoyed the challenge of solving difficult problems and asking intriguing questions. His love for this challenge started with math, then with chemistry later in high school. However, during his introduction to the field of biology, Dr. Silva initially felt that the subject was a little out of reach understanding-wise. Introductory biology classes are usually based on the memorization of concepts rather than complete comprehension. Dr. Silva believes (and I agree) that no high school student, not even a Nobel Prize Winner, can really wrap their head around what it means to go from a cell to an entire functioning organism, with trillions of hormones, neurotransmitters, and cell membrane receptors operating in harmony (homeostasis). 

Several faculty in high school urged Dr. Silva to pursue biology at the college level, where some of the big questions he was pondering in high school could begin to be tackled. So, not really seeing himself becoming a mathematician or a chemist, Dr. Silva decided to become a biology major at the University of Sao Paulo in Brazil. Coincidentally, it was around my age where he also joined a lab. The lab was focused on the characterization of antioxidant enzymes and his project as an undergrad was to study the regulation of the proteasome, which is an organelle involved in protein degradation. His time in the lab, similar to our experience in BSURF, was the first time he was exposed to the life of a scientist. He thought, “Oh this is really cool. People are going to pay me to come up with interesting questions and try to answer them.” 

When he finished his undergraduate degree along with his lab project, Dr. Silva knew he wanted to expand beyond the proteasome and look at stress in the context of the whole cell. Once he learned more about ubiquitination and the integration of cell’s stress response, his career became geared towards cell biology. After he received his Ph.D. from the University of Sao Paulo, he went to NYU for his post-doc. Interestingly, staying in the US was never originally planned. When he was finishing his Ph.D., Dr. Silva knew that having international experience would be beneficial for his career. Thus, his original plan was to learn from the experience, grow as a scientist, and then return to a faculty position back home. However, when he arrived in the US, his science took off. His projects were going well and the ideas he had were working. He realized the value of continuing his career abroad. This realization, in combination with the economic crisis in Brazil, led to the decision to stay here.

So what led him to Duke? In short, it was the complete package. Duke had a lot of quality resources his lab could benefit from, a lot of good students at both the graduate and undergraduate level, and the potential for industry connections in the Research Triangle. Reflecting back on the birth of the Silva Lab, Dr. Silva believes the lab has been able to do most of what he originally had planned. Of course, the more you research, the more avenues open up, taking you and your lab on a multitude of different and unexpected pathways. For example, the current work being done in the lab with mammalian cells has already evolved far past the original plans. As the lab continues to explore different avenues of the cellular response to oxidative stress, Dr. Silva hopes to continue to do what he loves most, ask interesting questions about the natural world and seek their answers. 

Dr. Gustavo Silva (Photo from https://sites.duke.edu/silvalab/people/)

This summer I will be investigating Rad6’s role in the fermentation and cellular respiration metabolism of yeast. Now, you may be wondering how this is relevant, why this is interesting, or why you should care. Let me give you some background.

As a reminder, I am working at the Silva Lab which focuses on protein and ribosomal ubiquitination in response to stress! Ubiquitin is a very important and highly conserved small protein in the cells of all eukaryotic organisms. Ubiquitin’s function is well…ubiquitous. It has many functions in many places throughout the cell, but the one you have probably heard of is protein degradation, where ubiquitin is used to flag proteins that need to be degraded by the proteasome, perhaps because they have been damaged or are no longer of use to the cell. Findings suggest that a large array of neurodegenerative diseases show aggregates of ubiquitin-flagged proteins (Tai and Schuman 2008). This indicates how important ubiquitin pathways are for the function of neurons! Ubiquitin is an incredibly important protein and it serves a prominent role in translation (one the lab is focused on elucidating) when cells are exposed to oxidative stress. 

One of the lab’s main proteins of interest is called Rad6 in yeast. Rad6 is a ubiquitin-conjugating enzyme or an E2 (for those that are relatively familiar with ubiquitination). It’s involved in all sorts of pathways, such as chromatin silencing at telomeres, protein degradation, histone ubiquitination, and DNA repair. Its homolog in humans is called UBE2A and its malfunction is linked to several diseases, including X-linked intellectual disability type Nascimento and Late-Onset Parkinson’s.

Back to my research project. Why am I investigating Rad6 in the context of yeast? Well, yeast are one of the few organisms that prefer fermentation over cellular respiration. They can switch their cellular metabolism to cellular respiration depending on their environment. Before I came along, the lab found some interesting and unusual observations (more on that in my upcoming chalk talk) in the rad6∆ strain of yeast when it is in fermentation. In addition, mutations in the human UBE2A protein that lead to X-linked intellectual disability type Nascimento were found to affect the function of mitochondria in neurons (Verstreken et al. 2013). As you know, mitochondria are pretty important for cellular respiration! My job will be to take these initial observations and see if I can try to figure out what is going on here. Whatever findings result from this project, I am truly excited to contribute to the lab’s overarching goal of investigating Rad6 and ubiquitination during oxidative stress!

The Ubiquitin-Proteasome System (UPS) from Tai and Schuman 2008

After the blur that is the end of spring semester, I find myself writing on Duke’s now-silent East Campus, with weeks of research ahead of me. This summer, I am continuing my work at the Silva Lab, which focuses on elucidating ubiquitin’s role in the cellular response to oxidative stress. Oxidative stress often underlies disease, so understanding how cells respond, specifically through mapping relevant cellular pathways, could lead to the development of tools that promote the wellbeing of cells. Yeast is the primary model organism I will be working with.

My goals and expectations for my time at the Silva Lab focus mainly on my development as a scientist. Firstly, I hope to become a more independent, contributing member of my lab. Seven weeks is a very, very short amount of time in the world of biological research. Nevertheless, I look forward to investing time and energy in a project that I can be proud of and perhaps continue during my time at Duke. Secondly, I want to become a more confident lab member, from my understanding of the literature I read to the quality of the data I produce. Lastly, I want to become a more critical and questioning scientist. In many introductory STEM courses, we tend to accept teachings as absolute truths. In the world of research, scientists must be critical of everything they observe and must support their findings with as many different methods as possible. Through BSURF, I hope that I can continue to shift my mindset from passively accepting to engaged and skeptical.

The prospect of full-time research is both exciting and daunting. As I look to the summer with the excitement that comes from potentially learning something new about the natural world, I worry about whether or not I will be good at it or how this experience will shape my career. For the time being, I hope to put these worries aside and focus on personal growth. When I think of the scientist I hope to become, these are the qualities I most hope to embody: independence, confidence, collaboration, and skepticism. If I can make concrete steps towards becoming this scientist, I will consider BSURF a success.