My presentation focuses on research I conducted using immunohistochemistry to identify proteins in insulin stimulated pathways. My research contributed to the lab's ability to analyze p300 and CBP proteins for their use of acetylation. p300 and CBP often are talked about hand in hand as two proteins that function together to induce and carry out acetylation of other proteins. Generally, you can think of them as a messenger, carrying the information from one source to the next in a long line of messangers. Phosphorylation has long been seen as the primary driving force of downstream signaling. Our lab examined the importance and underestimation of acetylation through these two proteins. Initially, the lab used Western Blotting to look for protein presence, but the results we obtained were faint and difficult to analyze. I was assigned the task of trouble-shooting a protocol for the staining of these two proteins. I sliced tissue, plated it, stained it, and calculated the changes between knockout- and wild-type tissue. I was ultimately successful in identifying p300 but statistically unsuccessful in identifying CBP due to a technical error. My research showed a much more reliable way of identifying at least 1 of the proteins rather than Western Blotting techniques. This has a broader implication that there will possibly be more accurate or reliable research done on these p300 and CBP proteins, and acetylation as a primer for downstream pathway messaging done in the future.

Today, volatile gas anesthetics are commonly used in medicine for various procedures. While they are effective and safe for most adults, multiple or long exposures of anesthesia to young children and infants is thought to carry the risks of neurodevelopmental defects due to the well-established neurotoxic effects of anesthetics on the developing nervous system, referred to as anesthesia induced neurotoxicity (AIN). If the mechanisms of anesthesia and AIN can be studied and better understood, strategies to prevent defects associated with AIN can potentially be developed. At the Seattle Children’s Research Institute, I am assisting as an undergraduate researcher in a project to better understand AIN. My project specifically involves using C. elegans, a laboratory invertebrate species, to study the effects of anesthesia on neurotoxicity in specific developmental stages, including programmed cell death (apoptosis). The short life span and rapid reproductive cycle of these nematodes make them great models for this study because isoflurane exposure has been observed to induce AIN in these organisms. I am working to research the specific developmental stages of C. elegans that isoflurane affects the most. In lab, I am culturing worms to known stages of development, using green flourescent protein (GFP) reporter strains as markers to study apoptosis (specifically in the apoptoic proteins ced-3, ced-1, and ced-10), and exposing worms to isoflurane during these stages. Using PCR, western-blotting, and imaging techniques, I am analyzing the proteins, neurons, and developmental stage(s) impacted by AIN. This should provide new insight into the proteins and specific neurons that anesthesia induced neurotoxicity targets. By carefully analyzing the mechanisms of AIN, I hope to gain knowledge about how it can damage normal neuronal function in C. Elegans that can eventually be applied to humans.

Studies have shown that isoflurane, a common anesthetic agent, can negatively impact lifespan in Caenorhabditis elegans, and repeated or extended isoflurane exposure causes neurotoxicity and neurological defects in mammals, including mice and human. In an effort to counteract the effects of anesthesia, rapamycin, one of the most popularly studied drug in ageing, may provide a good candidate. Its inhibition of the mechanistic target of rapamycin (mTOR) has proven to modulate pathways in ageing and mitochondrial diseases, and recent data from the Morgan and Sedensky laboratory at Seattle Children’s Research Institute suggests it may prevent the detrimental effects of isoflurane on neuronal function. We are studying the impact of isoflurane treatment and rapamycin using C. elegans, a multicellular eukaryote that has proven to be an exceptional research model in the study of lifespan and health during aging. C. elegans has been used to study pathways involved in aging for decades, and the first genes regulating lifespan in mammals were discovered in these microscopic worms. C. elegans can be grown on solid media plates and their lifespan measured through synchronized growth and plate counts. We treated wild-type and various GFP reporter worm strains with isoflurane and/or rapamycin and assessed their effects on lifespan using various techniques to measure age-related changes, including fluorescence microscopy for the accumulation of autofluorescent pigment lipofuscin, western blotting, and lifespan curves. We anticipate that these experiments will open the door toward further studies on the effects of anesthesia on aging.

Aneuploidy is an irregular number of chromosomes caused by errors in cell division. Down syndrome is a common disease resulting from aneuploidy, where there are three copies of chromosome 21. I am in interested in why these errors occur and the mechanism that corrects them. Prior to anaphase, an attachment of microtubule fibers to chromosomes must occur to allow chromosomes to segregate properly. The attachments form by trial-and-error. Microtubule fibers will continue to attach and detach to chromosomes until stability is achieved. In the 1960s, Dr. Bruce Nicklas manipulated individual chromosomes using a microneedle to physically misalign chromosomes and pull on chromosome attachments to create artificial tension. The Asbury Lab is interested in recreating his experiments using tools that were not available at the time, such as small molecule inhibitors, to determine whether certain molecules such as Aurora B kinase are necessary for chromosomes to correct themselves. I use drugs such as ZM, an Aurora B kinase inhibitor, and Taxol, a microtubule polymerizer and common chemotherapy agent. I treat live cricket cells with drugs such as these, and record the treatment’s effect on meiosis. One common phenotype we are expecting is a delayed transition from metaphase to anaphase due to inhibition of a protein required to allow the cell to continue into anaphase. Once my research determines whether the drugs delay cell division and the effective concentrations, the lab can test the importance of the drug’s molecular targets in the error correction mechanism. Further research into the mechanisms of chromosome segregation has implications for understanding what causes aneuploidy, the mechanistic and biophysical mysteries of cell division, and the rising of cancerous cells.