For the Natural Science & Mathematics Colloquium on March 7, College alum Randall Reif ’06 presented a new method of analyzing apoptosis, or programmed cell destruction, in his lecture “Microfluidic Investigations of Receptor-Mediated Apoptosis.”
Associate Professor of Chemistry Randy Larsen introduced Reif and the lecture, presented in the Schaefer Hall lecture room to professors, students, and community members at 4:40 p.m. According to Larsen, who was Reif’s SMP mentor before he graduated in 2006, Reif published eight journal articles during his four-year journey to a Ph.D. at Texas Tech University. Reif is currently at the University of Kentucky following postdoctoral work at the University of Cincinnati.
“Apoptosis plays a role in several things in the body,” said Reif to begin the lecture, “including brain development, aging, fetal development, and menstrual cycles.” Problems in apoptosis can lead to equally major problems, including viral infection, cancer, heart attacks, and autoimmune disease.
Apoptosis is often a method of programmed cell death, through which the cell makes morphological and chemical changes that cause it to die. This is most often initiated due to some damage to the cell, including damage to its DNA, that cannot be efficiently recovered.
One biochemical method by which a cell initiates apoptosis is the caspase cascade, also known as the damage response pathway. After a relatively complex interaction of novel proteins produced by the cell, caspase 3 is generated, which induces apoptosis.
“Activation of caspases leads to chromatin fragmentation, phosphatidylserine (PS) externalization, and [cell] death,” said Reif.
A chemistry major while at St. Mary’s, Reif investigated microfluidics as a method of analyzing apoptotic cells. A microfluidics device possesses small chambers that can catch cells, and has small drainages for fluids to pass through. Reif modified the traditional microfluidics device by coating the inner chamber with polydimethylsiloxane (PDMS), bovine serum albumin (BSA), and biotin.
Then, he exposed the device to biotinylated antibodies for apoptotic proteins, meaning that the antibodies would mount themselves onto the biotin molecules in the chamber and catch any cells expressing particular proteins on their surface that they would only express during apoptosis.
Using this method, Reif could induce apoptosis in cells and then pass them through the microfluidics device, catching apoptotic cells and flushing out non-binding (non-apoptotic) cells. This would allow him to analyze not only properties of those isolated cells but also understand the rate at which apoptosis was induced in the cells, offering a time-based understanding of the caspase pathway.
This method serves as an alternative to flow cytometry, a commonly used technique in which cells can be individually analyzed and categorized.
“Microfluidics allows for controlled timing of apoptotic induction, and maintains viability of the cells,” said Reif. The method also allowed him to expose the cells to various reagents relatively easily.
Reif also discussed the intricacies of this work, including fluorescence-based methods of analyzing rates of apoptosis in cells, assays designed for detecting PS on the surface of apoptotic cells, and how he used ImageJ (a microscope imaging software) to measure cell fluorescence. His future goals include understanding the relationship between apoptotic receptor activation and the temporal dynamics of apoptosis.
Reif concluded the lecture by discussing some of his other post-undergraduate projects, including using engineered RNA nanoparticles (small RNA molecules artificially designed) as a method of molecule and drug delivery to specific tissues. This involved a discusion of three-way junction (3WJ) motifs, RNA molecules with three “arms” arranged in the shape of a T that serve as a very stable, specific method of molecule delivery. Such structures can be easily absorbed (endocytosed) by cells, making them important areas of research in the pharmaceutical sciences.
“I was impressed at how far we’ve come in nanotechnology,” said senior computer science major Rowan Copley, who attended the lecture.
Reif also answered some questions about the transition from undergraduate to graduate school. “After undergrad, there are no boundaries,” he said. “It’s about how your experience and what you know fits with a principal investigator’s work.” According to Reif, the biggest advantage of St. Mary’s was learning how to write from a science perspective, a skill not as common at the graduate level.
“Thanks for paying attention,” said Associate Professor of Chemistry Paul Blanchette in response to Reif’s concluding remarks. Blanchette taught Reif Physical Chemistry I and II during Reif’s undergraduate career.