During neonatal transport, specialized pediatric transport teams closely monitor the status of critically ill newborns. Hyperspectral imaging, a method of manipulating light, can be used to measure the vital signs along with video of the patient’s physical appearance for remote monitoring. Appropriate light intensity is critical for clear visibility of the newborn and hyperspectral imaging accuracy, but this must be balanced with safety for sensitive eyes. In June 2022, an experiment using light bars and a photometer was conducted at Seattle Children’s Hospital using a newborn manikin in a transport incubator to measure the amount of light needed to view the manikin and the potential light exposure to the eyes of the newborn. Eleven images depicting the visibility of the patient model in the incubator were taken in controlled amounts of light. Upon my work analyzing the experimental results, preliminary light testing in the range of light intensity (0.5 - 1400 Lux) showed that the amount of light that reached the patient’s eyes was significantly lower than the maximum intensity of the light source and did not increase linearly with the increasing light intensity. I found that visibility of the patient relied on the light level, increasing as the light measured increased. My research of optimizing light levels for visibility and safety will inform approaches to remote patient monitoring during neonatal transports. Next steps include: to determine minimum acceptable lighting conditions for patient visibility, to establish minimum lighting conditions for hyperspectral imaging, to compare the spectral properties of measured light with existing data from ophthalmology literature on the safety of the light for newborn eyes to regulate the safest amount of light required for visibility.

The Ubiquitin Proteasome System (UPS) is a molecular recycling machine, responsible for  proteolysis and protein activity regulation. The components of the UPS attach a small protein ubiquitin onto other proteins which directly affects their activity or serves as a signal calling for modification or lysis. Ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3) are two classes of proteins that determine which proteins are tagged. Ube2W is an E2 with a unique function—it is the only E2 that places ubiquitin onto disordered N termini and amino acylated side chains. In this study we aim to elucidate the mechanism of reactivity and specificity of the enigmatic Ube2W. What structural and chemical features are responsible for its one-of-a-kind functionality? What does this imply about the role of this E2 on the cellular level? We designed a set of Ube2W mutants that had various putatively important features removed or changed to analogs from different E2s. We performed mutagenesis PCR followed by reactivity assays in the presence of known Ube2W substrates. We plan to collect NMR data for the Ube2W-substrate and Ube2W-ubiquitin interactions. We hope to determine which features are critical for this unique E2’s function by following the changes in reactivity when they are removed or altered. The interactions of substrates with these critical residues will help draft an outline for the precise mechanism. Improving our mechanistic understanding of Ube2W will pave the way for being able to control when and under what circumstances this unique biochemical reaction is used by the cell. This work aims to expand the current understanding of the UPS and aid in taming UPS-related diseases.

Microfinance institutions are considered pivotal in addressing poverty in low- and middle-income countries and providing financial inclusion through loans, savings, and insurance to those typically excluded from traditional banking. Most microloan recipients are women who own small businesses, and thus microfinance is regarded as pivotal to increasing women’s economic status. Some studies, however, highlight the rather lacking ability of microloans to improve household and business outcomes, as well as measures of women’s empowerment. My research explores if targeting such an agenda is the most effective way to uplift women and therefore help economic conditions in middle-income countries. I do this by examining the impact of microfinance loans on women’s autonomy in Vietnam by constructing a panel dataset from the 2006 and 2010 Vietnam Household Living Standards Surveys and designing a causal model. One channel through which microfinance empowers a woman is through her ability to financially contribute to the household, which in turn increases her decision-making ability, and thus changes gender relations in the household. In order to test increased decision-making, I calculate the share of resources in the household that are allocated to each woman and estimate changes in resource shares as a result of receiving a microloan. I expect results to show little to no increase in resource shares as the outcome of a household obtaining a microloan. In order to test changes in gender relations, I compare the previous results across Vietnam’s eight regions, which display varying levels of intimate partner violence. I expect regions with high rates of intimate partner violence to be negatively correlated with increased resource shares for women as compared to regions that have low rates of intimate partner violence. By addressing both the economic and social status of women, I illustrate a more complete picture of the efficacy of microfinance.

