Author Archives: Moreen Njoroge

Farewell

Research is to see what everybody else has seen, and to think what nobody else has thought. -Albert Szent-Gyorgy

Approximately 8 weeks ago I was sitting in an airport in Hyderabad, India wondering how I was possibly going to catch up on a week’s worth of lab experience that I missed. I was worried that 7 weeks was not enough to get any data and that I might have just missed the most important week of the program. Well I sort of did, I missed a wonderful Opening breakfast and faculty talks. However, my first meeting with my mentor allowed me to understand the pace at which the rest of my summer would go as she set out the goals she has for my project. Considering I was still very jet lagged, all I heard was kinases, PfPK9, and Pf…something and phosphorylation. I had a feeling it had something to do with malaria considering this was the main research interest of the Derbyshire lab. I immediately realized that I eventually had no option but to read the folder of papers sitting in my inbox. This opened up a whole new perspective for me. On top of the daily lab work, reading the papers gave me insight to the way scientists think, perform experiments, and present their work. In some papers, I even felt their passion for research and especially treating malaria through their words.

After becoming fully engaged with the literature, the lab’s goals, and my own project, I hit my first stumbling block. For a whole week’s worth of work, none of my experiments worked. I questioned my competence but most of all, the process of science. It took a while to understand that I can allocate all these resources and time to an experiment and it is still very possible, in fact likely, that I will end the week at the same point in which I started. My fear came true, however, as this was the result for the remaining 7 weeks. Often times, it seemed like a tug of war where if I express PfPK9 well then somehow cleaving it would destroy the protein. Despite this, there were many small victories and these are what made my entire experience worth it. Being able to purify just a few more microliters than the week before, seeing cleaner and darker bands on a Western blot, getting the chromatography machine to work in order to batch-bind overnight, and laughing our way through the mistakes with my mentor.

I though BSURF would solidify and reaffirm my career choice. Though this would have been the ideal outcome, I am leaving BSURF with more questions than answers. I am more sure than ever that I want to pursue an MD degree but now I am faced with the knowledge that I enjoy asking questions and I can see myself entering the world of translational research. Dr. Lefkowitz said, we should look forward to not knowing where life will take us and that is what excites and motivates me.

I would like to thank Trinity College for funding my summer research experience, Dr. Grunwald and Jason for ensuring everything ran smoothly, my mentor, Amber Eubanks and the Derbyshire lab for being patient with me, and lastly my peers in the BSURF program.

Faculty Talks: Dr. Raphael Valdivia

We have all once experienced a fork in the road. I have always heard that the difference between those who are successful and those who are not is how much one is willing to overcome certain obstacles. Dr. Raphael Valdivia truly drove this message home as he emphasized the importance of never giving up in the midst of adversity, allowing creativity and passion to guide you, and always giving back to society. Not only did those words resonate with me, but I was able to relate to his story regarding his upbringing. Dr. Valdivia was born in Lima, Peru and his father always stressed the importance of receiving an education. Because of this, he was motivated to find any way possible to leave his native country and pursue higher education in the United States. After attending UWC USA for the remainder of his high school career, Dr. Valdivia attended Cornell University where he had wonderful mentors who shaped his career as a future scientist. Here at Duke, I have been able to find advisors who have encouraged me in the same manner so I was able to understand when he said “find someone who’s career you admire.”

Although Dr. Valdivia did not have a successful first round of attempting to apply to graduate school, his determination and persistence as well as knowledge of what his true passion is kept him going. After getting accepted to Stanford University, Dr. Valdivia was able to work on a project dealing with bacteria for his doctorate degree. It was, again, because of a very involved mentor, that he was able to successfully enter the field of science with full force. After a while, Dr. Valdivia came to work at Duke University where he now studies Chlamydia, a pathogen that is now associated with sexually transmitted infections. His research focuses on the pathogenicity of Chlamydia and how a cell is unable to perform apoptosis or cell death when infected with Chlamydia. I found the videos of this process to be very interesting and informative.

Dr. Valdivia spoke on the importance of hard work, serendipity, and service to society. I was inspired by his willingness to continue mentoring others as he realized the impact his mentors had on him. Currently, Dr. Valdivia is a Vice Dean and he helps counsel students as well as faculty on effective learning methods. He also works closely with legislature in improving the way lawmakers view discovery science research in hopes of increasing the number of grants that are given out to scientists. Overall, every faculty talk has truly taught me a lot. I have been inspired by female scientists such as Dr. Williams and her work on neuroplasticity and aerobic exercise and I am privileged to have been granted such an opportunity.

