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This summer has been one of the most challenging, yet rewarding experiences I have ever had. The internship at the Columbia University Medical Center has taught me to think independently as a scientist and realize how fun science can be! Thanks to the Noyce Internship, I have greatly expanded my technical skills in the laboratory. I have learned not only the protocols for assays, but also the purpose of each. The assays I have practiced include genotyping using polymerase chain reaction, immunohistochemistry, fluorescence and H&E staining, Ligand and Carbohydrate Engagement Assay (LACE), and DNA cloning methods. However, the greatest lessons I learned where when the results of each were not the same as our hypothesis. I have had to repeat many experiments and troubleshoot when our hypothesis was not supported by the results. This has taught me to be more patient, perseverant, and meticulous. I have learned to analyze where in the protocol the errors could have come from and adjust the changes for the future experiments, until they work. These qualities of being detail-oriented and persistent are those that I will continue to implement in my role as a student. Whether it is in lab or in the classroom, these qualities will change how I deal with shortcomings. Furthermore, this experience has taught me to become a better critical-thinker. Whether it is designing the experiment or analyzing and contextualizing the data, the internship has helped me understand how to think like a scientist. Research involves so many factors, some that are within our control and some that are not. For instance, after weeks of staining, I could not find the phenotype that we had seen in our previous stainings. This was frustrating and neither my mentor nor I could understand why that was the case. We hypothesized that during sectioning the region of our interest may have been missed. As a result, we implemented minor changes such as to section the entire eye, instead of just the retina in the back of the eye. This allowed us to finally see the phenotype, much to our relief. Seeing the phenotype once again was very exciting and rewarding. I realized not to overlook any steps and work-hard to fix any mishaps in each step. This has helped to critically analyze my own work and become focused when carrying out any procedure. In addition, one of the most important skills I have learned is how to communicate research work to an audience. I presented my results at our weekly lab meetings to the principal investigator and other post-doctoral researchers. This was essential in receiving feedback on the work I had done and presenting to a scientific audience. As part of the Summer Research Institute, I created a poster, detailing our work and present the poster at the Poster Session. Creating a poster allowed me to reflect on the overall goal of this project. I synthesized the information and presented the material on the poster so that it could be accessible to understand for all. I also had to think about how to orally present the poster at the session, without using jargon and clearly demonstrate and explain what the research was about. This was a gratifying and challenging process, as well. I learned to represent scientific data, using imagery and diagrams so that it could be informative and easy to understand. Presenting my work has helped to become comfortable speaking in both a scientific and general audience, both of which were much different. The internship primarily helped me understand the importance of having a good mentorship. My positive experience in the lab has primarily been the result of the cordial relationship I have developed with my mentor and the other researchers in the lab. My mentor has guided me throughout the entire summer at every step, from explaining the basic biological concepts to performing the experiments. He has also challenged me to think critically about our project and encouraged me to become more and more independent in the lab. This guidance was so essential in becoming comfortable with using techniques that I was not familiar with before. Moreover, my mentor and the other researchers have spoken of their experiences in becoming a researcher. Speaking to them every day has helped me realize how the scientific industry works. This has given me insight into the hard work and struggles of becoming a researcher, but also has inspired me to consider pursuing a career in research at some point in the future. This guidance is a factor that is greatly essential in STEM education. The STEM field requires extensive mentorship, which can inspire the person to work harder. STEM education can be dramatically altered if students can see the rewards of working in such a field and have someone to look up to when there are facing struggles. I have been lucky enough to find such a laboratory and hope to guide other students as well in the future. Despite the end of the Noyce Internship, I hope to continue to work at the Columbia University Medical Center, throughout the academic year. I wish to apply the skills and lessons I have learned to become a better student and educator.
  Preparing LACE staining. In the past few weeks of the internship, I have continued to learn and improve on new lab procedures. I have been able to see the progression of our larger project, as we enter the second phase, which involves new assays and concepts.