 Historical discourse has dangerously misrepresented and normalized the positioning of Black women in various states of vulnerability. Such discourses have then been embodied by society and further perpetuated within institutions- shaping the realities of Black women. This study explored how social services at a Seattle-based women’s shelter affect the experiences of Black women utilizing social services, while also understanding how Black women thrive utilizing such services. In this community-based qualitative research project, I used ethnographic methods to conduct semi-structured interviews with Black women utilizing social services at a Seattle-based womens shelter. I also conducted participant-observation methods to collect data on the shelter’s environment and the dynamic between Black women seeking services and employees providing services. Finally, I reviewed relevant theoretical frameworks such as intersectionality, the criminalization of poverty, and racial capitalism to assist in my analysis of how Black women seeking services at a Seattle women’s shelter have been positioned to experience vulnerability. Key findings include 1) social services have been socially constructed to sustain the experience of vulnerability within the Black community, specifically by Black women utilizing services; and 2) through various levels of gratitude, Black women find spiritual peace and grow new resiliences to systemic challenges. These findings draw awareness to the ways social services have deepened Black women’s dependency to them. Such awareness could serve as a critical framework for reshaping the way social services within the greater Seattle area assist Black women to promote prosperity, dismantle racist rhetoric, and aim to eradicate, not perpetuate, homelessness.

When the cell undergoes stress, it leads to an increase in protein instability and misfolded states that prevents proper cell functions. Small Heat Shock Proteins (sHSPs) are molecular chaperones that work to maintain a healthy proteome by associating with misfolded proteins to delay aggregation under stress conditions. Other chaperones and co-factors will then refold or degrade the misfolded protein client. HSPB5 is a human sHSP ubiquitously expressed throughout the body. A HSPB5 disease mutant, where arginine is mutated to glycine at residue 120 (R120G), is a defective chaperone associated with cataracts and desmin-related myopathy. HSPB5 comprises three domains but only one domain—the alpha-crystallin domain (ACD)—is folded. My research aims to understand the effect of the R120G mutation on the folded ACD’s structure. HSPB5 creates a dimer through electrostatic and hydrophobic interactions between ACD at the dimer interface. This network of interactions causes the dimer interface to be highly sensitive to electrostatics, working like a sensor for environmental charges. Wild-type HSPB5 is more positive at its dimer interface, likely facilitating interactions with negatively charged compounds. In the R120G mutant, the loss of arginines at the dimer interface site causes it to be less positive, hypothetically lowering HSPB5’s affinity for these compounds. Through site-directed mutagenesis, I obtained HIS-tagged cleavable constructs for wild type and R120G B5 ACD that allowed for easier purification. Using these constructs, I grew isotopically labelled N15 B5 ACD in minimal media and purified my protein sample through nickel affinity, size exclusion and anion exchange chromatography. Through NMR titration experimentation, I investigate how amino acid identity at the R120 site will affect ACD interactions with charged molecules in R120 mutant of HSPB5. Learning how mutations at the R120 site affect protein dynamics and client interactions will be a step forward in understanding the sHSPs’ overall chaperone mechanism.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool for detecting unpaired electrons in molecular systems. By attaching two spin-labels which contain unpaired electrons to different regions of a protein, the distance between the spin labels can be measured, making this technique particularly useful for studying protein structure and dynamics. The signal sensitivity of these measurements is especially sensitive to protons on amino acids adjacent to the spin label. While it is known that the presence of neighboring protons to the spin label decreases the signal intensity, the magnitude in which specific amino acids affect the signal is not well understood. My research aim is to determine how specific neighboring amino acids affect the EPR signal. Here, I design model systems in which spin labels are placed on various parts of maltose-binding protein (MBP) to construct a sample set that contains the spin label in diverse amino acid environments. These spin labels are placed on MBP through a process called site-directed spin labeling. MBP is mutated through site-directed mutagenesis using specific primers, the final plasmid transformed into competent E. coli cells. Through collecting EPR data of these MBP mutants, we can gain insight to which amino acids neighboring the spin label most affect the signal. This project will help us understand how to determine spin-labeling sites to result in maximum EPR signal intensity. Maximizing this signal intensity will enable us to use EPR to study biological systems that could not previously be studied due to lack of sensitivity, such as membrane proteins.

