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.

Acute myeloid leukemia (AML) is a type of blood cancer. This disease is treated by chemotherapy and bone marrow transplantation, but mortality rates remain high and new therapies are needed. Because gene expression in AML differs from person to person, it is possible that personalized therapies can be designed by understanding how gene expression predicts drug sensitivity. Previous studies tested the sensitivity of 14 human AML cell lines and 30 primary patient samples to 160 chemotherapy drugs, and utilized computational approaches to identify genes that are correlated with the sensitivity to specific drugs. In my project, I am evaluating several of these predictions by silencing gene expression in AML cell lines with a tool called short hairpin RNA (shRNA), which is used to bind and degrade the expression products of genes called messenger RNA (mRNA). I will then test the ability of drugs to inhibit growth in cells that have undergone gene silencing versus control cells that have not undergone gene silencing. If we successfully validate these gene expression-drug sensitivity relationships, they could lead to personalized clinical treatment approaches in AML.

Cardiac arrest is a serious and often life-threatening event that can result in devastating outcomes. Targeted temperature management, or TTM, is a treatment utilized after cardiac arrest which improves patient outcomes. Determining the best treatment temperature and combination of sedative and paralytic medications, whose purpose is to slow the brain’s metabolism and maintain brain tissue oxygenation, will improve this beneficial treatment for future patients. The purpose of this study was to compare the time to ‘reawakening’, defined as the patient following commands, between post-cardiac arrest patients who received different combinations of paralytic and sedative medications and who were treated at 33°C or 36°C during TTM. Time to ‘reawakening’ after TTM is a critical factor in this patient population in terms of each patient's prognosis. The sub-aims of this study were to determine if time to ‘reawakening’ was influenced by: 1) cooling patients to the target temperature of only 36°C, and 2) the patient’s temperature when signs of shivering occurred during the TTM protocol. We were interested in studying these two aims because cooling patients to the target temperature of 36 degrees is expected to decrease the incidence of shivering and therefore less sedative and paralytic will be needed. Additionally, knowing what patient temperatures increase the incidence of shivering will better direct the dosing and timing of medications. Data for this study was retrieved through a retrospective chart review of 200 patients who received TTM treatment at Harborview Medical Center. Once we determine which treatment temperature and medication protocol results in the fastest reawakening and least amount of shivering, the results may influence future TTM protocols and could influence the care that future post-cardiac arrest patients receive. TTM could change in a way which ensures patients have the best possible outcomes after experiencing a traumatic event like cardiac arrest.

 The development of drugs is essential for human health. Approximately 40-60% of all currently prescribed drugs target a specific type of membrane protein called a G-protein coupled receptor (GPCR). In the human body, the binding of a neurotransmitter or hormones to a GPCR transmits signals across the membrane to bring about an intracellular response, which ultimately permits cells to communicate with each other and regulate body physiology. The Hague lab studies the molecular mechanism of drugs targeting GPCRs. Currently, we are investigating an important structural portion of certain GPCRs called the PDZ binding motif, which is a site for protein-protein interactions. My role in the lab focuses on the somatostatin (SST) receptor subfamily, which regulates the endocrine system and neurotransmission by inhibiting the release of secondary hormones produced in the gastrointestinal tract and pituitary gland. A drug that targets SST receptors is octreotide. This drug inhibits growth hormone and insulin release, and is FDA approved for the treatment of growth-hormone producing tumors and GI tract disorders. Interestingly, all five SST GPCRs contain this PDZ-binding motif, and my research examined the importance of this motif using a variety of biochemistry techniques that quantified the expression, localization, signaling, and protein-protein interaction network. I created SST receptor mutants lacking the PDZ-binding motif using polymerase chain reaction and then transfected the SST mutants into human cells. These novel clones were subjected to plasma membrane assays, confocal microscopy, and mass spectrometry analysis. I found that removing the PDZ-binding motif had minor impact on overall receptor function, but more studies are necessary to determine the role of the PDZ-binding motif for SST receptor function. Our ultimate goal is to identify novel PDZ-interacting proteins for GPCRs, and then disrupt or enhance this interaction with novel small molecules to treat disease.

