The tools of mathematical modelling have been applied to many sports where athletes are searching for strategies to improve their performance. Downhill mountain bike racing is a sport that could benefit from mathematical modelling. My research is aimed at developing a computer algorithm that uses mathematical modelling to simulate downhill mountain biking. In a mountain bike race, racers compete for the fastest time to individually ride down a trail with turns and obstacles. The fastest riders are the most efficient at converting their potential energy to kinetic energy. These riders accomplish this by optimizing the amount of their energy expended on braking and friction so they are just below the borderline of crashing throughout the entire trail. The finishing times of the top 10 riders are often very close, indicating that they are approaching a physical time limit to complete the trail. By using mathematical modelling and the GPS profile of a downhill trail as input, I aim to develop an algorithm that optimizes braking at every point on the trail and outputs the minimum time to complete the trail. The results from this algorithm would provide insights into how a rider could improve their performance. In my work, I show that mathematical modelling can accurately simulate the motion of a downhill rider, explain the challenges in developing a computer algorithm that optimizes braking, and discuss the results of an experimentally tested algorithm.

In the last 30 years temperature has risen 0.6°C per decade in high latitude regions, twice as fast as the global average. This extreme warming is causing perennially frozen ground (permafrost) to thaw, thereby changing subsurface hydrology and exposing previously stored, deep millenial-aged soils to microbial activity. These changes are stimulating greater organic matter mineralization and emissions of potent greenhouse gases (GHG), carbon dioxide and methane (CO₂ and CH₄). The magnitude of soil carbon mobilization is poorly contained, in part because it is unclear what fraction of GHGs are emitted to the atmosphere directly, versus released to above ground aquatic networks. To better define the role of streams in the changing arctic carbon cycle, we explored headwater stream carbon chemistry in 10 individual catchments situated in a remote and understudied subarctic landscape of interior Alaska. We found an unexpected, positive relationship between CO₂ and CH₄ across streams, with concentrations peaking in the summer for CO₂, and fall for CH₄, suggesting stream emissions peaked when soil active layers were deepest and permafrost carbon layers were most hydrologically engaged. The positive relationship between surface water temperatures and the concentration of each gas reflected these strong seasonal shifts in stream GHG content. Organic carbon content in stream water was also linked to CO₂ but not CH₄, indicating potential differences in sources and sinks of each GHG that are currently being explored with ongoing stable isotope analyses. Taken together, our findings show that closer-than-expected coupling of CO₂ and CH₄ may make some streams much greater emissions hot-spots than others, and that accounting for seasonality is critical for understanding the greenhouse gas budget of individual streams. 

While there is an abundance of computational tools for protein design, the vast majority of them focus on static structures. Data for the most probable rotamers of an amino acid are often obtained from mining its appearances across crystal structures, which do not take into account the native dynamics or conditions of a protein. Additionally, conformational secondary structure propensities are often derived from the frequency that an amino acid appears within a certain range of dihedral backbone angles in static structures. However, these data do not emulate realistic conditions, because proteins are dynamic and surrounded by solvent; thus, incorporation of dynamic data should improve the design process. Wrangler, our in-house software for peptide design, integrates dynamic data for both rotamers and secondary structure propensities. The data are obtained from molecular dynamics (MD) simulations in explicit water, taking into account interactions with surrounding solvent to better sample the most probable conformations. Several amyloid peptide aggregation inhibitors were designed using Wrangler’s scoring metrics to validate the software. These inhibitors were shown via MD simulations to better retain secondary structure than our current standards. Future work involves synthesizing the peptides in the wet lab to determine whether they experimentally perform better than our current inhibitors.

The Lyman-alpha power spectrum has previously been used to constrain the Universe’s initial conditions and particle constituents (such as the amount and mass of the dark matter) and the temperature of intergalactic gas (which constrains reionization processes). To further improve these constraints, we use another Lyman series transition (Lyman-beta). The Lyman-beta absorption cross-section is lower than that of Lyman-alpha so it probes the intergalactic medium at higher densities where Lyman-alpha features are saturated. Therefore, the Lyman-beta forest allows for a better measurement of the slope of the temperature-density relation, allowing additional constraints on reionization and the subsequent thermal evolution. In this work, we present an analysis of the Lyman-beta power spectrum using the VLT/XSHOOTER XQ-100 Legacy Survey.

Wildfires play a major role in the structure and composition of landscapes and the general ecology of the Pacific Northwest. The Klamath-Siskiyou eco-region in northern California and southern Oregon has been experiencing an increase in the frequency, scale, and intensity of wildfires in recent years. Understanding the effects of wildfires on small mammal communities is an important, yet understudied, aspect of the response of wildlife to wildfires. In 2014, two wildfires burned areas on and adjacent to a long-term study area of wildlife. We will investigate the effects of these wildfires on the occupancy of small mammals such as Neotoma fuscipes, Glaucomys sabrinus, and Tamiasciuris douglasii, using occupancy analyses of data collected during fall field seasons using track plate stations. The longitudinal data set that we have includes years of data before the fires occurred, allowing us to disentangle any effects of the wildfires from any naturally occurring variation. This research is important in determining what lasting effects these increases in wildfire frequency are going to have on predator-prey dynamics.

Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The genome of the model plant Arabidopsis thaliana has more than 30 GRX genes, but the biological function of most of these GRXs is unknown. We previously found that a small group of Arabidopsis GRX genes is specifically activated by nitrate, a common source of nitrogen in the soil. In order to better characterize the function of one of these nitrate-regulated GRXs, AtGRX660, we generated transgenic Arabidopsis plants that continuously overexpress the AtGRX660 gene. We isolated RNA from 12 independent transgenic lines and quantified AtGRX660 mRNA levels via real-time reverse transcriptase PCR. Three elite lines displaying >100-fold increase in basal AtGRX660 transcript levels were selected for further analysis. AtGRX660-overexpression lines and wild-type plants were grown on soil in a controlled environment growth chamber for characterization of shoot phenotypes, and on vertically-orientated plates of plant growth media for characterization of root phenotypes. All AtGRX660-overexpression lines displayed a dwarf shoot phenotype, with significant reductions in shoot biomass, total leaf area, and silique length compared to wild-type plants. In addition, root system architecture was highly altered. While primary root growth was normal in the transgenic plant lines, lateral roots were almost completely absent. Phase contrast microscopy demonstrated that lateral root primordia develop in the transgenic lines, but these primordia do not elongate and emerge from the primary root. Overall, these results suggest that AtGRX660 acts a negative regulator of shoot organ development and inhibits lateral root elongation. Our findings could have agricultural relevance in plant drought tolerance, since AtGRX660 differentially affects primary root system growth (root system depth) and lateral root system growth (root system breadth).

A person’s age is one of the greatest risk factors for many age-related diseases, such as Alzheimer’s disease, heart disease, and cancer. Developing interventions that target this common risk factor may allow us to slow down the progression of all age-associated diseases. One promising target for aging research is the mTOR (mechanistic Target of Rapamycin) signaling pathway, which is a master regulator of cell growth that allows the cell to fine tune its response to various environmental conditions. Inhibition of mTOR pathway has been shown to extend lifespan in yeasts, worms, fruit flies, and mice. In addition of being a target in aging research, the mTOR pathway is also a well-known effective target in some human cancers to prevent cancerous cells from dividing uncontrollably. We have previously developed a yeast-based system to identify mTOR inhibitors by comparing the growth of yeast strains that are differentially sensitive to mTOR inhibitors; a wild type strain, a tor1Δ mutant, and an fpr1Δ mutant. However, the system is not able to identify a few known ATP competitive inhibitors that have been shown to inhibit mTOR in human cells. This could be due to dissimilarities between yeast and human mTOR protein kinase domains. To address this issue, we are developing a humanized strain of yeast that should allow for identification of additional mTOR inhibitors that are effective in human cells. I am developing a novel hybrid gene containing the human mTOR kinase domain fused with the N-terminus of the yeast TOR2 gene. This new humanized strain will be utilized in our yeast-based assay with the goal of first detecting mTOR inhibitors known to be effective in human cells but unable to inhibit yeast mTOR. Successful completion of this goal will facilitate high-throughput screening of novel compounds that may have mTOR inhibitory and cancer fighting effects.

Long non-coding RNAs (ncRNAs) play a significant role in transcriptional regulation; therefore, mutations in their sequences can lead to human disease. An example is provided by the promoter associated non-coding RNA (paRNA) that, when bound with Argonaut 1 (Ago1) and a miRNA, plays a key role in coordinating gene silencing of the tumor suppressor CDH1 in epithelial cells. The paRNA expression can be implicated with cancer. This is due to a single nucleotide polymorphism (SNP) at positions -160(C/A) relative to the CDH1 promoter. The -160(A) isoform favors a unique secondary structure of the paRNA, distinct from more common -160(C), which favors over-suppression of CDH1 and leads to increased cancer risk. To better understand how the -160(A) isoform mechanistically drives increased suppression of CDH1, our lab is applying NMR-based methods to determine the 3D structure of the paRNA. However, the T7 RNA polymerase used to synthesize RNA is prone to producing heterogeneous products in longer RNAs such as this one. Having exact lengths of the transcript is important for improving signal-to-noise and peak sharpness in NMR spectra, which is critical for 3D structure determination. To overcome this problem, I designed a pUC19 plasmid containing the paRNA -160(A) sequence with a downstream self-cleaving hepatitis delta virus (HDV) ribozyme. Following transcription, the HDV ribozyme undergoes self-cleavage, producing homogenous ends at the 3’ end of the paRNA. My results show that HDV incorporation produces a single species of RNA with no 3’ overhang and improves NMR spectra quality. This cloning tool is easily adaptable to other large RNAs, facilitating data collection for other large RNAs used in our lab. Ultimately, our ability to predict the 3D structure of this paRNA could someday lead to its use as a potential drug target.

