Author Archives: Tamanna Srivastava

Endings and Beginnings

When I first applied to BSURF, I had no idea what to expect. I had no experience working in a research lab, and the thought of dedicating my entire summer to it seemed daunting, to say the least. I felt thoroughly unprepared for what was to come (and that feeling probably carried on with me until Week 3). But as the program started and I began immersing myself more in it, my fears were replaced with enjoyment. I am confident to say that these past couple of weeks have been amazing, and I would not have wanted to do anything else with my time.

From technical laboratory skills to presentations of research, the program has allowed me to learn a lot on my path to becoming a scientist. Under the guidance of supportive mentors, I have gained skills like learning how to run an ITC or plating yeast colonies. Besides technical skills, working in a lab this summer has taught me several life skills as well. The most important one is patience. Sitting for hours on end, waiting for a centrifuge to spin a sample down to a desired volume is a boring task for anyone. Having to do that for every single protein purification made me realize the importance of being patient in science. Results or methods do not appear or finish immediately. Yes, I had to wait more than 6 hours for it to get to the desired volume, but hey, it ended up being worth it as my purifications were successful. Another valuable life skill that I learned was accepting failures. For the past 6 weeks, the data that I was getting from the ITC (calorimetry) instrument was not usable. Sharp changes in a baseline that should be flat, small differences in values between the blank and sample runs, and overnight equilibration that resulted in no run were some of the setbacks I experienced when using the instrument. While I was not able to get the data I needed to get this summer (I’ll try again during the year!), each setback allowed me to learn more about potential problems in our method and potential solutions. Under the guidance of one of my grad students, we were able to fix some problems and have been able to get it to somewhat work recently. Finally, I think this experience has given me direction. I was not 100% sure about wanting to work in a lab in the future, but after this experience, I have realized that research is for me. Whether in industry or academia, I want to continue on my path to become a scientist. This summer was a rigorous, but enjoyable experience that reinforced my goals of being a scientist.

The past few weeks have been a whirlwind of disappointments (but productive ones!), discoveries, and delights. I am sad that the experience is over, but I feel like I have a lot more direction on what I want to do in the future thanks to the program. So while this summer is ending, my time in research is only beginning.

Hearing about HIV

In the past couple of weeks, we have had a lot of amazing, accomplished speakers, all at different points in their careers, come talk to us about life and science. Each talk has been fascinating, with a new life story and lens to look at research every time.

Of all the talks, the one that stuck with me most was given by Dr.Mary Klotman, as she gave some really interesting insights into HIV research. Previously, when learning about HIV, I always looked at it from a social perspective, rather than a scientific one. Thus, her talk taught me a lot about the mechanism of the disease. For example, I did not know that the virus uses kidney cells for long term storage. Additionally, I did not realize that the virus actually integrates into a hosts genome, making it difficult to find a cure. It was really fascinating to hear information about the symptoms and pathway of the disease from someone who has seen it in both a clinical setting and laboratory.

I think her experience with seeing patients living with HIV really made her talk stick with me. Her anecdotes about the experiences of patients, from those being taken care of by their mothers to those who killed themselves, made the talk even more impactful. She did research on HIV back when it was a guaranteed death sentence, and hearing how that impacted patients along with the science behind it made the talk a good blend of social and scientific.

Overall, Dr.Klotman’s talk on HIV was fascinating and informative. It was really a privilege to hear from someone with such breadth of experience as both a doctor and a scientist.