Chronic kidney disease is the ninth leading cause of death in the United States. The current process for pathological diagnosis involves pathologists manually reviewing histochemically stained tissue slides. This analysis is also used to inform further treatment and is therefore critical to patient outcomes. In this project, we aim to improve the robustness of the diagnostic process by utilizing machine learning models to identify and classify features indicative of kidney disease on whole slide images. We manually annotate Masson’s Trichrome stained kidney tissue images from our collaborators at the University of Illinois for three functional structures (tubular cytoplasm, tubular basement membrane, glomerulus) and three indicators of damage to the kidney (fibrosis, edema, and inflammation). These annotations are used to train our VGG16 convolutional neural network model to classify patches of unmarked whole slide images into the four categories: tubular cytoplasm, fibrosis, inflammation, and glomerulus. We also address data variability that often comes from differences in the histochemical staining procedure across labs resulting in inconsistency across stains/imaging that can typically affect the generalizability of deep learning models. To address this, we are training a CycleGAN for image-to-image translation as a method of stain normalization and investigating the effect on the accuracy of our VGG16 model. Additionally, I will be training a model to identify the cortex versus medulla regions of the kidney to add to the pipeline for area-specific evaluations. Our research with integrating machine learning models within renal pathology aims to decrease the time and manual labor needed in the process and increase the accuracy of diagnoses.

In the ubiquitin-proteasome system, the E2 enzymes are involved in the second step of transferring the ubiquitin (Ub) to a substrate. Specifically, an E2 enzyme receives the ubiquitin from an E1-Ubiquitin(E1-Ub) conjugate and becomes an E2-Ub conjugate. Then, an E3 enzyme can deliver the ubiquitin from the E2-Ub conjugate to a substrate to form a substrate-Ub conjugate. Most of the E2s attach Ub to a substrate lysine residue, but Ubc6, an E2 from yeast, also seems to react with substrate hydroxyl groups on serine/threonine/tyrosine. This project is divided into two parts: first, validate the reactivity of Ubc6 in different amino acid (serine/threonine/lysine/cysteine/tyrosine) conditions. Second, apply a proper machine model to predict the reactivity of Ube2J2-Ub, a mammalian homolog of the Ubc6-Ub conjugate. The quantification analysis on the Ubc6 charge/discharge assays can reveal the rate of the reactivity of the Ubc6 in different amino acid conditions. After validation, three types of E2s with known reactivities: Ubc2D(1/2/3/4)-Ub, Ube2L3-Ub, and Ubc6-Ub, can be used as training sets for the machine learning model. Once the model predicts the reactivity of Ube2J2, the prediction can be validated by performing assays on Ube2J2. We expect that Ubc6 reacts fastest with Cysteine, followed by Threonine, Lysine, Tyrosine, and Serine. Since Ube2J2 is a human homolog of Ubc6, we predict that Ube2J2 has the same reactivity as Ubc6. The implication of this project is whether machine learning can assist with finding the reactivity of a protein enzyme.

Under cellular stress conditions, proteins are destabilized, often causing them to misfold, and in certain cases aggregate. Without protective systems in place, significant cellular dysfunction occurs, often resulting in cell death. One mechanism of molecular stress response is increased expression of small heat shock proteins (sHSPs). sHSPs are ATP-independent chaperones that are thought to keep proteins in a refolding-competent state during stress and work with other chaperone proteins to rescue proteins from irreversible aggregation. Members of the sHSP family are defined by their shared structure: a conserved α-crystallin domain (ACD) flanked by unstructured N-terminal (NTR) and C-terminal (CTR) regions. Transient interactions between the three domains are important regulators in oligomerization. The structured ACD is the building block of sHSP oligomers and provides a framework for studying oligomerization. The ACD dimer contains three grooves into which the NTR and CTR can bind. Though it is clear the ACD plays a major role, the regulation of these domain interactions underlying oligomer formation is not well-defined. HSPB5 and HSPB1 are two human sHSPs that are ubiquitously expressed throughout tissues and can form homo- and hetero-oligomers. My experiments were aimed at studying the effects of mixing the isolated ACD with the full-length protein. To analyze the domain interactions and oligomer size changes upon mixing, I used size-exclusion chromatography (SEC). In these mixing experiments, addition of the ACD to different oligomers increased the number of available grooves with each titration point. The experiments presented here provide glimpses into the hierarchical organization of homo- and hetero-oligomers of HSPB1 and HSPB5. My results indicate that not only does the number of grooves matter, but also their identity. My working hypothesis is that the identity dictated the strength of the interactions between the different binding components and led to differences in the propensity for subunit exchange in oligomers.