Imidacloprid (IMI) may be a viable alternative to carbaryl for controlling burrowing shrimp (Neotropea californiensis) that destabilize sediments resulting in poor survival and low yields of the commercially harvested Pacific oyster (Crassostrea gigas). Previous laboratory tests indicate the shrimp are overtly affected (immobile, cannot burrow) when exposed to IMI in sediment pore water, but are not killed. These results contrast sharply with observed efficacy in the field, i.e. mortality 72-96 h post application and subsequent reductions in burrow counts. Understanding the factors governing efficacy in the field may improve control. We exposed 11 adult female shrimp that had burrowed within 30 cm of native sandy sediment to each of four concentrations of IMI (Nuprid® 2F) for 96 h: 6, 30, and 150 ppb active ingredient. We compared the response of these shrimp to that of 10 negative controls. Surface water (10 cm) was exchanged with clean seawater (salinity=30 ppt) 6 h post dose and then twice daily simulating tidal exchanges. Behavior and water quality were monitored once daily. All shrimp in the IMI exposures were overtly affected and mortality was dose dependent: 0 ppb (0%), 6 ppb (9%), 30 ppb (27%) and 150 ppb (82%). The LC50 was 51 ppb (95% CI=24-146 ppb). Mortality at 150 ppb occurred 30-54 h post dose; mortality at lower concentrations occurred between 54-78 h. Shrimp that survived the highest doses were closest to the surface when immobilized where dissolved oxygen (DO, % saturation) was greatest. Immobilized shrimp were unable to maintain their burrows, the burrows subsequently collapsed, separating them from the DO within the surface water, resulting in asphyxiation. Results suggest the shrimp can tolerate very low dissolved oxygen within sediment without IMI, but their sensitivity to IMI increases due to factors associated with their impaired ability to maintain burrow integrity and circulate water.

Poliovirus (PV) remains endemic in three countries and is responsible for sporadic outbreaks in several others. Clinical surveillance for acute flaccid paralysis (AFP) is the gold standard for PV surveillance. However, as few as 1 in 100 to 1 in 1000 PV infections will develop AFP, particularly in well-vaccinated populations. Environmental surveillance of wastewater using a bag-mediated filtration system is currently being investigated to detect sub-clinical circulation of poliovirus. However, the filter samples will be collected remotely and evaluated centrally, which requires a shipping period. During this shipping period, bacterial and fungal growth may degrade PV on the filter surface. Inhibition of fungal and bacterial growth is necessary to ensure PV survival on filters during transit from field collection to the processing lab. Shipping on icepacks at 4°C is costly, thus finding an alternative is ideal. Preservative agents were investigated as an alternative to reduce microbial growth while not affecting PV recovery from its performance methods. Preservatives investigated included sodium azide, calcium propionate, o-phenylphenol, and sodium benzoate. Preservative solution or PBS control was pumped into the positively charged Virocap filter and eluted with beef extract, pH 9.50. After 1.5 hours, the eluate was pumped out, pH adjusted, and dosed with 10^2 PFU/mL PV1. Filters were run in duplicate and samples were plated on BGMK cells for enumeration by plaque assay. 0.0195% sodium azide eluate had a 64% recovery when compared to PV expressed in the PBS control eluate. 0.1% calcium propionate had a 102% recovery, 2% sodium benzoate had a 101% recovery, and 0.02% o-phenylphenol had a 111.1% recovery. Sodium azide is not recommended as a good preservative for PV due to low recovery, but 0.1% calcium propionate, 2% sodium benzoate, and 0.02% o-phenylphenol should be investigated further for PV preservation and recovery in wastewater over time.