It’s been a long time coming: The joys and woes of research

 

World without malaria

Well, it turns out this is not an easy task. Everyday is not sunny, but everyday is certainly a small adventure in itself. Looking back, I was very naïve about the process of scientific research. I had always heard that scientists experience failure and they learn from it, that is what makes a good scientist. It all sounds very pleasant and ideal, until I was the one experiencing the failure. At the moment, it does not feel as though I am learning from it. Whenever I have finished expressing the PK9 and UBC13 and I am well on my way in the purification process, my heart drops every time I see a truncation of the protein in a Western Blot. This usually means two things: we need to start over which means preparing 4 more liters of media, incubating overnight, inducing with IPTG, batch binding overnight, etc. Though tedious, I have repeated this process so many times that I now do not even think twice when I see that we do not have enough protein to begin an assay with.

Recently, however, we had a small breakthrough in our experiments due to changing various conditions in our buffers, IPTG, as well as incubation conditions. A few weeks ago, I would never think I could celebrate at the sight of just a few more microliters of protein. (And by this I mean microliters of protein when diluted). I have also found it very difficult to make certain choices such as whether to use a protease and cleave the protein in order to get rid of any non-specific protein. However, this has sometimes resulted in losing more protein than intended. On the other hand, not cleaving could mean ending up with unpurified protein which would bring us back to the same point. This checks and balances system is quite frustrating.

In the next two weeks, I hope to be able to finally purify enough protein to be able to begin an ATPase assay where I would be able to determine whether the phosphorylation of UBC13 is being affected (by measuring the amount of ATP being converted to ADP). I am extremely hopeful that I will be able to get some results in time for the poster session. This summer has been quite a rollercoaster and although I am sad to see it come to a close, I am excited to continue my project in the Derbyshire lab during the Fall semester.

Chalk talks: Scientific crossroads

I thoroughly enjoyed listening to the chalk talks this week. From the lipid rafts in the cell to research on the Cryptococcus fungus and the Trehalose pathway, I was able to truly gain a deeper understanding of a broad variety of research interests within BSURF. Coming into this program, I was under the assumption that most of us would be researching very similar topics and therefore the integration of biology with engineering and ecology has been especially refreshing.

I found Ajile’s topic especially interesting as she took the time to explain the role of antibodies in HIV research in a manner I had not heard before. According to her research, bnAps are potent antibodies that have the ability to neutralize the HIV virus. The problem with bnAps is that they do not last for a long time in the body as they tend to aggregate and they do not have the ability to remove antigens from the body. Basically, she studies the effect of variant pH levels in different buffers. If the bnAps are able to withstand the conditions within the buffers, this would elude to the fact that they might be able to last for a longer time in the body. The importance of her research on a broader scale is that these antibodies can be used to create therapeutic drugs that slow down the progression of HIV. Malaria and HIV continue to be the leading causes of death in Sub-Saharan Africa, a region I call home, and therefore the progression of research on both fronts is not only impressive, but encouraging for the future.

I was overall very impressed with everyone’s chalk talks and I would love to learn more about each project especially as we begin to receive results or come up with conclusions. The implications of these projects are wide and it is an honor to work with people who are passionate about scientific inquiries that are yet to be answered.

A Day in the Derbyshire Lab

My typical day begins at 8:15am on Mondays and Fridays with a debriefing session with my mentor. We usually go over the order of business for the day which includes the tasks that should be accomplished that day. Most of the time, we have had overnight cultures growing in one of the Shaker rooms and I begin by completing the prepping for the cultures so we can begin our experiments. As described by the purpose of my project, we are trying to determine whether the compounds that were screened by a researcher in our lab are directly or specifically targeting PfPK9. We can determine this by matching the IC50 value of the cell-based assays with our assays in vitro. My day occurs in the following steps:

  1. Expressing PfPK9 and its substrate PfUBC13. Essentially, I want to find the optimal conditions so that I can get the most amount of pure protein.
  • In expression, I have a plasmid with a GST site linked to our PfPK9 site and an ampicillin resistant site. So I use high expression cells BL21 in order to ensure I have an adequate amount of PK9 to purify.
  • After growing the cultures overnight with Lbamp, I induce the lac operon with IPTG which mimics the allo-lactose sugar that turns on the DNA to allow for expression and I begin the purification process.
  1. I then use three main methods of purification: a GST column, gel filtration, and an anion exchange column
  • In the GST (glutathione s-transferase), I use an affinity purification method where I bind the GST-PfPK9 protein to the resin which has glutathione. I then wash to remove non-specific binders and then compete off with an excess of glutathione so that the protein diffuses off the resin more pure. I then further purify the elute
  • Another method I use for purification purposes is gel filtration which is a form of separation based solely on size. I still use resin however, these beads have pores through which the large proteins cannot diffuse through therefore the larger contaminants will flow through the fastest and the smallest proteins come off last. Since I know the size of PfPK9, I can then speculate at which point it will flow through the resin, but I also collect fractions from the whole column elution and test it for PfPK9.
  • In the event that we have not achieved optimal purification, I then use an anion exchange column in which I know that the resin is positively charged while our protein is negatively charged. Therefore, we can load at low concentrations and then gradually increase the salt concentration which allows the negative chloride ions to slowly build up and compete off proteins.
  1. In order to check whether we have purified enough protein to begin an assay, I perform a gel electrophoresis use coomassie and SDS/PAGE buffer. I then perform a western blot by using various imaging buffers in order to ensure that I can clearly visualize the bands. Darker bands at the right size indicate that we have purified enough protein to begin an assay. To confirm, we run MALDI or check our values using a nano-drop machine which is similar to a Bradford assay.
  2. Although I have not yet reached this step, after obtaining enough pure protein, the next step would be to perform an ATPase activity “hydrolysis” assay. This in vitro assay will allow me to determine what the level of inhibition is. I expect that there should be more ATP hydrolysis in the presence of UBC13 because it should be phosphorylated by PK9.

After a long day of purifying protein (successfully or not), there is a new to-do list on the board for the next protein purification steps for the next day. As a side note, we do not simultaneously purify UBC13 with PK9. Once we are at the purification stage for PK9, we begin expressing UBC13.

Plasmodium protein kinases

Under the supervision of Amber Eubanks, a third-year graduate student and I have been studying the biochemistry of kinases in Plasmodium, the parasite that causes malaria. Malaria kills hundreds of thousands of people each year across the globe. The primary classes of patients affected are pregnant women and children under the age of five and primarily affects Africa, Southeast Asia and South America. It is already known that kinases make excellent drug targets in Plasmodium because they are often unique and many do not have human host homologs. This means that I can specifically target parasite kinases over human kinases. Compounds that fit this profile will make viable drug candidates because they will not have off-target effects in human patients during treatment.

In addition to making excellent drug targets, kinases are involved in many of the critical biological processes within organisms, however, the roles of many kinases in Plasmodium are unknown. In fact, over 40% of the parasite genome has unpredicted functions at present. Our aim is to elucidate their functions/roles within the parasite to better understand the Plasmodium biology, develop probes for further studying the parasites and also to lend more information about pathways that can be targeted in the parasites.

More specifically, we are working with PfPK9 and its substrate, UBC13 (ubiquitin-conjugating enzyme 13) which are very specific to the Plasmodium species. Essentially, the goal is to test whether PfPK9 phosphorylates UBC13 since previous experiments have shown that phosphorylation of UBC13 negatively regulates its activity (Haystead). We are currently working with IPTG which mimics allo-lactose to turn on the lac-operon in order to begin translation of DNA. However, since IPTG is only a mimic, it cannot be hydrolyzed by and Beta-galoctosidase therefore the concentration of IPTG stays the same and this leads to more production of PfPK9.

Fig-5-PfUBC13-is-a-PfPK9-substrate-and-its-phosphorylation-suppresses-the-E2's

Fractions show the gradient

Additionally, we use protein purification methods such as batch or column binding with glutathione to link the GST-PfPK9 to a sepharose resin in order to separate using affinity. Collecting the fractions that have the highest peaks indicate the most probable locations of PfPK9. I then perform gel electrophoresis and Western blots to see whether PfPK9 has been successfully isolated from contaminants in order to then be able to begin a hydrolysis assay.

IMG_4387

Derbyshire Lab material

Although isolating one kinase seems nearly impossible, PfPK9 has no known homologs therefore we can use gel filtration or an anion exchange column which separates the positively charged protein from the negatively charged sepharose resin in order to isolate PfPK9. This is a very general overview of my project in the lab and I hope to be able to create a stronger foundation within research as I advance in my chemistry and biology courses at Duke and with additional lab experience

Interview with Graduate Student, Amber Eubanks

I have had the pleasure of working with and interviewing [Future] Dr. Amber Eubanks who is currently a third year PhD candidate at the Derbyshire Lab in the Department of Chemistry. As a graduate student, Amber spends countless hours in the laboratory but yet she always welcomes anyone who walks into the lab with a warm smile. In her undergraduate career, Amber attended Appalachian State University and was a Chemistry major and Biology minor. Just like many students at Duke, Amber began her college years as a Premed student but after shadowing and having other life-altering experiences, she decided that patient contact was not necessarily the only way to help others. In her sophomore year, Amber began working in a Pharmacology and Biochemistry research lab and it is after this that she decided that she would further pursue her interest in research. I felt a lot more at ease when she told me that she was a “lab rat” in her first lab position as most of her tasks included mostly making buffers. Because of Amber’s family background, she was motivated to continue onto graduate school as she would be one of few in her family to earn a graduate degree. Amber’s love and passion not only for learning, but for teaching, is evident in her everyday attitude with the undergrads in the lab as well as her colleagues. Similar to many of us in BSURF, Amber did have any previous research experience but this did not stop her as she was able to form meaningful and especially impactful relationships with her PI and upperclassmen who greatly influenced her career path.