Sarcopenia, or age-related of loss of muscle mass and function, is associated with a decline in quality of life for elderly populations and few effective treatment options. Sarcopenia is linked to mitochondrial dysfunction and elevated mitochondrial oxidant production. We are investigating the role of mitochondrial oxidative stress in sarcopenia using a mitochondrial targeted therapeutic and a mouse model of accelerated sarcopenia. SS-31 is a mitochondrial targeted peptide that associates with cardiolipin, decreases oxidant production, and increases ATP production. Superoxide dismutase 1 knockout (SOD1KO) mice lack superoxide dismutase 1 (an enzyme that converts the oxidant superoxide into hydrogen peroxide and molecular oxygen) resulting in an accelerated sarcopenia phenotype. We are testing whether treatment with SS-31 preserves muscle function in the SOD1KO mice. We hypothesize that improving mitochondrial function with SS-31 treatment will delay the decline in muscle function in the SOD1KO mice. To test this, we are administering SS-31 to SOD1KO mice through surgically-inserted osmotic pumps for 8 weeks between 3 and 5 months of age (the published timeframe for the onset of skeletal muscle decline in SOD1KO mice) and performing in vivo muscle function measurements of the gastrocnemius before pump insertion and monthly after pump insertion for 3 months. We compare muscle functional measurements with histological and biochemical analyses of mouse tissue samples upon euthanasia and determine skeletal muscle fiber type, metabolite and protein concentrations, and muscle fiber respiration and oxidant production. We expect SOD1KO mice with SS-31 to have a lower rate of decline in muscle force production and increased fatigue resistance over time, higher max ATP production, and decreased oxidative stress. The effect of SS-31 on muscle function, mitochondrial quality, and redox homeostasis has exciting potential as a translational therapeutic treatment for human sarcopenia.

In recent decades, increased nitrogen (N) pollution in coastal aquatic ecosystems caused by increasing agricultural and urban activities has led to extensive habitat degradation and loss, change in the structure of aquatic food webs, and higher frequency of hypoxia. Nitrogen is ubiquitous in the biosphere, entering coastal environments through multiple pathways including ground- and surface waters, plus atmospheric deposition. The fate of much of the N entering coastal environments is not well established. In particular, the magnitude of N consumed at coastal margins, versus exported to downstream estuaries is poorly constrained. It is widely recognized that inland wetlands are important N sinks, as they are sites of fixation of reduced N to inert dinitrogen gas, however the role of coastal wetlands as N sinks is more difficult to establish due to the dynamic nature of these tidal environments. Here, to better understand the role of coastal wetlands in the global N cycle, we established a high-resolution budget of nitrate (the most abundant form of reduced N) from 9/12/2017 to 10/12/2017 at First Mallard Slough within the Suisun Marsh complex, a brackish tidal marsh in the San Francisco Bay Estuary. We modelled nitrate concentrations at 15-minute intervals using a submersible ultraviolet nitrate analyzer (SUNA), and matched these estimates with simultaneous hydrodynamic measurements of water flux. Data were synthesized in Python to establish 15-minute resolution estimates of nitrate mass exchange, showing that the wetland exported a net total of 2.54 Mg of N as NO₃^2- over the complete measurement period, or 84.6 kg N per day. Contrary to other studies showing wetlands are important nitrate sinks, our result revealed that the Suisun Wetland complex was an important N exporter to the San Francisco Bay Estuary, at least over the period measured here. Longer-term observations are needed to confirm this pattern at a complete annual scale.

Alzheimer’s disease (AD) is characterized as an irreversible, progressive brain disorder that gradually destroys memory and thinking skills, and eventually the ability to perform simple everyday tasks. Despite many decades of research focus on AD, the cause and mechanistic understanding of the progression remains enigmatic. Identification of amyloid beta plaques and hyperphosphorylated Tau in post-mortem analysis of brain biopsy is viewed as definitive for diagnosis of AD. Dr. Su-in Lee’s lab has developed a machine learning method that integrates brain tissue pathology metrics with gene expression analysis from the same patient samples. Their analysis identifies genes and pathways that are statistically-associated with the concordance of pathological AD phenotypes. Working with researchers in the Kaeberlein lab, we are using the nematode C. elegans to directly test the impact that these genes have on the progression of human tau-induced neuronal dysfunction and death. The Kaeberlein lab previously identified a subset of genes in complex I of the electron transport chain (ETC) that are highly correlated with human amyloid beta-induced paralysis. We are now extending that work by examining the relationship of these ETC genes in the lifespan and healthspan of human Tau-model nematodes. Our preliminary results suggest that reduction of Complex I activity increases the lifespan of human tau-models nematodes. We are conducting further studies to determine the replicability of these findings as well as disrupting ETC function with RNAi from other genes. Through directly testing nematode orthologs of human genes that are statistically-associated with AD neuropathology we will generate a better understanding of the cellular mechanisms that influence normative aging and Alzheimer’s disease.