 The development of new medications is critical for treating disease and extending life. Approximately 40-60% of drugs target a specific type of membrane protein termed G-Protein Coupled Receptors (GPCRs), which permit cells to communicate with each other (by binding neurotransmitters) and regulate body physiology. The Hague lab studies the molecular mechanism of drugs targeting these receptors. Specifically, they examine a small, yet important structural portion of the receptor - the PDZ binding motif - which acts a protein-protein interaction site. Interestingly, of the ~800 different GPCRs in the body, 30 have this PDZ binding motif, including those activated by the neurotransmitter 5-hydroxytryptamine (5-HT), which is commonly known as serotonin. Serotonin regulates many key CNS processes including mood, sleep, and appetite. Current 5-HT medications that are on the market are used to treat depression, generalized anxiety disorder, and social phobia. Two common antidepressants that affect serotonin GPCRs are Selective Serotonin Reuptake Inhibitors, or SSRIs, and Monoamine Oxidase Inhibitors, or MAOIs. My goal was to examine the necessity of the PDZ binding motif for 5-HT serotonin receptor function in human cells. To do this, HEK293 cells expressing the wild-type 5HT receptor (WT) or mutated 5-HT receptor missing the PDZ binding motif (ΔPDZ) were subjected to diagnostic assays including: microscopy, protein biochemical analysis, proteomics, and cellular signaling. In summary, the data suggest the PDZ-binding motif is not essential for 5-HT serotonin receptor function in HEK293 cells, although further experiments are necessary to solidify this conclusion.

“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.

Plasmonic nanoparticles (NPs) have been reported to improve the performance of organic solar energy harvesting devices. Gold (Au) and silver (Ag) are two of the more commonly researched plasmonic nanoparticles. The plasmonic effect occurs when incident light induces a fluctuation of electron clouds within a metal. This phenomenon resonates at a specific wavelength depending on NP size, geometry and the dielectric constants of the metal and medium. Previous publications have shown that inclusion of combined Ag and Au NPs increases light absorption and can enhance the power conversion efficiency by 20 percent. This study evaluates the effect of varying concentration of Ag and Au NPs on power conversion efficiencies within a bulk heterojunction (BHJ) organic solar cell, and examines the mechanisms behind the enhancement. Lumerical FDTD Solutions was used to model scattering and plasmonic resonance in OPV devices with different combinations of 40 nm Ag and Au NPs within the PEDOT:PSS charge transport layer. Light enhancement and absorption was modeled as a function of wavelength and spatial arrangement. The results indicated that the absorption profile for the mixed NP device was dominated by the contribution of the Ag NPs, which prompted the investigation into the ideal NP concentrations/ratios. OPV devices were constructed on an ITO substrate with a PTB7:PC70BM active layer. Power conversion efficiencies were determined experimentally as a function of open-circuit voltage, short-circuit current density, and fill factor. Device morphologies were evaluated using atomic force microscopy (AFM). The absorption profiles of the devices were measured using UV-Vis spectroscopy. The optimal concentrations of Ag and Au NPs were determined. Future research on this topic should focus on optimizing particle geometry, ligands, and dielectric NP coatings.

Across the United States, there is a paucity of data about food insecurity among university students. For this project, we are defining food insecurity as a lack of access to nutritious food and reduced intake due to reduced income or household demand. Currently, UWT does not know if there is an issue with food insecurity among students nor the reasons if insecurity does exist. This research used mixed methods which included a catalyst survey of the entire UWT student body to document the prevalence of food insecurity. The second method is a community mapping about food resources near UWT. Various types of food resources exist; however, we hypothesize that there are few affordable meal resources within a 10-minute walking distance of the UWT neighborhood causing barriers to accessibility. We defined a meal as hearty and filling food providing enough energy, strength, and nutrients to sustain through short periods of time (1-3 hours), an entrée (consisting of more than one food group) and beverage ranging from $6.00 - $8.00. We analyzed data to create user-friendly tools such as maps, databases, and websites for the UWT student population to access affordable food resources. By doing this research, our hope is to generate awareness of the need for more food resources and provide resource information for affordable food within the UWT community. UWT is projected to grow to 7,000 students by 2020; therefore, the availability of affordable food resources will impact the academic performance of the University of Washington Tacoma students. Each UWT student’s journey to success can be supported by the availability of affordable food resources that impacts their health (mind, body, spirit); as a result, students will not have this barrier in achieving their potential and UWT will successfully meet its mission and values.

