The vast majority of current protein-based drugs involve binding a therapeutic target with an exogenous molecule that neutralizes or blocks its function. A major drawback of this approach necessitates a stoichiometric number of binding sites be administered to the patient, resulting in many caveats such as high dosage, stability, and immunogenicity. Enzymes represent an underexplored avenue for protein therapeutics with vastly enhanced potency. Proteases are essential and ubiquitous enzymes that catalyze peptide bond cleavage. While there are multiple therapies available derived from naturally occurring proteases, currently there are no novel, engineered protease therapeutics. In this work, we seek to establish a highly specific and processive protease system with traits desirable for use as a protein therapy, such as high solubility and thermostability. For this purpose, we have chosen to engineer the small ubiquitin-like modifier (SUMO) protease. The target of this protease, SUMO, is a small protein important for cell cycle regulation in eukaryotes. The mechanism for SUMOylation is analogous to the ubiquitin regulation system; these small proteins are adducted to other molecules and serve as signals for degradation or cellular localization. Removal of these tags is essential to the regulatory process, and is mediated by the highly specific SUMO protease. This inherent specificity makes the SUMO protease an ideal engineering platform. Here, we take the first steps toward unlocking the therapeutic potential of the SUMO protease by enhancing the stability and solubility of the yeast homologue. To accomplish this, we re-engineered surface residues with the Rosetta software suite. Furthermore, we have developed a novel Förster resonance energy transfer assay to allow us to monitor protease activity in real time. In addition to laying a foundation for the creation of highly selective therapeutic proteases, our work has immediate implications for the production and purification of recombinant protein in a laboratory setting.

“Permaculture” refers to a design approach for creating resilient and integrated systems using principles and tools grounded in ecology. Permaculture design is usually applied to agriculture and food production, but recent permaculture-based projects in economics, urban planning, and governance have emerged as alternative applications of this design approach. However, there are few examples of applying permaculture to education systems; a gap in the general knowledge of the permaculture community and the field of education reform that I hope to help fill. This study examines how the ecological principles demonstrated in permaculture design can be used to address the problems that standardization has produced in K-12 public education in the United States. Standardization has emerged as the dominant approach of education reform, most visibly manifested in No Child Left Behind and the Common Core State Standards initiative (U.S. Department of Education, National Governors Association). However, as recent research has shown, standardization as a paradigm in education causes more harm than good for students, teachers, and schools (Robinson, Ken). Through research in educational philosophy, curriculum and instruction, development and learning, and systems theory, I justify how permaculture design principles can be applied to educational systems. From this I develop an original synthesis of education theory and permaculture principles as an alternative to standardization. My argument is presented in a three-part paper: a review of the critiques of standardization as a dominant paradigm for education; explanation of the twelve permaculture principles in the context of education; and a demonstration of applying these principles in the re-imagining of American schooling.

The Hashemite Kingdom of Jordan is currently working to develop a domestic nuclear power capability, to which they are entitled under the Nuclear Non-Proliferation Agreement that they signed in 1970. Jordan’s economy is rapidly expanding and their current path of importing energy is costly and unsustainable. While nuclear politics in the Middle East are complicated, and efforts to build nuclear power plants that do not produce weapons are viewed with suspicion by the world, Jordan generally occupies a favorable view among the Western world, following decades of security cooperation with the United States and their signing of the 1994 peace treaty with neighboring Israel. However, Jordan’s bold advances have been led by a strict monarchy that has been criticized for not representing the people. Furthermore, regional conflict has led to an enormous refugee influx which now makes up the majority of the population. Political upheavals in the region has made others worried if the country has enough stability to insure that nuclear infrastructure always remains in trusted hands. My research includes extensive firsthand experience from a semester in Amman, where I surveyed Jordanians, both inside and outside the nuclear profession, regarding their opinions of progressing nuclear energy in the country, taking into account the regional challenges. The rapid change in Jordan’s demographics and in the security situation of it’s neighbors makes it necessary to constantly reexamine this country. Given the overwhelming focus of Western attention towards hostile regimes, such as Iran, and their nuclear ambitions, this research covers an often overlooked area of global nuclear development.

My project is a study of urban form, not as it exists in any particular real place, but as an amalgamation of realism and idealism. The goal of this work is to depict a snapshot of an imaginary city, its buildings, people, spaces, and history. In creating this project, I did an extensive amount of research into the urban form in action, relying on direct observation, images from the internet, and studies of cities from architecture, urban planning, and design. The City of Atlas Bay is not a real place, but it does include references to diverse locations such as Seattle, New York City, Chicago, Los Angeles, Philadelphia, Washington D.C., London, and Shanghai. Despite the final product’s large scale, the work is also meant to be examined in fragmented sections, a half-way intentional product of the way it was created, which was in fragments. Just as one would not immediately grasp a large city all at once, the work is best viewed in fragments, through examination of its individual and distinct constituent neighborhoods. To foster this, I have included a large amount of detail in every part of the work, encouraging the viewer to linger on the streets as if in a real place. What impression the work leaves with the viewer depends on how the viewer interacts with urban life, whether it be a fast-paced inventory of major artifacts, or through a leisurely stroll examining their surroundings. This project is related the studies in my major, Community Environment and Planning, a bachelor’s program in the Department of Urban Planning, College of Built Environments.