Pacific sand lance (Ammodytes personatus) are an important forage fish in the Salish Sea that spend large portions of their time buried in sediment. Five important A. personatus habitats, including San Juan Channel (SJC), Sucia Island (SI), North Peapod Island (NPI), Salmon Bank (SBnk), and Iceberg Point (IP), have been documented by students in the Pelagic Ecosystem Function apprenticeship at Friday Harbor Labs since 2010. Continued sampling efforts have led to extension of an important time series at these sand wave fields throughout the San Juan Archipelago, as well as three new sites. New sites include Hein Bank (HB), North Waldron Island (NWI), and Puffin Island (PI). Both HB and NWI had A. personatus present, while PI did not. A synthesis of data from previous years showed sediment type at Sucia Island and North Peapod Island are most similar to each other, while San Juan Channel and Salmon Bank both have a large variation in sediment type. Shell hash is a prominent portion of many A. personatus habitats. Previous studies of these wave fields have treated shell hash as analogous to typical geologic sediments, however this study aimed to examine the divergence of shell hash physical dynamics from what is generally expected. The irregular shape of these biologically-derived sediments leads to a relationship between diameter and minimum settling velocity that is the opposite of what is expected for geologically-derived sediment particles. Of the shell hash tested (n=70), the maximum velocity was relatively consistent, and differences in settling velocity between diameters was seen in minimum velocities. These results have implications for understanding net transport throughout the system where different sediment types are present, understanding how sorting and packing of shell hash impact the habitat quality for A. personatus, and how differing amounts of these materials may distinguish these important benthic habitats.

The use of targeted microbubbles to image the molecular expression of vascular factors is an active area of ultrasound research. Combining the imaging advantages of ultrasound (e.g. cost, ease of use, availability) with the potential of molecular imaging makes targeted microbubbles especially attractive for studying the expression of vascular factors. During imaging, signals from molecularly attached microbubbles need to be separated from signals of non-attached free-flowing microbubbles in the vasculature. Thus far, different indirect approaches have been used to isolate stationary microbubbles. In this work, we present a direct approach to classify bound microbubbles in the presence of free-flowing microbubbles by processing nonlinear Doppler acquisitions. Nonlinear Doppler sequences are programmed on a research platform where Doppler processing separates low frequency stationary microbubbles signals from high frequency flowing microbubbles signals. In-vitro experiments are conducted by imaging stationary microbubbles surrounded by free-flowing microbubbles in a dialysis tube. In-vivo experiments are conducted by applying this approach to image the extent of inflammation associated with spinal cord injury (SCI), which plays a critical role in progressive tissue loss after injury. Both targeted and non-targeted microbubbles have been imaged in a rat SCI model. Targeted microbubbles were made for the inflammation marker p-selectin. Our in vivo results show successful separation of a limited number of non-targeted microbubbles adhering around spinal cord contusions. We believe this may be due to interactions between microbubbles and activated leukocytes. We expect to observe increases in bubble adherence and differences in the spatial distribution in using targeted bubbles, hopefully elucidating the extent of inflammation due to SCI. This work demonstrates the potential to separate bound targeted microbubbles from of free-flowing microbubbles to image a vascular factor for inflammation, which demonstrates practical pre-clinical ultrasound molecular imaging and opportunities for broader applications.

Spinal cord injury (SCI) is often a life changing and debilitating condition, where the loss of sensory and motor capabilities can be accompanied with bladder, bowel, respiratory and other dysfunctions. It is known that traumatic SCI causes an almost a complete loss of blood flow at the site of injury, as well as significant hypoperfused regions surrounding the injury, resulting in progressive cell death referred to as secondary injury. Counteracting secondary injury of spinal cord tissue, referred to as "rescue-able" tissue, is an active area of neuroprotective research. Surprisingly, there are no existing techniques to detect and assess contused spinal cord tissue at risk for secondary injury clinically or pre-clinically. In this work, we present an approach to visualize and quantify the blood flow changes after SCI by imaging microbubbles, an intravascular contrasting agent, with ultrasound following intra-venous injection. Nonlinear Doppler sequences were programmed on a research platform where Doppler processing separates microbubbles in the vasculature from background tissue signals. Our preliminary data demonstrate the ability to visualize changes in blood flow resulting from SCI in a rodent model. We will present results characterizing differences in blood flow associated from different injury severities. The nonlinear Doppler sequences are used to quantify the different characteristics of low velocity blood flow changes in the smaller vasculature and higher velocity blood flow changes in the larger vasculature. In addition, the passage of a bolus injection of microbubbles also highlights differences in blood flow in the contused and surrounding spinal cord tissue. Once translated, this ultrasound imaging technique could assist in detecting and monitoring local tissue perfusion at the injury site, ultimately improving SCI patient outcomes.

