This past year I was able to conduct research examining the immune response of the nematode C elegans when infected with a model organism of M tuberculosis called BCG. A key focus of my research depended on the gene daf2, which is involved in the insulin and immune pathways of C elegans. Expression of daf2 leads to the sequestering of daf16 transcription factors in the cytoplasm of cells. Daf16 is a forked-head transcription factor involved in the p38 MAPK pathway, a pathway involved in the response to pathogens. It has been shown that daf2 mutants are stress-resistant and long-lived, and also resistant to killing by Gram-negative bacterial pathogen P aeruginosa. Interestingly, daf16 is required for this resistance and studies reveal that its downstream effectors, notably lys-7, are anti-microbial genes. I am especially interested in the role of daf2 and daf16 in the immune response, especially considering reactive oxygen species (ROS) interactions. It has been shown that glutathione (GSH) is a thiol-based detoxification molecule used in the human immune response to protect host tissue from oxidative stress. GSH is also able to mediate BCG killing within granulomas in the lung. GSH is also produced within the nematode, and may be involved in the immune response when infected with BCG.
On April 6th, I had the opportunity to present my research thus far at the Eastern Colleges Science Conference at Manhattan College. At this conference I presented my data in a poster and was able to express my goals and future directions with professionals as well as other students. Having the ability to speak about my work as well as learn about other’s was extremely rewarding and was a great introduction into the research community outside of Pace University. I couldn’t believe some of the projects I saw were done by students my age in other undergraduate institutions and honestly motivated me to further my work and expand the project.
One of the biggest lessons I’ve learned from this research initiative is that scientific research is not done alone. Great insights and discoveries stem from research teams that devote so much time and energy into their work. I would have nothing without the help from my faculty mentors as well as partners on my research team. Another lesson I learned is that time management is an extremely tough skill to master and takes true trial and error. Juggling research, course work, and involvement in outside organizations was a struggle I faced this past semester. As I stated in previous blog posts, I learned the hard way that organization and sticking to a tight schedule are essential while in this program. Following hard deadlines and remaining on track is easier said than done, and it takes hard work and dedication to maintain.
Scientific research is based both on literature review as well as laboratory work. Literature review can be done from the comfort of your own home and occur at any time of the day. In contrast, working in the laboratory requires time and patience. Not every experimental design will give the expected results, or give any results for that matter. Over the past year I’ve learned that when performing experiments, you should not only be prepared to perform Plan A, but also B through Z. These experiments are more times than not built off the backs of experiments done before and data found from previous studies, which again supports the need for thorough and extensive literature review. You cannot move forward in a project if you have a hazy view and understanding of what you are doing. Thus, you must be constantly reading articles and journals pertaining to your project. The more you understand the easier it is to create experiments and correct mistakes in the trials that you performed.
I believe this year was the perfect time to begin my own research project and be a part of this program due the cultivation of information I learned in my previous courses as well as the current courses I am taking. Being knowledgeable in areas such as signal transduction and cellular processes during the immune response helped me not only understand information within the literature, but also to create my own experiments. Besides course work, my research advisor Dr. Marcello and professor Dr. Marcy Kelly were the underlying support system of my research. Both were available at every turn during my project and always so eager to lend a helping hand. With the guidance they have given me I look forward to continuing my work as well as pursuing a future career in the laboratory.
Recently my research lab has unfortunately experienced contamination of the machinery responsible for creating the NGM plates used in my project. The machinery was contaminated with E. coli, which is the bacteria used in C. elegans research so fortunately the source of contamination was discovered. While the equipment was being sterilized, my project consisted of increasing amounts of literature review. I wish to understand the nematode life cycle and metabolism as best as I can, as well as known interactions between the worm and M. tuberculosis. One paper, entitled “The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific ordorants” has given major insights into the way the nematode reacts to specific volatile organic compounds (VOCs) produced by M. tuberculosis. The researchers plated a sample of worms onto a plate containing a sample of a known VOC produced by the bacteria as well as a control substance where the effect on the nematode is known. The behavior of the nematodes were then tracked for an hour. The data consisted of reactions to different concentrations of four know VOCs as well as a calculated chemotaxis index value. A follow up experiment I plan on performing is observing the effect the VOCs have on nematodes lacking the gene responsible for olfaction.
Working with Dr. Marcello has been an amazing experience. Fortunately our schedules are very compatible so we are able to meet very frequently. Any questions that arise while I’m in lab are quickly answered and serve as a fantastic learning experience that will assist me in my future career. Dr. Marcello has also ordered C. elegans mutants, one of which lack the daf2 gene and the other will lack a key gene in the olfactory mechanism. These mutants are key to my project and I cannot wait to begin experiments.
I am fortunate this semester to have had the opportunity to submit a research abstract to the Eastern Colleges Science Conference. Attending this conference will serve as an experience opportunity that will aid me in my career as well as a catalyst for my research.
In order to perform research in a scientific field, you must have extraordinary patience and expect the unexpected. I had the pleasure of learning this lesson this semester navigating the trials and errors of my experiments. In order to begin the extent of my research, I must first prove that the nematode C. elegans will ingest the bacteria involved in my study. To do this, I performed several feeding assays. This entails placing equal amounts of E. coli, the nematode’s bacteria of choice, and the experimental bacteria on opposite sides of a NGM (nematode growth medium) plate. I then placed a sample of worms into the middle of the plate and monitored them for 4 hours, checking the movement of the worms every half hour. Two plates were made for each experiment for accuracy. Although tedious, I was able to observe to which side of the plate the worms traveled and if they preferred one bacterium over the other. It may sound extremely simple, but nevertheless errors were encountered. For example during my last experiment, the worms did not make any significant movements and therefore I was unable to detect any preference for either bacterium. This raised many questions, especially since I had followed the same procedure as previous experiments. I am currently looking into this event. I also found that maintenance of the worms before performing the experiment requires precise timing and ensuring you have enough worms to place on each plate. Dr. Marcello is extremely helpful in this aspect due to his extensive knowledge of C. elegans.
Half way through the semester, Dr. Kelly was able to order a mutated strain of the experimental bacteria that contains a plasmid causing it to fluoresce. This was a huge step in my research because it would allow me to visualize the bacteria in the worm gut and observe how it interacted with the intestinal epithelia. Unfortunately, an error occurred in growing the mutated bacteria and we were unable to use the cultures. I believe the organization from which we received the bacteria recommended the wrong antibiotic to include in the cultures, which was insufficient in keeping the bacteria alive. We are currently in contact with the organization to receive more bacteria as well as updated procedures. This was definitely a setback in my research, however it taught me the importance of maintenance of the organisms I’m working with as well as performing extra research to understand why I am performing such procedures.
The title of my project is Mutations in DAF-2 pathway affect the immune response in C. elegans when infected with M. avium. Caenorhabditis elegans is a model organism for studying infection with Mycobacterium avium due to the worm’s transparent, streamlined body as well as the similarities between its immune response to that of humans due to similar epithelial intestinal cells. The purpose of my research is to study the immune response in C. elegans from infection with M. avium. The DAF-2 pathway is involved in both immune response and aging. Worms genetically engineered to not produce the proteins involved in the DAF-2 pathway will be created and infected with the bacteria to study exactly how these proteins protect the worm from infection. The results from this research will give helpful insights to how homologous proteins in humans work to protect the body from M. avium infection.
What I hope to take away from this project is a better understanding of the research process and how to balance between academic work and personal work. I am passionate about performing research and I’m extremely excited to produce data from an experiment completely created by myself and to be able to showcase this data to the scientific community.