 The mitochondria are cell organelles that perform important cellular functions such as energy production, regulation of the cell cycle, and cell death. Therefore, proper mitochondrial function is critical for healthy aging of an organism. Paradoxically, attenuation of mitochondrial function in the nematode worm Caenorhabditis elegans has been observed to extend lifespan. It has been proposed that induction of the mitochondrial unfolded protein response (UPRmt) is responsible for this enhanced longevity. The UPRmt is a response to proteotoxic stress in the mitochondria that results in induction of nuclear-encoded mitochondrial chaperones, proteins that stabilize and fold misfolded and aggregated proteins. However, the mechanism(s) by which proteotoxic stress is recognized in the mitochondria and signaled to the nucleus, resulting in the UPRmt response, remain largely unknown. To further understand the UPRmt pathway, we carried out a genome-wide RNAi screen to search for RNAi clones that specifically reduce UPRmt signaling under conditions of mitochondrial stress. To do this, we used a mutation in the electron transport chain (ETC) gene isp-1 to cause constitutive activation of the UPRmt GFP reporter, hsp-6p::gfp and screened for RNAi clones that reduced GFP fluorescence. We expect that positive hits in our screen will target signaling components of the UPRmt. In total, we identified approximately 60 RNAi clones that reduce the UPRmt response in isp-1(qm150) worms. Currently, we are re-testing positive hits after sequence validation and determining if the targeted genes specifically signal the UPRmt or non-specifically affect expression of GFP reporters. Once we identify genes that are specific to the UPRmt, we plan to test the role of these genes in mitochondrial function and lifespan. A better understanding of the UPRmt pathway could provide significant insight into mitochondrial biology, which impacts healthy aging and several age-related diseases.

Mitochondria, the energy producing organelles in eukaryotic cells, play a critical role in cell metabolism and regulation. Interestingly, mitochondrial dysfunction promotes lifespan extension in Caenorhabditis elegans. One proposed mechanism is that the induction of mitochondrial unfolded protein response (UPR^mt) increases the expression of mitochondrial chaperones, nuclear-encoded enzymes that assist in folding of misfolded or aggregated proteins, to maintain mitochondrial protein homeostasis. In an effort to elucidate genes involved in the UPR^mt pathway, we performed a genome-wide RNAi screen in transgenic animals expressing mitochondrial chaperone GFP reporter hsp-6_pr::gfp. Unexpectedly, we found that knockdown of genes involved in the pentose phosphate pathway (PPP) activates hsp-6_pr::gfp expression. The PPP is a metabolic shunt off of glycolysis that produces NADPH, a reducing equivalent used for glutathione reduction and lipid metabolism, and ribose 5-phosphate for nucleotide synthesis. In addition, knockdown of transaldolase, a gene in the reductive phase of the PPP, increases the lifespan of C. elegans. Through lifespan epistasis analysis, we found that transaldolase deficiency promotes longevity by a mechanism that is genetically distinct from known longevity factors such as the insulin-like/IGF-1 target (DAF-16), the hypoxia inducible factor-1 (HIF-1), AMP kinase (AAK-2), the UPR^mt transcription factor (ATFS-1), or the germline signaling (GLP-1). This suggests that the PPP inhibition promotes longevity through an uncharacterized pathway that may respond to mitochondrial dysfunction. In addition, we found that transaldolase knockdown alters expression of fatty acid desaturase, an enzyme that oxidizes fatty acid chains. We hypothesize that dysregulation of lipid metabolism by inhibition of the PPP may cause a defect in mitochondrial lipid composition, which may increase mitochondrial proteotoxic stress, inducing the UPR^mt. Understanding the genetics behind the UPR^mt and the PPP’s role in aging has enormous benefits to human health as both the PPP and mitochondrial dysfunction are highly correlated with many age-related diseases including cancer and neurodegenerative diseases.

Neurological outcomes for premature infants with cerebellar hemorrhages are poor, but the mechanisms of injury are not understood. We hypothesize that the addition of blood to cerebellar slices would decrease external granule layer (EGL) proliferation and impair migration of cells leading to developmental disruption. Our objective is to develop a slice culture model for understanding the effects of cerebellar hemorrhage on cerebellum development. Postnatal day six mice were decapitated and trunk blood obtained using a heparinized syringe. Whole brains were removed and 200 µm thick sagittal cerebellar slices were obtained using a Vibratome. Slices were placed on a cell culture insert with serum containing media. Trunk blood (4µL) was applied directly to treatment slices. Slices are incubated for 48 hours. Media samples were collected for lactate dehydrogenase (LDH) assays. BrdU was added 24 or 2 hours before fixation. Slices were fixed in 4% paraformaldehyde overnight. Immunohistochemistry for calbindin, BrdU, GFAP, PCNA was performed. Slices were processed with LacZ and Nissl staining. Images were taken on a confocal microscope and analyzed for EGL cell counts and thickness. LDH, a marker of cell death, increased in the media of blood-exposed slices than control slices for 4, 24, and 48 hours. Blood-exposed slices have thinner EGL (control 141 versus 120 µm blood-exposed). Bergmann glia were more disorganized after blood exposure and lacked the radial appearance of control slices. Purkinje cell arborization was decreased after blood exposure. Cell proliferation was lower in blood-exposed slices than in control slices (control 77 versus 26 blood-exposed for PCNA; control 80 versus 55 blood-exposed for BrdU). In conclusion, the addition of blood to neonatal mouse cerebellum slice cultures decreased proliferation of cells in the EGL after 48 hours. Additional experiments are underway to better characterize the mechanism of action.