I have had quite a few embarrassing moments in the lab such as not balancing the centrifuge and having an extremely heavy machine rattle so much so that it interrupts everyone’s work. Or when I am told exactly where something is and I still go to the wrong place when asked to find it. When asked about her most embarrassing lab moment, Amber told me of when she was once using fluorescence probe in a minus 20. When she went to get it out of the minus 20 and she started weighing things, the scale was not going over a few micrograms even though she placed all the material on the scale. She went to her PI and she found out she was weighing packing material that was really light.

Despite the embarrassing moments, Amber had the opportunity to work on an extremely interesting project in her undergraduate career after being granted summer funding from NASA. Her original project was studying the c.elegan and specifically its mode of drug transport but she had to somehow align this with NASA’s interests. Since drug efficacy is not the same for humans when they go to space, Amber began growing liquid cultures of c.elegans on a microplate which no one had tried before. She was able to suspend liquid cultures on rotators which allowed the liquid cultures to grow in space-like conditions. Surprisingly, she was convinced all her specimen died but after a short break, she found that they were all alive and the microgravity was not impairing their ability to move. The c.elegans that were in space-like conditions were longer, skinnier, and they moved in different patterns which meant that the change in drug transport was a result of the change in their physiology. I found this story especially inspiring as it is a clear depiction of the type of dedication and persistence seen in successful scientists.

According to Amber, science is always challenging and the only way to find meaning is to keep in touch with one’s purpose. For her, biochemistry and specifically proteomics is what has kept her in the lab thus far. Although she cannot see herself doing anything else, she hopes to enter a postdoctoral fellowship where she could focus more on Cell Biology or Proteomics. Her advice to undergrads is that it is never too early to start and I believe we have had a head start with BSURF. I am excited to spend my summer as well as the fourth-coming years assisting Amber in her research as well as hopefully beginning my own projects as a future medical scientist.

I always thought malaria was all about mosquitoes

           Well I was wrong. I have been ill with malaria countless times while living in Kenya and I never blamed anything but the obvious culprit, the mosquito. My expectation when I entered the Derbyshire lab was that I would be standing at a lab bench dissecting a ton of mosquitoes in order to study the plasmodium parasite. However, what I encountered instead were the intricacies and nuances involved in studying this disease that kills nearly 214 million people annually.

Upon entering the lab, I was overwhelmed by the sheer amount of equipment that was before me. I had never before worked with anything other than plastic pipets, a weighing machine, and a centrifuge in a biology lab. The fact that there were various refridgerators with varying temperatures, autoclaving machines, state-of-the-art PCR machines, and centrifuges bigger than the ones I had ever seen made me nervous. Would I ever acclimate to such an environment where everything seemed to be so meticulous? I was not sure whether I trusted myself and I certainly questioned why I was given such an opportunity in the first place. However, this fear was quickly replaced with an overwhelming sense of gratitude at the fact that someone believed in me enough to allow me to discover my interest in biochemistry research. The magnitude of the task in front of me seemed daunting and I struggled with suppressing any self-doubt when my mentor was explaining my project to me with much excitement. Although I was suffering a bad case of jet lag after returning from India the day before, I could not help but feel energized by the passion behind the research.

Why was I afraid of uncharted territory? My mentor repeated many times that this project is one that no one in the lab has previously worked on before. I immediately pictured spending hours in the lab only to end up with inconclusive results. However, I now understand that even if this happens, it will be a step in the right direction. I am looking forward to many failed attempts, not because I am pessimistic, but because I know that I will learn the most from knowing what did not work and the reason behind it. The fact that I will be studying a disease that has affected my family in unfortunate ways adds positive pressure and I am more motivated to learn the biochemistry involved. By the end of the summer, if all I will have learned are basic science skills, that will be more than enough to propel me toward an exciting future in tropical disease research. I am more than confident that after seven weeks, however, that I will have attained a greater confidence not only as a scientist in training, but as an African female in a STEM field.