Many communities of color have been disenfranchised as a result of interactions with the criminal legal system. While many studies have shown that access to higher education reduces recidivism and encourages upward mobility, a very small percentage of this impacted population are actually able to access institutions of higher education. Therefore, the purpose of this research is to address the disparate representation of system-impacted individuals in higher education. This project aims to answer and begin to respond to the following questions: 1) What are the challenges and outright barriers for system-impacted students who wish to continue their education at UW? 2) What can UW do to make it a feasible destination for students who are system-impacted? This research project includes: the collection and analysis of existing data on system-impacted individuals and access to higher education; interviews with UW administrative offices that may play a role in the access to higher education for system-impacted individuals. Thereafter, a working group consisting of: system-impacted individuals; system-impacted students; students interested in criminal justice reform; and UW faculty interested in criminal justice reform; will form and implement strategies on how to lower institutional barriers and create clearer pathways to the UW for system impacted-individuals. By building partnering strategies with the UW community, the goal of the Pathways to UW project is to develop more clearer and transparent pathways for individuals who have been system-impacted to enroll on UW campuses. I want to ultimately build a community and a working network on campus that supports access and welcomes individuals impacted by the criminal and immigration system. Currently, there is no system in place on campus that supports such a marginalized population. To have such a system in place would address many racial and class disparities among the marginalized communities these impacted individuals traditionally come from.

Ion Mobility-Mass Spectrometry (IM-MS) is an analytical technique that is useful for analyzing large biomolecules with minimal disruption to their natural structure. One of the most common methods of introducing proteins to the gas phase in IM-MS is electrospray ionization (ESI), whereby a large voltage applied to a sample induces a spray of droplets that quickly evaporate and leave the desired analyte as a gas-phase ion. It is established that the high voltages of this process lead to a buildup of charge via electrochemistry in the sample solution, which may cause changes to the pH of the solution. However, this phenomenon has primarily been characterized in systems with continuously replenishing samples that form a steady state between excess charge formation and incoming sample flow. This work establishes a method to identify these changes in small, non-replenishing systems, which have not yet been characterized and are the standard practice for IM-MS analysis of proteins. Using SNARF-4F, a pH-sensitive fluorescent dye; a series of filters and a camera; and Python scripts for image processing; the rates and spatial position of pH changes under non-equilibrium conditions were determined with high accuracy. Additionally, several methods of preventing or slowing pH changes are examined. These include the use of buffers such as ammonium dihydrogen phosphate and ammonium bicarbonate, or periodic cycling between positive and negative electrospray. Preliminary findings indicate that these buffers can affect pH change, but at high enough concentrations, may also reduce the quality of mass spectra.

Alzheimer's disease (AD) is the most common cause of dementia, a general term for memory loss and other cognitive abilities. Although this disease has been a major research focus since the 1980s the pathologic mechanisms are still not understood, and therapeutic interventions have been ineffective. The most definitive method for classifying AD is through identifying accumulations of toxic proteins amyloid-beta and tau proteins in post-mortem brain tissue. Dr. Su-in Lee’s lab has developed a machine learning method that integrates the pathological tau phenotypes with gene expression levels in the same brain tissue. This analysis highlights the genes with expression level changes that correlate with the pathological protein aggregation phenotypes. For this proposal I will directly test the impact of these candidate genes on cellular pathologies resulting from aggregating human tau protein with a new C. elegans AD model in which human tau is expressed in the worm’s muscle. This tau expression will likely result in premature paralysis because previous nematode AD models with human amyloid-beta have shown this phenotype. The results of my genetic screening will lead to a better understanding of the human genes that are dysregulated in human AD brains and provide a basis for genetically-dissecting the pathways that influence the mechanisms of tau toxicity.

Recently, microfluidic technologies have been developed to allow higher throughput collection of yeast replicative lifespan data. Adoption of these devices has been limited, in part, due to the high cost of the motorized microscopy instrumentation from mainline manufacturers. Inspired by recent development of open source microscopy hardware and software, we developed minimal-cost hardware attachments to provide long-term focus stabilization for lower-cost microscopes and open-source software to manage concurrent time-lapse image acquisition from multiple microscopes. This functional 3D printed scaffold along with attached electronics has shown to capture accurate yeast lifespans without costing as much as other third party autofocusing system. We hope that this can help spur the wider adoption of microfluidic technologies for the study of yeast replicative aging.

Neurodegenerative diseases, such as Alzheimer’s (AD), Parkinson's, and Huntington’s, affect millions of people. In AD, prior studies indicate the formation and accumulation of amyloid-beta proteins may play a crucial role in the pathology of the disease. The Herpes Simplex Virus (HSV-1) encodes an alkaline nuclease (UL12.5) known to cause degradation of the mitochondrial genome. HSV-1 infection has been previously associated with AD brain pathology. We hypothesize that UL12.5 activity in the brain may predispose an individual to amyloid-beta aggregation and AD neuropathology. Here, we controlledl the amyloid-beta protein aggregation using a degron attached UL12.5, which is induced by the plant hormone auxin through a molecular signaling pathway known as auxin-inducible degron. We have engineered an auxin UL12.5-degron construct in order to precisely control the temporal and cell type expression of UL12.5 in Caenorhabditis elegans (C.elegans). This construct was microinjected into the worms and by using auxin, we controleld the expression of UL12.5 and tested its effects on amyloid-beta and Huntington protein aggregation. Here, we have elucidated the relationship between HSV-1 infection, UL12.5 expression, and neurodegenerative disease which may form the basis of novel treatments.