Hsp Regulation by PI3P is important for Plasmodium Falciparum

Throughout the world millions of people are affected by Plasmodium Falciparum, a parasite responsible for the transmission of the deadliest form of malaria.  Several molecules in the parasite work to keep it alive during transmission and infection; PI3P is one such lipid necessary for parasite growth but with an unknown signaling pathway. Previous work by the Derbyshire lab indicated that PfHsp70– a parasite heat shock protein– was a potential candidate for binding to PI3P in the parasite life cycle. Within PfHsp70 is a LID domain, usually responsible for binding to anionic lipids like PI3P. We believe that deletion of the LID domain in PfHsp70 will decrease its binding affinity to PI3P. To test this, both the Wild Type and LID-deleted PfHsp70 proteins were expressed (in yeast) and purified. Isothermal Titration Calorimetry will be used to quantitatively assess changes in binding affinity. In the ITC both the purified Wild Type and LID-deleted proteins will react with PI3P to produce a binding curve with heat released/absorbed plotted against concentration of PI3P. If a lower binding affinity is measured in the LID-deleted interaction, we will potentially know more about PI3P signaling and ways to disrupt PI3P synthesis to cause parasite death.

What do I do?

Through the past couple of weeks my routine in lab has kind of been all over the place. Depending on the goal for the day, I alternate between the third, fourth, and ultimately the fifth floor (where my lab is located) of the French Family Science Center.

So why does my daily routine potentially have me going all over the place?

I always start my day in the fifth floor, leaving my backpack and checking what I have to do for the day. Sometimes I will have to check in with my grad students to see if they have something they want me to do for them. but usually, I will have some kind of plan before-hand for what I could be doing. If it’s a protein purification, I get started immediately on thawing the pellets in ice (from the fourth floor) and preparing my buffers. Protein purifications generally take 3 days so, when I need to start them, I will come into lab at 8 am in order for the sample to lyse, clean, and spin down to be injected into the chromatography system at a reasonable time. (Because spinning down the sample takes such a long time, I usually will load the sample on the second day). If it’s a protein expression, I’ll come into lab at around 10am, start making the medium, and then deliver it to the fourth floor to be autoclaved. After being autoclaved, I inject the sample and wait for incubation, finally harvesting them once incubation is done.

While I wait for incubating, centrifuging, or autoclaving (I’m usually waiting for one of these things) I’ll be at my desk area reading up on papers that I’ve been given or surfing the internet. Sometimes I’ll go on a walk. (Recently, though, I’ve been working on some image analysis for one of my grad students) Once my waiting is done, I will usually be at the bench finishing up preparation. Sometimes, I will need to set up a chromatography system or gel running in the background.

So far, though, I’ve only covered the fourth and fifth floors. Where does the third floor come in? Well, the Nano ITC I’m using is in the Hargrove Lab on the third floor so throughout my day, I go back and forth cleaning the ITC and then loading samples to run and doing my work in lab on the fifth floor. I’ll usually run the ITC while I am doing other experiments and check in on it after an hour and a half.

My daily routine varies depending on what I’m doing. I rotate between floors, wait a lot, and run different kinds of experiments. Overall, I love what I’m doing and working in lab.

Alphas and Betas and Low-Ranked, Oh My!

I would like to start this post off by saying that all of the chalk talks given this past week were absolutely amazing! They all dived into complex topics like genetics and neurobiology in a simple way that made understanding them much easier. Seeing the research drawn as a visual tool made the talks more dynamic and really simple to follow. As someone with extremely limited biology experience, I really enjoyed that I could follow along and learn so much.

As someone who loves animals and grew up watching documentaries on their behavior, I found Christine’s talk extremely interesting. I knew that there were dominance hierarchies in baboons, but I had no idea there were distinctions between the types of stress that a baboon experienced depending on where was in the hierarchy. It was really interesting to learn about the distinction between energetic stress (more dominant in Alphas) and psychosocial stress (more dominant in low rankings males). I think the most fascinating thing for me was that stress levels could actually be quantified through measuring glucocorticoids in the feces. (Who knew poop could be so useful?) It is also really fascinating to think that you can tell what a dominant source of stress for a group of baboons is by measuring certain hormone levels and not just observing their interactions in the wild; I had no idea the level of T3 secretion indicated a certain type of stress (energetic) experienced. Additionally, it surprised me that just by moving down one rank– from an Alpha to a Beta– a baboon’s stress levels would decrease significantly! Overall, the talk gave a really interesting insight into the stressors of baboons in hierarchies from a hormonal standpoint. Christine did a great job in explaining her project in a clear, fascinating way. 

