The Leonard M. Miller School of Medicine at the University of Miami represents a beacon of innovation poised to pursue breakthroughs in medical research. In efforts to rid society of harmful and complex illnesses such as cancer, stroke and heart disease, the Miller School faculty is granted more than a quarter of a billion dollars from agencies to fund projects meant to improve human health. Among numerous facilities responsible for providing world-class care, the Bascom Palmer Eye Institute stands out as the best-recognized in its field. Ranked best in ophthalmology in the nation for more than a decade, Bascom Palmer’s physicians and scientists also direct research labs which investigate methods of improving clinical care. With assets like these, University of Miami undergraduate students demonstrating determination, motivation and enthusiasm are provided the opportunity to work with researchers to implement the institute’s mission.
One of these undergraduates is Areeba Imam, a pre-medical student majoring in biochemistry at the University of Miami. For the past year, she has been working in Dr. Sanjoy Bhattacharya’s lab. His lab focuses on research involving the neurodegenerative diseases of glaucoma and multiple sclerosis. Glaucoma — affecting more than 3 million Americans — is a condition in which increased internal eye pressure results in damage to the optic nerve, leading to severe vision loss and blindness. Multiple sclerosis is an illness in which the immune system attacks the myelin sheaths of nerves within the central nervous system, resulting in broken communication between the brain and body.
Imam’s research experience began with observing Bhattacharya as he conducted several simple protocols. These protocols included the methods to passage cells, cell media changing, Western and dot blots and multiple protein extractions, as well as other techniques involving immunohistochemistry. Initially, like her mentor, she focused on experimental procedures concerning the interaction of cochlin with TREK-1 potassium channels.
However, Imam recently began working on her own independent project concentrated on examining the order of protein expression in human trabecular meshwork cells. Prior to experimentation, however, she realized that “a foundation of knowledge had to be established.” Intensive research done by a previous lab member revealed a model analogous to the restriction modification system in bacteria. His hypothesis stated that, under increasing levels of internal eye pressure (IOP), protein expression is altered in trabecular meshwork cells, resulting in the pathogenesis of glaucoma. “With this assumption, further research identified 10 proteins of focus for my project,” Imam said. “For the last couple of weeks, I have been experimenting to determine the baseline level of expression for each of the proteins of focus.” The procedure involves RNA extraction from normal trabecular meshwork (NTM-5) and glaucomatous trabecular meshwork (GTM-3) cells, followed by first-strand cDNA synthesis, and, finally, amplification by standard PCR.
The process begins by identifying 30 proteins that are up- and downregulated with respect to the trabecular meshwork stretching. These collections of proteins are then assessed through two databases: NCBI Nucleotide Database and the UCSC Genome Bioinformatics; these search for and detect the promoter region. “I then use an algorithm called the Basic Local Alignment Search Tool, or BLAST, which helps find the common regions between the two sequences of each protein sensed by the databases,” Imam explained. This algorithm is also very useful in identifying the putative promoter region. This particular amino acid sequence is copied into the Genomatix, allowing her to compile a list of transcription factors found in the promoter regions of each protein. According to Imam, understanding these transcription factors and their characteristics is crucial to the study because they can have a connection to stretching and pressure.
Her experiment focuses on studying a situation analogous to the restriction modification system in bacteria NTM-5 and GTM-3 cells to determine the changes in protein expression as a result of stretching in the trabecular meshwork. And after classifying and sorting the proteins through all of these mechanisms, only 10 are being focused on. These proteins are up/down regulated with respect to changes in lipid and prostaglandins. Previous research has shown that topical application of lipids and prostaglandins have lowered IOP in glaucomatous patients and cochlin is chosen as a part of these 10 proteins since it is linked in glaucomatous tissue. From this background, Imam hypothesized that, at different levels of IOP, the expression levels of protein in NTM-5 and GTM-3 cells will refashion due to similarities in their transcription factors. A baseline level of protein expression is first established with reverse transcriptase PCR with the following approach:
mRNA → cDNA → amplification → measure
The NTM-5 and GTM-3 cells are then placed under varying pressures to simulate increasing IOP levels in patients. The expression levels are then quantified using real-time PCR and subsequently compared with baseline levels in order to analyze the results. “Changes in protein profiles with increasing pressure may contribute to glaucoma,” Imam hypothesized.
Imam hopes to continue working on the project throughout her time at the University of Miami. From her research so far, she has learned that the practical application of science solidifies concepts that cannot be taught through only a textbook. “It is a consistent learning experience through which Dr. Bhattacharya and his team have taught me to be patient, yet diligent in my work.” Imam intends to continue her work on the project in order to gain a better understanding of the pathogenesis of glaucoma.