The lysosome is a critical organelle affected by the aging process. Changes to lysosomal physiology during aging and their effects on cellular function are poorly understood. Here, we study the lysosome-like vacuole of the budding yeast during replicative aging. Using a microfluidic device, we trap hundreds of mother cells and visualize them over the course of their replicative lifespans. Using strains with fluorescent protein tags we investigate protein abundance, intracellular physiological conditions, and organelle morphology. We find that during aging, the vacuole becomes progressively less acidic. Using a vma mutant as genetic model of vacuolar pH dysfunction, we find that loss of vacuolar acidity triggers a loss of respiratory capacity, loss of iron-sulfur cluster protein activity, and iron starvation response. We find that iron supplementation rescues both the loss of respiratory capacity and shortened replicative lifespan of vma mutant cells. We see that during aging, a similar iron starvation response occurs, correlated on a single-cell level with the loss of vacuolar acidity. We propose that the loss of iron-sulfur cluster synthesis capacity due to loss of vacuolar acidity is a conserved process underlying multiple aging phenotypes.

Cellular growth and aging requires constant replication of the genome. The processes of genome replication and segregation must be carefully regulated to prevent DNA damage which can cause cell death or oncogenic transformation. Damage can arise from errors in cellular processes, as well as environmental factors such as UV radiation or chemical agents. In mammals, the protein Retinoblastoma (Rb) prevents cells from beginning the process of DNA replication for cell division if internal conditions are not suitable for replication. This function is disrupted in virtually all cancers, and mutations in Rb have been widely studied as a result. Whi5 is the functional analog to Rb in S. cerevisiae. Whi5 activity is modulated by phosphorylation at 4 different sites, triggering Whi5 export from the nucleus and allowing transcription of DNA replication genes. Previous research suggested Whi5 export initiates irreversible progression through cell division. However, using single-cell observation technology, we show that Whi5 can re-enter the nucleus prior to cellular division, arresting the cell cycle. Cells that undergo Whi5 re-entry events exhibit a longer average replicative life span, counted in number of divisions. Additionally, cells without a functional copy of Whi5 have a reduced replicative lifespan. Our results suggest Whi5 may play a role in arresting the cell cycle in response to DNA damage to prevent the cell from making fatal errors during replication and ensure healthy aging. To investigate the role of Whi5 in response to DNA damage, I am using CRISPR gene editing technology to modify the phosphorylation sites on the Whi5 gene, modulating Whi5 activity. The ability of modified Whi5 strains to recover from UV induced DNA damage and their relative replicative life spans will clarify the role of Whi5 in DNA damage response. Our results may help us better understand the interactions governing Rb dysfunction in cancer cells.

For many globular proteins, the sequence and native structure are known. However, less is understood about how a string of amino acids folds into a functional protein. Experimental study of folding presents challenges due to the transience and variability of folding/unfolding transition states and intermediates. Alternatively, computational study of unfolding can provide significant insight into folding. Here, molecular dynamics simulations have been used to study the unfolding pathways of the SH3 domain structural family and to investigate the factors that determine the path and outcome. To separate folding determinants from amino acid sequence, 17 SH3 proteins were chosen with an average sequence identity of only 27%. Six unfolding simulations were performed for each protein, and the unfolding transition state ensemble was identified by locating the large, rapid conformational changes that signal the start of unfolding. Contact analysis was used to characterize the structure of the transition states ensembles. Two general pathways at the transition state were identified, distinguished based on the specific β-sheet structure lost at the transition state. In the first, more populated pathway contacts in the β-sheet containing the N- and C- terminal β-strands were lost while the second pathway was defined by structure loss in the other β-sheet. Though many of the investigated proteins went through both pathways in different simulations, most showed a clear bias towards one pathway. This work demonstrates that similar protein structures can fold through different pathways. The bias of many SH3 proteins towards one folding pathway also suggests the presence of some elements of primary structure that direct folding. Further investigation of the SH3 domain may yield ‘rules’ that determine the structure and folding pathway of the domain, and these rules may inform the study of other, similar proteins.

Alzheimer’s Disease (AD) leads to a progressive loss of neuronal function and ultimately death. Age is the greatest risk factor for AD; thus, a compound that slows down the aging process can help delay AD. In the lab, we use a Caenorhabditis elegans (C. elegans) model of AD in which human amyloid beta (Abeta) is expressed in body wall muscle cells resulting in age-onset paralysis. When combined with a robotic image acquisition device (the WormBot), we can analyze 144 different treatments simultaneously. The drug Istudying is Alpha Lipoic Acid. It is an antioxidant and cofactor for metabolic reactions required for oxidative metabolism. It has been previously reported to increase lifespan in C. elegans. My project focus on determining if Alpha Lipoic Acid reduces paralysis in the Abeta model. I investigated the mechanism of action of Alpha Lipoic Acid using RNAi knockdown of specific genetic pathways that are known to modulate aging. Ultimately, I have determined the optimal dose and a mechanism of action.