All the chalk talks this past week allowed me to dive deeper into fields with which I was unfamiliar. I really enjoyed learning more about each of the projects everyone else was working on and seeing common threads that linked them together. So far, BSURF has taught me so much and the talks really enriched my learning in a fun, simple, and visual way. Thanks everyone!

Interviewing Dr.Derbyshire!

Dr.Emily Derbyshire started out as an undergraduate at Trinity College, a liberal arts college in Connecticut, where she studied Chemistry. As an undergraduate, she always knew that she wanted to pursue research; she had gotten some glimpses of it in high school and worked in a lab as an undergraduate (even dedicating her summers to research!). She loved the bench work and problem-solving aspects in research, however, at that point she did not think she would have ended up as a professor.

After her undergraduate studies, she then went on to pursue her PhD in Molecular Biology at UC Berkeley. As a Post-Doc, she realized she wanted to orient her research to a more disease-related area, so she began working in Chemical Biology with a focus on malaria at Harvard Medical School.

When asked about her goals in college and their evolution, Dr. Derbyshire responded that her primary focus as an undergraduate was to graduate and get her degree. She worked three jobs to get herself through college! She decided that she wanted to go to graduate school after an internship in a company one summer where she saw the different degrees of freedom people had with just a Bachelors degree versus a PhD. She wanted a higher level of intellectual stimulation, so she decided in her junior year to get a PhD.

Looking at research specifically, Dr.Derbyshire mentioned that her goals were dictated mostly by the labs in which she worked. In her words, “they were very chemical and molecular.” Her research “zoomed out” over time. Studying the conformational changes from a molecule binding to a protein evolved into diving deeper on how molecules inhibit an organism (which is similar to what we are doing in lab!). So far, her favorite part about doing research has been gathering data to learn something new both personally and in the scientific community.

In terms of teaching, Dr.Derbyshire has enjoyed the experience, describing it as “fun”. As an undergraduate and graduate student, she had done some TA work, but what she did was dictated by the faculty member with whom she worked. As a professor here, however, she has been able to teach topics that really interest her within Chemical Biology. As a professor, she enjoys most when students come to her with a question that lets her know they were thinking about what she said and its application in other aspects. She likes to see students not just memorizing, but taking the time to synthesize what they’ve learned and pushing forward by asking questions.

Science in general has been a focus for Dr.Derbyshire due to its problem-solving nature and thinking involved. It is a path that answers important questions while help others at the same time, as exemplified by studying malaria, a complex disease that affects millions.

For future scientists, Dr.Derbyshire says “follow what excites you and make sure that you’re interested in it. That’s the most important thing!”

What is going on?!

In the Derbyshire Lab, my project focuses on studying the interaction between a Plasmodium falciparum parasite protein, PfHsp70-1 and a phospholipid, PI3P.

Plasmodium falciparum is a unicellular, protozoan, parasite species that infects humans. It is transmitted though mosquito bites and results in falciparum malaria—the most malignant form of this disease. When a bite occurs, sporozoites (forms of Plasmodium falciparum that result from a division of the cyst containing the parasite zygotes) that have clustered in a mosquito’s salivary glands enter into the bloodstream. From there, they enter and infect liver cells and multiply, rupturing to produce merozoites. Merozoites invade and infect red blood cells initiating the symptoms of malaria.

Heat shock proteins within P. falciparum act as molecular chaperones, helping in the folding/unfolding of other macromolecules. The Hsp70 family is believed to be essential for the life cycle of this parasite, as proteins in the family help mitigate the effects of changing temperature (and other physiological stressors) when the parasite is passing from the cold-blooded mosquito to the warm-blooded human host. It also facilitates invasion of host cells (by potentially trafficking parasite proteins), and increases heat resistance/resilience in the blood stage.