Over the past 20 years, the capital city of Indonesia has sunk four meters into the ocean and continues at a rate of approximately 11 inches per year. At this rate, more than 95% of Jakarta, the capital city, is predicted to be completely submerged by 2050, inevitably drowning the city if stricter policies are not set in place for groundwater extraction. Due to the population’s poor access to piped water, private businesses and the public have resorted to illegal groundwater extraction, which has caused extensive land subsidence. The combined effects of regulated and unregulated groundwater extraction and the lack of equitable water access to the population have worsened the situation. Jakarta is not alone; other countries like China, Thailand, Vietnam, and the United States are also experiencing land subsidence due to extensive groundwater extraction. My literature review will demonstrate the effects of groundwater extraction and land subsidence in Indonesia by using a comparative public policy analysis on the different policies and management approaches of countries in Southeast Asia and North America. I will be comparing the policies in use by these different countries to determine the necessary policy reforms to create a more effective approach to solving the groundwater issue in Indonesia. I establish that the Indonesian government’s short-term plans to overcome land subsidence is not a sustainable solution to the damage that has been done to the city of Jakarta. Rather, the policies set in place need to be reformed to ensure that equitable water is supplied throughout the population and groundwater extraction is done safely and in moderation. This work is important not only to prevent the further sinking of Jakarta but also to overcome the long-term environmental impacts of groundwater extraction in Indonesia and other countries.

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. TB is typically diagnosed by analysis of sputum, which can be difficult and uncomfortable to produce, especially for those living with HIV and children. Improved means of case finding are essential for disease detection and control. Oral Swab Analysis (OSA) is an alternative processing method for detecting Mycobacterium tuberculosis (Mtb). Oral swabs can be collected easily from everyone, including those who have difficulty producing sputum, but OSA requires further optimization to reach the level of sensitivity of current sputum analysis methods. Over the past year I have been working with high capacity foam swabs to optimize processing methods for Mtb detection. Foam swabs collect more biomass and Mtb material but also greater amounts of Polymerase Chain Reaction (PCR) inhibitory material from the tongue. I explored various DNA extraction techniques to minimize the impact of inhibitors. The GeneXpert, a cartridge based nucleic acid amplification test, has yielded the most promising results. I have developed an optimized protocol that has improved the efficiency of the Xpert machine when tested with contrived foam swab eluates. I will validate my findings with a paired analysis between the current “gold-standard” of oral swabs, the Copan FloqSwab, and foam swabs, using clinical samples collected from TB patients by our collaborators in South Africa. I anticipate the foam swabs will detect a greater number of weakly-positive samples as well as yield stronger PCR signals than their FloqSwab pairs. The global burden of TB remains incredibly high and there is an urgent need for improved case finding methods. Diagnostics that are effective in the absence of symptoms, safe and implementable in low resource settings are of the utmost importance. This research has the potential to improve upon existing clinical testing and increase detection of TB disease worldwide.

Pathogenic bacteria often promote their growth by introducing proteins into host cells they are infecting. Some of these introduced proteins hijack host cell ubiquitin signaling pathways. Ubiquitin (Ub) is a small protein that is involved in many signaling pathways within eukaryotic cells. In ubiquitin-mediated degradation, the degradation of a target protein is promoted by the attachment of chains of ubiquitin onto the target protein. The attachment of poly-ubiquitin chains is facilitated by a class of proteins called E3 ubiquitin ligases. Salmonella Typhimurium secretes into host cells an IpaH/SspH class E3 ubiquitin ligase called SspH1. My research investigates the mechanism for how SspH1 modifies its target protein, Protein Kinase N1 (PKN1), with poly-ubiquitin chains. I am investigating whether ubiquitin is transferred sequentially onto PKN1 or if a long poly-ubiquitin chain is first formed on SspH1 and then transferred en-bloc onto PKN1. Using biochemical methods, I will compare whether the rate of poly-ubiquitin chain synthesis is faster starting with unmodified PKN1 or mono-Ubiquitinated PKN1, an intermediate in the sequential addition pathway. I hypothesize that the formation of poly-ubiquitin chains onto mono-Ub-PKN1 is slower than the formation of poly-ubiquitin chains onto free PKN1. This would suggest that the mechanism does not occur through the sequential addition of ubiquitin. Learning more about the mechanism of SspH1 will allow us to both better understand the IpaH/SspH class of proteins and better understand how ubiquitin can be transferred onto protein substrates.