My project has been focusing on testing our hypothesis, which is that proteoglycans, specifically heparan sulfates, interact with the Crb1 protein in retinal degeneration. These past few weeks, I have been using the genetic model to find conclusions to see if the heparan sulfates have an impact on the phenotype of the eye in mice. I have employed several crucial techniques in this regard. Firstly, once the eyes of the mice are fixed first in 4% PFA and Tissue Tek, I have made cryo-sections, which allows us to cut through the different layers of the eye. Once the sections have been dried, I have performed two types of staining: immunohistochemistry and LACE, both of which I have done in the initial few weeks. Ligand and Carbohydrate Engagement Assay (LACE) detects the interaction between receptor and ligand and allows us to see the presence of heparan sulfates throughout the eye. Immunohistochemistry involves the use of specific antibodies that stain for certain types of cells and regions, such as the photoreceptor cells and the outer limiting membrane. I have learned to increase the efficiency and quality of these two types of staining and there was significant improvement in the specificity of the fluorescent signals. The results further elucidated our hypothesis and allowed us to enter the second phase of our project. In order to analyze the signal and convey the results of the staining, it is necessary to have clear and aesthetic images, taken under the microscope. In the first weeks, my images were blurry and sometimes missed the target region of the eye, due to the cutting of the eye during cryo-sectioning. Since the last report, I have done staining numerous times. I have learned to use the microscope to adjust the slides and various settings on the software used in our lab to create better images. In the final upcoming weeks of the internship, I hope to have excellent pictures to display at the Summer Research Institute Poster Session. I will be presenting the results of these staining in our weekly lab meeting, with the other post-doctoral researchers. The project has also entered a second phase, turning to focus on the Crb1 protein itself and the possible interaction it may have with heparan sulfates. In order to test our hypothesis, I have been introduced to a new technique, DNA Cloning/ recombinant DNA. By analyzing the genetic sequence of the cDNA of the Crb1 protein, my mentor has identified the regions that we need to target for our hypothesis. Based on this information, we have created primers that will cut the DNA along specific sites. This will allow us to use clone fragments of the Crb1 cDNA that are of interest to create bacterial vectors. These vectors will allow us to see protein expression in a host cell. This aspect of the project has been absolutely fascinating!! Having learned the concept of restriction enzymes and recombinant DNA in previous biology courses, the ability to apply and actually conduct these assays in a molecular lab has been exciting. We are currently testing our primers and are in the initial stages of these functional experiments. In the coming weeks, I hope I can produce good results from these assays and understand and improve the way I carry out the protocol. Furthermore, I will be focusing on synthesizing these two aspects of our project. I want to condense the concepts in a concise manner, in order for me to clearly communicate the theory and results of the project for SRI presentation. As the internship enters the final weeks, I have been reflecting on the importance of having a supportive and collaborative mentorship. When I first started in the lab last year, I was initially nervous, for it was my first experience in research. I felt overwhelmed at the time, having not taken any biology course in college yet. It was the patience of my mentor that has allowed to grow as a researcher and as a critical thinker. He has taught me the concepts and the techniques and encouraged me, when many of my experiments did not work. He has challenged to me to think as a scientist, which has been invaluable in making my research experience rewarding. This nurturing mentorship is a key concept that we can apply in the field of STEM education. For many, having an individual to guide them can truly change the way they look at STEM. No matter how daunting a challenge can be, having the right motivation and support directly affects our interest in the project at hand. I have been lucky to have positive interactions with the other researchers in the lab as well. They have been sharing their experiences and have helped me understand the challenges in research. I have had the chance to engage with other researchers as well from other research labs in the Department of Ophthalmology in an informal setting. Talking to others has been itself a learning experience and I forward to engaging with the other researchers for the duration of my internship. In the upcoming weeks, I hope to have my abstract and poster ready for SRI! I look forward to speaking about my project and learning about the other great research projects conducted