The Miocene epoch represents a hotbed of cetacean evolution; during the epoch, whales underwent a large radiation of morphological forms and ecological specialization. The superfamily Platanistoidea experienced high diversity, especially during the Miocene, with 47 extinct and extant species currently known to science. However, of these 47 species, the Ganges River Dolphin is the sole extant member of this once large clade. Here in Washington State, the University of Washington Burke Museum (UWBM) specimen 87105 is a probable representative of the Platanistoidea in the Pacific Northwest. The specimen consists of the posterior third of the skull, along with some non-diagnostic ribs and vertebrae. It is tentatively a member of the Squalodelphidae, and shares morphological similarities, namely thickened supraorbital bosses, with other members of the Platanistoidea, such as the Platanistids found in the Astoria formation in Oregon and in the Calvert Cliffs formation in Maryland. This project's goal is to describe UWBM 87105, and place it as either a member of the Squalodelphidae or the Platanistidae.  Research into the distinguishing synapomorphies of the Platanistoidea and its families is ongoing; from these synapomorphies, I am deriving a character matrix for UWBM 87105. UWBM 87105's place within the whale phylogeny will expand what we know about the cetaceans present in the Pacific Northwest during the Miocene. In addition, studying this specimen will shed light on the diversity of the Platanistoidea, diversity that is no longer present in the oceans today.

An efficient and environmentally benign means of creating semiconducting polymers is necessary for the commercialization of organic electronics. Furthermore, it is crucial that as much control as possible be exerted over the polymerization process. To these ends, two polymerization methods are useful – C-H activation and living polymerization. First, C-H activation is a collection of synthetic techniques where C-H bonds undergo direct catalytic alteration to yield new homo- or heteroatom linkages. Whereas organometallic reagents were traditionally used to achieve these results, C-H activation allows for shorter syntheses utilizing less-hazardous reagents and broader functional group compatibility. Living polymerization is a form of addition polymerization and in our case, the number of chains is constant during the reaction and dependent on the catalyst-to-monomer ratio. This allows for much narrower mass distributions of the desired polymer and more consistent growth. To achieve this, benzoxazole-based monomers containing a C-O electrophile are to be reacted in the presence of a zero-valent nickel species with electron-rich phosphine ligands. By increasing the electron density on the metal center, we aim to facilitate a “ring-walking” mechanism at higher temperatures than previously thought possible. Monomer synthesis and polymerization results are detailed.

Solar energy is arguably the most promising source of renewable energy because it is thought to be both the cleanest and most abundant source. Several types of photovoltaics are now available, including inorganic, organic, and hybrid organic-inorganic. While inorganic photovoltaics have high efficiencies, good charge carrier mobilities, and relatively long charge carrier times, their widespread implementation has suffered from expensive production and materials competition with the microelectronics industry. Organic photovoltaics are solution processable and applicable to flexible substrates; however, their output has been hindered by their low charge carrier mobilities and short exciton diffusion lengths. Hybrid organic-inorganic photovoltaics have been introduced in an attempt to overcome the shortcomings of both organic and inorganic photovoltaics. Inorganic semiconductors are used in hybrid photovoltaics as the electron acceptor due to the ease of changing their size and shape, and therefore the bandgap, of these materials, which allows them to contribute to the photocurrent of the cell while being solution processable and potentially enhancing the conductivity. Conjugated polymers are used as electron donors due to their large absorption cross-section and solution processability. This project addresses the synthesis of hybrid cadmium selenide (CdSe) nanotetrapods, which we believe will facilitate control of the direction of electron motion in the active layer of the cell. Amines are needed for CdSe tetrapod functionalization with semiconducting polymers according to our reaction scheme. Tetrapods tend to convert into quantum dots because dots are thermodynamically favorable; our approach to have kinetic control, and thus have the tetrapods retain their shape, has involved synthesizing the tetrapods with amines as ligands. The reaction time, growth temperature, ligand type, and monomer concentration are used to control the synthesis, and the effects of each variable on the size and shape of the tetrapods will be studied via transmission electron microscopy (TEM).

