The 2016 ChEM-H Undergraduate Scholars and their postdoc mentors. Left to right: Dr. Saad Bhamla, Aanchal Johri, Dr. Monica Olcina, Ryan Kim, Dr. Karen Mruk, Patrick Piza, Dr. Raul Navarro, Nate Hansen. Not pictured: Dr. Jennifer Cao and Trevor Mileur.
Research Project Descriptions
Developing a low-cost, electricity-free centrifuge as a novel tool for point-of-care diagnostics
Undergraduate Scholar: Aanchal Johri, Mathematics Major Postdoc Mentor: Dr. Saad Bhamla Research Group: Prof. Manu Prakash, Department of Bioengineering
Some of the most pressing problems in global health today demand innovative solutions that are scalable, accessible, inexpensive, and efficient. The purpose of this project was to design healthcare tools for extremely resource-constrained settings, especially in the field of global health and point-of-care disease diagnostics. In particular, the goal was to create a low-cost, electricity-free centrifuge. The centrifuge is a staple component of laboratories and is commonly used to process biological samples, such as blood. Furthermore, the centrifuge isolates key biomarkers from patient samples, which can then be used to accurately diagnose various diseases and conditions. The project employs a diverse set of engineering and design skills, including 3D printing, laser cutting, and rapid prototyping. The first rendition of the device was constructed simply out of paper ("paperfuge"). After many rounds of prototyping with various types of materials and designs, similar devices were created using acrylic, wood, tape, and PDMS. The devices were then tested with parasitic blood and were shown to successfully isolate malaria parasites and filariasis worms from patient blood. Ultimately, combining interdisciplinary fields of biology, physics, and engineering can lead to the development of innovative tools to solve global health issues!
Read more about Aanchal and Saad's work.
Characterizing zebrafish models of injury using light-activated ablation
Undergraduate Scholar: Patrick Piza, Chemical Engineering Major Postdoc Mentor: Dr. Karen Mruk Research Group: Prof. James Chen, Departments of Chemical & Systems Biology and Developmental Biology
Human spinal cord injury (SCI) has debilitating and lasting effects on the whole central nervous system (CNS), such as paralysis. SCI is characterized by two types of injury: primary, which occurs at the impact, and secondary, occurring minutes to days following the injury. The mechanisms of secondary injury are a strong research interest because they occur in the therapeutic window and are, what many believe, the cause of the lack of regeneration in mammalian neural cells. Secondary injury causes cells to die via different mechanisms and an efficient model for these mechanisms in SCI is needed. Zebrafish are ideal organisms for modeling SCI response because of their unique ability to regenerate. We caused death in neural cells in a spatiotemporal specific manner using a genetic method of light-induced cell ablation. Other methods to model injury suffer from a lack of cell specificity and reproducibility. By developing stable lines of zebrafish encoded with toxins onto the CNS we induced specific cell death to neural cells throughout the spinal cord. Using experiments to screen fish to determine death rate and the presence of different chemical markers, specific mechanisms of death can be characterized for different toxins, completing a model. These fish will serve as a resource for studies investigating cell death and regeneration in zebrafish, and will become a useful tool in modeling spinal cord injury.
Targeting innate immunity to improve cancer treatments
Undergraduate Scholar: Ryan Kim, Biology Major Postdoc Mentor: Dr. Monica Olcina Research Group: Prof. Amato Giaccia, Department of Radiation Oncology
Targeting components of the immune system is emerging as an important treatment strategy for cancer with some remarkable results being achieved in patients with tumors that have traditionally been very difficult to treat. Despite these encouraging results there is still a significant fraction of patients that does not benefit from these types of treatments, highlighting the need to increase our understanding of regulation of the immune system in cancer. The complement system is an important pathway in innate immunity that is dysregulated in cancer. This dysregulation has been suggested to provide a survival advantage to the tumor. The mechanisms behind the dysregulation of this pathway and how to target it to improve cancer treatments, however, remain incompletely understood. With the help of a ChEM-H undergraduate scholar we have worked towards uncovering the mechanism of dysregulation of a critical component of this pathway, the receptor for complement factor C5a. We have found that this receptor is regulated at a transcriptional level by physical components of the tumor microenvironment such as the low oxygen regions found in tumors. In order to study the function of this receptor we have used CRISPR/Cas9 technology to knockout this gene in cancer cells. We will further investigate how best to target this gene in the context of other commonly used cancer therapies such as radiotherapy and emerging immunotherapies.
Genome-wide screen to identify regulators of GSH metabolism
Undergraduate Scholar: Trevor Mileur, Chemical Engineering Major Postdoc Mentor: Dr. Jennifer Cao Research Group: Prof. Scott Dixon, Department of Biology
Cellular redox imbalance is associated with several pathologies such as cancer and neurodegeneration. Reactive oxygen species (ROS) generated in part as by-products of aerobic metabolism alter cellular redox status and affect signaling pathways involved in cell proliferation and cell death. Our study aimed to uncover novel genes that regulate the metabolism of the most abundant cellular antioxidant, glutathione (GSH). We utilized a human haploid genetic screen coupled with fluorescent activated cell-sorting (FACS) to identify single genetic mutations that result in relatively high or low levels of GSH. A promising candidate gene is ABCC1, which encodes for the multidrug resistance protein (Mrp1). Using CRISPR-Cas9 to knockout the gene, we show that ABCC1 knockouts lead to an increase in GSH levels relative to a control population. Furthermore, we observe that both genetic and chemical ablation of Mrp1 function confer resistance to ferroptosis, a non-apoptotic cell death triggered by the loss of GSH function. These results suggest a link between Mrp1 and ferroptosis through the modulation of cellular GSH levels.
Publications:
A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity. J.Y. Cao, A. Poddar, L. Magtanong, J.H. Lumb, T.R. Mileur, M.A. Reid, C.M. Dovey, J. Wang, J.W. Locasale, E. Stone, S.P.C. Cole, J.E. Carette, S.J. Dixon. Cell Reports. 2019, 26, 1544–1556.
Ubiquitin chains and protein stability: Why does K48 linked ubiquitin signal degradation?
Undergraduate Scholar: Nate Hansen, Biology Major Postdoc Mentor: Dr. Raul Navarro Research Group: Prof. Tom Wandless, Department of Chemical & Systems Biology
In order to remain viable, cells must sustain a constant balance between protein synthesis and degradation. When this balance is perturbed, the regulation of protein levels in the cell is disrupted. In this way, breakdown of key protein quality control elements can lead to the improper disposal of damaged proteins and the aggregation of misfolded proteins. Protein aggregation is associated with the pathogenesis of a number of deadly neuronal disorders as well as several types of cancer. A key regulatory pathway that cells use to eliminate these potentially toxic aberrant proteins is the ubiquitin proteasome system (UPS). Damaged or misfolded proteins are tagged by a string of ubiquitin molecules and are directed to the 26S proteasome for degradation. Ubiquitin proteins may be linked together through bonds between different lysine residues – leading to a wide variety of possible “linkage topologies” that may be created. In order to probe the effect that lysine-48 linked ubiquitin chains have on the degradation of a tagged substrate, we implemented a biochemical approach to systematically prepare protein substrates that bear different, yet defined, ubiquitin topologies. We are currently optimizing this reaction scheme and are investigating methods to assess the thermodynamic stability of substrates tagged by a lysine-48 linked ubiquitin chain. We hypothesize that in addition to recruiting the proteasome to a particular protein substrate, this configuration of ubiquitin molecules also influences the folding state of the attached substrate, such that it independently promotes the unfolding of the protein.