throughout the summer. In the first two weeks of my internship, I have been honing wet-lab techniques and understanding the theory and goals for my project. Although I have been working in the Zhang lab at the Columbia University Medical Center part-time since June 2016, working full-time is a new experience that is rewarding, yet challenging. The internship requires me to recall and apply the concepts I have learned in previous biology courses, such as cell signaling pathways, genetics, and basic biochemistry, in a new, dynamic context. This application of class material to real-life research has been a great experience thus far. Dr. Zhang’s lab focuses primarily on the mechanism of the fibroblast growth factor (FGF) signaling pathway on retinal development, as well as the role of proteoglycans in the retina. Research is conducted on the interactions between various proteins and other signaling pathways with FGF-signaling. The impact of these interactions is analyzed at different stages of development of the neural retina, starting at the embryonic level. This will help us understand the genetic basis for various diseases of the eye and possibly find cures or treatment for these diseases in the future. My project focuses on the interaction of proteoglycans and the crumbs homolog 1 (Crb1) protein in Leber’s congenital amaurosis (LCA) and some forms of Retinitis pigmentosa (RP), both of which occur at an early-onset. LCA and RP are genetic degenerative disorders that cause progressive destruction of the photoreceptor cells in the retina. Starting from a young age, an individual who has inherited this disease progressively loses vision from the peripheral side of the eye to the entire eye. Prior research has demonstrated that several mutations in the Crb1 gene are associated with LCA and RP. The project that my mentor, a post-doctoral researcher, and I are working on seeks to understand if and how proteoglycans - specifically, heparan sulfates -interact with the Crb1 protein in these disorders. I have, thus far, been trying to analyze the phenotypes for various genes that are known to be involved in RP by using mouse models to look for any similarities and differences. Furthermore, I have been reading literature and trying to understand the theoretical basis for my project. This literature review has been essential in seeing how my project ties into previous research and the new avenue in which our project is taking the field of ophthalmology. Within the duration of the summer, I wish to produce conclusive results that provide more information about our hypothesis. I will be presenting my results on a weekly basis in our lab’s group meeting along with the other post-docs in the lab. I have learned a wide array of wet-lab techniques in the lab, thus far. Firstly, I genotype newly born litters, through the process of polymerase chain reaction (PCR) and seeing the results on gel electrophoresis. This is crucial in understanding whether the phenotypes we see in the mice are in correspondence with our prediction based on the genotype. Following PCR and gel electrophoresis, I have learned to do cryo-sectioning. My mentor fixes the eyes that are removed from mice in a solution overnight, which makes a block with the eye in the center. In order to analyze these eyes, it is necessary to make sections of these blocks on a microscopic slide. I have learned to do this through cryo-sectioning. It was initially a bit hard to get all the sections to have equal parts of the retina. By repeating the cryo-sectioning on more blocks, I have been improving my technique. Thirdly, I have been conducting various staining on these sections of the eye to understand the phenotypes that can be seen. For instance, I have conducted LACE staining, which shows the distribution of heparan sulfates in the eye. Furthermore, I have done numerous immunohistochemistry stains using several different antibodies, which stain for different types of retinal cells and structures, such as the photoreceptors and the outer limiting-membrane. I hope to improve on these techniques in the coming weeks and produce clear and precise images of the sections of the eye. The greatest challenge of working in a research lab has been improvising when the assays do not go in the direction you had hoped for. For instance, in many instances, the PCR and some of the staining did not work for me. This was disheartening initially because of the many steps in the experimental assays, especially for staining, which takes two days to see the results. However, from this comes the greatest reward of working in the research lab. I rationalize the possible causes of why the assay did not work and repeat the protocol with the new modifications. This allows me to understand how research works in a real-life setting. It does not go perfectly all the time, but it is part of the process in becoming a better critical-thinker and scientist. This is a lesson that I truly value. My experience in STEM research has been rewarding and I am excited to see what the rest of the summer brings!
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