Within the Hsp70 family is PfHsp70-1, a protein mainly found in the cytosol of the parasite cell. PfHsp70-1 is expressed in the blood stages of malaria and is believed to be a potential exporter of parasite proteins into red blood cells during infection. It has been proposed that PfHsp70-1 works with Hsp90 for parasite growth and development, possibly helps regulate the parasite’s protein translation, and trafficking proteins into the apicoplast, a parasite organelle. The linker interface on PfHsp70-1 is believed to be responsible for communication and interaction with Hsp40 for parasite development. Due to these properties, PfHSp70-1 has been studied closely in vaccine research.

PI(3)P is a phospholipid that regulates cellular functions and assists in vesicular trafficking of proteins. Plasmodium PI3-kinase has been shown to be essential for P. falciparum growth. During red blood cell infection, it is believed that P. falciparum forms a protein and lipid trafficking system by potentially synthesizing PI(3)P. According to Mbengue et al, PI3P works to transport parasite proteins from the Plasmodium endoplasmic reticulum, into the red blood cell during blood-stage infection. However, not much is known about the actual mechanism for trafficking, thus making it a target for vaccine research and development. It is important to study PI3P as it is a key component in the blood stage of falciparum malaria.

The Derbyshire lab has previously identified that the C-terminal domain of PfHsp70-1 may mediate its interaction with PI3P, which could play an important role in parasite survival. My project deals with the potential interaction between PfHsp70-1 and PI(3)P and the part of the structure involved with binding between the two. I will be determining the binding quantitatively via isothermal titration calorimetry.

So far, I have worked to induce protein expression (developed via yeast cell culture) and protein purification of Hsp70-1 WT. I am currently being trained on ITC so fingers crossed that I will be able to get some data on the binding affinity!

Lab has been an amazing experience these past two weeks, and I’ve been learning a lot under the guidance of some pretty great teachers. I’m looking forward to learning more and diving deeper into this project!

I have a lot to learn!

Over the course of the next 8 weeks, I will be working in the Derbyshire Lab, a biochemistry lab dedicated to researching malaria parasite biology. Through this experience, I hope to learn new research skills and understand where my research interests lie to gain some clarity as to what I want to do in the future.

Before getting into BSURF, I had no experience working in an academic, research lab. My wet lab experience was limited to classroom learning and summer internship in the Quality Control Department of a company. While in the classroom, wet labs gave me some technical skills and ideas about lab practice. However, I had no idea in what direction I wanted to apply these techniques. When I interned at the company, however, I began to realize that I potentially enjoyed working with supplements and drugs, meant to improve people’s quality of life.

Throughout this summer, I want to understand where my research interests lie while simultaneously expanding my knowledge on lab procedures and techniques that are required in research. My primary interest academically is Chemistry, so working in a Biochemistry lab will help me gain exposure to lab procedures important to both Biology and Chemistry research. Additionally, my overall knowledge of techniques associated with Biochemistry (eg., protein purification, etc.) is severely limited. In my first week, I have already been exposed to new lab procedures! I hope to learn a lot about how to conduct effective research through learning new techniques, while also getting some experience synthesizing and presenting data. Through learning these techniques, I also hope to strengthen my background knowledge of both Biology and Chemistry and dive deeper into the topic that I am researching. I want to learn more about malarial drug therapies and what direction research on the topic is taking now, and working in a lab will give me a unique, firsthand approach to becoming more familiar and involved with the research. Overall, I really just want to learn as much as I can while being taught by people who are in the forefront of their field.

Since I have never conducted academic research, I am also using this summer to figure out in what direction I want to conduct research in the future. Working in an industry lab gave me a general idea of a research topic I could be interested in (the study of molecules involved in infectious diseases in order to work towards therapies). However, since I have never done and kind of research before, working in the Derbyshire Lab will help me figure out if this is the type of research I want to do. It will also help me gain a little more clarity on whether I want to spend my life in academia or go into industry.

In conclusion, I want gain experience with actually conducting effective research, directed by focused research questions. This summer will be a fantastic learning experience and I look forward to being taught (and messing up!) a lot. I am really privileged to have been accepted into BSURF and I really hope that I can gain the most from it!

(Unfortunately, I forgot to take a picture of me in lab, so I will upload that as soon as I can!)

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