Previous research has established extracellular vesicle (EV) signaling as a ubiquitous cell-signaling modality used by all cells. In this modality RNA, peptide, and protein signals are packaged together in small vesicles and directed to specific downstream cell targets. In addition to being necessary for healthy physiology, EVs can also contribute to pathological processes such as neurodegeneration in Alzheimer’s disease through carrying toxic proteins tau, Aβ and β-synuclein between cell types. Recent research in the Kaeberlein and Mendenhall labs has shown that when human tau is specifically expressed in neurons of C. elegans, there is a significant increase in HSP90 chaperone and proteostatic stress reporter expression in the intestine. I hypothesize that the induced human tau neuron-to intestine stress signal is conveyed by the extracellular vesicles. To determine the signal pathway, extracellular vesicle signaling was disrupted specifically in neurons of a transgenic nematode line with neuron-specific human tau and fluorescent intestinal proteostatic stress reporter (daf-21::GFP). RAL-1 is a small GTPase that has been shown to be necessary for EV signaling in the C. elegans hypodermis and intestine. Therefore, RAL-1 function is disrupted though knocking down neuron-specific expression with either a double-stranded RNA ral-1 construct or a dominant-negative form of RAL-1 The degree of daf-21p::GFP expression was compared between the reporter lines and those with disrupted ral-1 function. Many studies utilizing C. elegans have shown that neurons send cellular stress signals to other tissues which strongly affect lifespan, metabolism, and proteostatic stress. Therefore, the results from my experiments contribute to determining whether EV-signaling carries neuronal stress-signals induced by human tau and establish a methodology for using C. elegans as a model for studying this important signaling pathway.

Alzheimer’s is a progressive, age-associated, neurodegenerative disease characterized by memory loss, deterioration in thinking and reasoning skills and gradual loss of executive function. Two well-documented biochemical hallmarks associated with disease progression are the accumulation of extracellular beta-amyloid (Aβ) plaques and intracellular neurofibrillary tangles found in the brains of Alzheimer’s patients. One theory of the disease holds that aggregated proteins found in these two deposits inhibit protein degradation by the 26S proteasome, a large protein that is one of the central components of the ubiquitin-proteasome clearance system. This system, of which the proteasome is a key component, executes most of the controlled protein degradation in the cell. Importantly, impairments in proteasome function are associated with multiple neurodegenerative diseases. The overall goal of this project is to determine whether proteasome stimulators can delay toxicity associated with Aβ aggregation in a C. elegans Alzheimer’s disease model. C. elegans is a powerful model organism in biomedical research due to its ease of culturing, well-described genetics, gene homology and availability of transgenic tools. My project utilizes a C. elegans strain that expresses the full-length human amyloid beta peptide (Aβ_1-42) in body wall muscle cells. These animals become paralyzed when up-shifted to a restricted temperature due to aggregation of Aβ proteins in the muscle. To test the hypothesis that proteasome stimulators delay the progression of Alzheimer’s symptoms, I will treat animals with four drugs and measure the time of paralysis. Using a high-throughput drug screening system recently developed in our lab, I will treat animals with four proteasome stimulators or vehicle controls, in triplicate, and determine the onset of paralysis for each drug. These results will help shed light on the involvement of protein aggregation clearance in treating symptoms of Alzheimer’s in this model organism, and help inform future treatments for this and other neurodegenerative disorders.

Aging is the single largest risk factor for many diseases, including Alzheimer’s disease (AD). Therefore, many of the molecular mechanisms that cause aging may also contribute to the onset and progression of aging-related diseases. Our approach to tackling the progression of AD takes a biology of aging standpoint, where screening compounds that impact lifespan may help identify therapies for AD. For this project, we are focusing on compounds that are either FDA approved or naturopathic. This is not only a novel approach but also has the potential to accelerate clinical translation. This study uses a human Amyloid-beta (Aß) protein expressing C. elegans strain that becomes progressively paralyzed with age. We are also utilizing a novel robotic imaging system (the WormBot) to explore the impact of these compounds on the healthspan of the AD model strain. Recording motility behavior of the nematodes under each compound condition and translating the time-lapse image data into quantitative healthspan curves, we will determine how each compound impacts the progression of paralysis. These results will provide insight into mechanisms of Aß toxicity and could lead to promising treatments that increase the quality of life for Alzheimer’s disease patients.