Interest in renewable energy resources has increased in recent years due to the rising demand and dwindling supply of fossil fuels. While inorganic materials, such as silicon, have traditionally dominated the solar energy market, the use of organic photovoltaics (OPVs) has grown more popular due to their potential to be low cost, light weight, and their ability to comply with flexible substrates. Poly(3-hexylthiophene) (P3HT) is a polymer that readily self-assembles into nanowires under proper conditions, resulting in nanowire domain sizes that are on the order of the exciton diffusion length. Directing polymer self-assembly provides an opportunity to control the structure property relationships of OPVs and can greatly improve charge transport within the active layer. The regioregularity of a polymer is a measure of the uniformity of its geometric isomers. Because individual P3HT chains begin to kink into S-like shapes during self-assembly, a correlation between the P3HT regioregularity and the frequency of resulting nanowire defects is expected. In an attempt to understand the process of self-assembly, we would like to determine if these defects are incorporated into the nanowires during self-assembly, or if they are excluded from their crystalline domains. To investigate this, P3HT nanowires were made from solutions of anisole and chloroform in varying ratios, heated at 70 °C and thoroughly mixed for 24 hours, then removed from heat and allowed to self-assemble for at least three days. These solutions were then filtered to separate the nanowires from the decanted solution, and analyzed separately. A variety of characterization methods were used to study the presence of nanowire defects as well as their effects, including nuclear magnetic resonance spectroscopy, gel permeation chromatography, differential scanning calorimetry, atomic force microscopy, and matrix-assisted laser desorption/ionization spectroscopy.

In the Hague Lab we study a class of proteins called G-protein coupled receptors (GPCR's). GPCR's are seven-pass transmembrane proteins that are involved in cell signaling. One GPCR we study is called alpha-1D adrenergic receptor. Adrenergic receptors bind the endogenous catecholamines adrenaline and noradrenaline. An important functional role of the alpha-1D GPCR in the body is to regulate blood pressure by affecting blood vessel diameter.  Our lab recently discovered that the alpha-1D adrenergic receptor's relatively long N-terminal domain is cleaved in human cells. We are currently elucidating the specific cleavage site - which we suspect is a GL matrix metalloprotease domain - and the physiological purpose of the cleavage. Over the last few months we have narrowed down the region of cleavage to approximately 15 amino acids. We also have a working theory that the N-terminal cleavage of the protein allows for better cell surface localization. We are currently examining how the cleavage event effects the signaling characteristics of the protein.

Trypanosoma brucei (T. brucei) is an ideal model system for the study of a diverse range of biological processes, a number of which are unique to the kinetoplastid pathogens. The ability to genetically manipulate these parasites allows us to investigate the molecular machinery and mechanisms underlying these major processes. We have focused specifically on two areas of study: first, the identification and functions of essential T. brucei kinases; second, the functions of a number of uncharacterized proteins that we predict are essential for precise RNA editing in the mitochondrion of T. brucei. We created conditional null cell lines in which both endogenous alleles of a gene of interest are eliminated and a tetracycline-regulatable copy is inserted. Through the presence or absence of tetracycline, we regulated gene expression and assessed the phenotypic effects. Using this method, we were able to identify a set of protein kinases essential for cell viability, potentially functioning in cell cycle regulation and in connection with other cellular processes. RNA editing is an essential process unique to kinetoplastids, in which multi-protein complexes called editosomes insert or delete uridine nucleotides to create functional mitochondrial mRNAs. The major components of editosomes have been identified, though our understanding of the functional and structural organization of these proteins is limited. This is especially true for a number of proteins with limited detectable homology to other known proteins or domains. By generating and analyzing conditional null cell lines, we will assess the functional and structural roles these proteins play within editosomes for precise RNA editing. We anticipate that these proteins are key to understanding why specific sites in mRNAs are edited by insertion, whilst others are edited by deletion of uridine residues, which is a major outstanding question in the field.

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.

 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.

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.

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.