The James Webb Space Telescope (JWST) has the power to resolve many unanswered questions in the field of astronomy. The new telescope, which has taken many years and large amounts of resources to build, will have a limited lifetime so it is imperative that it is used as efficiently as possible. The unprecedented power of the JWST means that for the first time it may be possible to detect the clustering of high-redshift galaxies, and this could help to clarify uncertain details about the early universe. The purpose of this project is to determine the ability with which the JWST will be able to detect the clustering of early galaxies and the most efficient survey strategies for realizing this capability. Using simulation data, we are able to take into account JWST detectability limits and analyze clustering at different redshifts (z=6, z=8, and z=10). By comparing the simulation galaxies to a random test case, we have come to the conclusion that the clustering of high-redshift galaxies will likely be detectable with the JWST. Simulating different survey strategies then allows us to determine how to maximize the JWST's efficiency. The amount of time that the JWST spends pointed in a specific direction determines the depth of the survey. For a given amount of time, a deeper survey will be able to pick up on dimmer galaxies, but it will cover a smaller area. Our next step is to determine an ideal combination of depth and of area for the detection of early galaxy clustering at different redshifts in order to best plan for the JWST's launch.

Prion diseases occur from the misfolding of the Prion Protein cellular form (PrP^C) under low pH conditions to the infectious scrapie species (PrP^Sc), which can aggregate further into insoluble fibrils. Previous studies have demonstrated that along with other amyloid oligomers, the prion scrapie oligomers cause neurotoxicity by disrupting the membrane, increasing its permeability and affecting calcium ion influx; however, the molecular mechanism for this effect is unknown. Molecular Dynamics simulations were performed to gain insight into the molecular mechanism of PrP^Sc-induced misfolding of PrP^C and oligomer toxicity in a membrane environment. The system was composed of the hexameric bovine PrP^Sc spiral model oligomer and the di-glycosylated human PrP^C attached to a POPC membrane via a glycophosphatidylinositol (GPI) anchor. Prior unpublished membrane simulations of this system have suggested that PrP^Sc induced PrP^C conformational changes as well as significant membrane disruption from oligomer-binding. Here we confirm and build upon these earlier studies demonstrating the reproducibility and robustness of oligomer binding affinity by varying the proximity of the oligomer to the membrane, providing key insight into infectious scrapie propagation and PrP^Sc cellular toxicity.

The DNA damage checkpoint (DDC) is critical for survival as it responds to damage such as mutations or chromosomal rearrangement, and prohibits the cell cycle from proceeding through a failed replication. One of the many responses DDC activates is histone degradation. When wrapped around histone proteins, DNA is blocked from repair mechanisms. By reducing histone and nucleosome density, the cell increases DNA mobility and allows the DNA to be quickly repaired via homologous recombination. The cell does this by uncoupling histone transcription from the S-phase of the cell cycle, which is normally confined to this phase. When DDC is activated outside of S-phase, the cell can no longer produce histones to replace those it consumed and ensure proper chromatin packing after the DNA is repaired. According to published literature, uncoupling histone transcription from S-phase increases lifespan but should compromise the DNA damage response. However, it is largely unknown how this uncoupling affects the DNA damage response in different mutants. Our goal is to explore how this affects mutants with reduced DNA damage responses. Our mutants of interest are 1) rad52Δ, which reduces homologous recombination, and 2) lif1Δ, which reduces non-homologous end joining (NHEJ). Our protocol involves yeast (Saccharomyces cerevisiae) spotting and exposing yeast to different durations of high-intensity UV energy. We will compare colony sizes across different strains, dilution factors, and UV-exposure degrees. We expect to see strains with rad52 or lif1 deletion to have a lower growth rate compared to the wild type. Following this experiment, we will explore how altering histone transcription interacts with, and may enhance rad52 and lif1 in DNA repair. We hope to apply our findings to mammalian and human genes, and help to decrease the onset frequency of age-related diseases such as cancer due to genomic instability.

As we look forward to a global increase in our aging population and rising burden of noncommunicable diseases worldwide, exploring future treatments for age-associated diseases such as cardiovascular diseases, neurodegenerative diseases, cancers, diabetes, and chronic respiratory disease are of increasing importance to public health. Some studies credit the increasing burden of age-associated disease, especially that in younger populations, to the abandoning of traditional cultural practices and their replacement by Westernized diet, lifestyle, and medicine. This study explores conserved pathways of aging in response to compounds and extracts recognized by indigenous and ancestral medicine from various cultures. Employing a novel image acquisition robot, lifespan and age-associated disease phenotypes of wild type C. elegans nematodes exposed to selected compounds and extracts are observed and compared to identify potential treatments for age-associated diseases. Through conversations with traditional healers, review of the literature, and exploration of historical texts 4 major medicinal plants of interest were identified. Bitter Melon (Momordica charantia), vijayasar (Pterocarpus marsupium), rooibos (Aspalathus linearis), and Alaskan blueberries (Vaccinium ovalifolium) are recognized in East Asian, Indian, South African, and Native American traditional medicine, respectively, as treatments for slowing aging and age-associated diseases. Our research investigates how these compounds affect the longevity of C. elegans, with a focus on identifying their interactions with conserved longevity pathways. Our results contribute independent data and analysis for underrepresented compounds in aging and longevity literature, inform future research on more complex organisms, and increase recognition of indigenous and ancestral knowledge in the field of aging and longevity research. In our evermore connected global society, recognizing and respecting knowledge from diverse and often marginalized cultures and sources is critical to progressing longevity research, promoting equity, and improving health in our communities.