DNA replication is the process by which genetic information is duplicated and passed from cell to cell. The process of DNA replication occurs with the assistance of various enzymes, including DNA polymerases, which synthesize new DNA molecules using template DNA and deoxyribonucleotides (dNTPs). Faithful DNA replication is vital for population survival and protected by exonuclease domains on the major DNA polymerases, which proofread replication errors. A secondary error-correction pathway known as mismatch repair (MMR) also scans the genome for replication errors. Defects in proofreading and MMR lead to "mutator phenotypes" marked by elevated mutation rates and increased cellular mutation burden.  In microbial populations, mutators spontaneously emerge and can increase adaptation to new environments by fueling genetic diversity. However, at extreme mutation rates, the accumulation of harmful mutations can also drive extinction of mutators.  Some mutator cells survive this "error-induced extinction" (EEX) by acquiring antimutator mutations that suppress the mutator phenotype. We have identified Saccharomyces cerevisiae strains that suppress elevated mutation rates through a screen for mutants that survive EEX. Using these eex mutant strains, we are currently mapping the genetic changes responsible for the antimutator phenotypes. Changes in mutation rates in haploid spores derived from these eex suppressor strains are being tracked to identify antimutator segregants, which are then being subjected to whole genome sequencing. As mutator phenotypes are important drivers in cancer, our analyses may reveal novel pathways that influence cancer evolution.

Ion Mobility Mass Spectrometry (IMMS) has become an important tool for separating small molecules such as peptides, glycans, and petroleum molecules. Ion Mobility is most often performed in He, N₂ or air. There is a great interest in using alternative drift gases to increase selectivity of ion mobility separations, specifically for complex mixtures like petroleum. In this study we measured arrival time distributions (ATD) of a set of double bond equivalents (DBE) in four different gases (He, N_2, CO₂, Ar) which span a range of masses and polarizabilities. Quinoline, isoquinoline, and their DBE were chosen because they are aromatic compounds that are easily protonated due to the presence of nitrogen and are similar to molecules found in petroleum. The measured ATDs were plotted as a function of reciprocal drift voltage; the resulting slope is inversely proportional to mobility. Using the mobility and the Mason-Schamp equation, we determined the collision cross section of each molecule with each drift gas. We also investigated the relationship between the dipole moment of an ion and its collision cross section. In general, collision cross sections increase with drift gas size and polarizability. Additionally some of the DBE were better separated with larger, more polarizable drift gases (N₂ and CO₂). We have shown that IMMS can be used with different gases to improve separation between ions of similar size and shape. This method can be used in future investigations of petroleum mixtures to potentially identify a wider range of molecules.

Biofilms are surface-associated aggregates of bacteria encased in a protective, extracellular matrix. Biofilm bacteria resist antibiotic treatment and killing by the host immune response, leading to persistence in a variety of chronic infections. Pseudomonas aeruginosa is an opportunistically pathogenic bacterium and a model organism for studying biofilm formation. One major factor that drives biofilm formation in P. aeruginosa and other bacteria is the intracellular second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP). Elevated c-di-GMP levels promote cell surface adhesiveness by up-regulating production of biofilm extracellular matrix components and down-regulating motility genes. In contrast, planktonic P. aeruginosa cells are known to have comparatively lower c-di-GMP levels than their biofilm counterparts. While factors contributing to the formation of mature biofilms have been well-characterized, early biofilm formation, when a bacterium first senses a surface and transitions from a planktonic state to a surface-attached state, remains largely understudied. Using a fluorescent, transcriptional reporter of intracellular c-di-GMP and confocal microscopy, we have monitored the dynamics of c-di-GMP production at the single cell level. Our major finding is that c-di-GMP levels are elevated in only a subpopulation of P. aeruginosa cells (30-69%) during this time. We have used flow-assisted cell sorting (FACS) to separate surface-attached cells with high and low c-di-GMP, to examine ways in which these populations are physiologically distinct. We have found by lectin staining and by qRT-PCR that surface-associated cells with high intracellular c-di-GMP produce more polysaccharide than their surface-associated, but low c-di-GMP counterparts. We hypothesize that the heterogeneity in c-di-GMP observed during P. aeruginosa surface sensing represents a specialization among the genetically homogenous population into subpopulations of non-motile, early polysaccharide producers and motile, surface-exploratory cells, both of which contribute to downstream biofilm maturation.

The University of Washington (UW) Seattle has over five writing centers on campus, where in-person tutoring is available for students. We hypothesize that certain obstacles reduce access barriers to writing center services for student populations, including those who experience disability, commute long distances, identify as ELL, or are non-residential. UW writing centers have seen an impressive increase in student use in the last 5 years. The overall increase in usage, coupled with changing student needs and resources has sparked an important conversation about the future of writing support services at UW. We hypothesize that online tutoring, in addition to the current model, could help solve issues of access. In order to explore this hypothesis we are have prepared a multi-phase, qualitative study, which is currently underway. Drawing upon the demographic and satisfaction data of current campus writing centers, we discuss what current gaps may exist and how online tutoring could potentially bridge them. The current tutoring setup, one-to-one in-person conversation, may also be excluding students who are not comfortable with this format. Through on-campus focus groups, interviews, and surveys we are triangulating on factors of the writing center experience most salient for different stakeholders. We also examine dimensions such as student's area of study, year in school, familial obligations, and current employment status as they may relate to writing center access. In a multi-factor perspective analysis, we gain perspectives of administrators, tutors, users, and potential users at writing centers on campus. In doing so, we pursue a better understanding of how writing centers could use online tutoring to provide services to students who would not have access otherwise. As students and writing center tutors we feel a deep, personal investment in this project and are excited to be a part of building a positive future for writing support at UW.

In the Pacific Northwest, partial-cutting techniques are increasingly being used as “alternative silviculture” or “ecological forestry” approaches to improving structural and compositional diversity in even-aged Douglas-fir plantations. The resulting canopy gaps vary in size and distribution of the overstory, thereby causing differences in the amount of light and moisture available in the understory environment. A better understanding of how different species respond to varying levels of light and rhizosphere moisture is critical for species selection and successful regeneration efforts. This study examined the early growth dynamics and tolerances of seven tree species native to the west side and five species or varieties of species native to the east side of the Cascade Mountains. The experiment followed a nested factorial design in which seedlings were grown in large pots with three light levels created by shade cloth and three distinct tapered irrigation regimes. This component study paralleled an in situ experiment conducted at field sites around the state. Seedling responses were assessed by repeated measurements of survival and aboveground morphology for growth response. After one year, seedlings were destructively sampled for measurements of leaf area and dry shoot and root weight. The majority of morphological responses for each species were significantly (p<0.05) affected by light availability. Light was the significant (p<0.05) driver of above- and belowground growth of Abies grandis, Larix occidentalis, Pinus monticola, Pseudotsuga menziesii, Tsuga heterophylla, and the eastside varieties of Pinus ponderosa and Thuja plicata. The interaction between light and water treatments had a significant (p<0.05) impact on above- and belowground growth of Alnus rubra and the westside varieties of P. ponderosa and T. plicata. The highest mortality rates were observed for Acer macrophyllum and the westside variety of P. ponderosa, suggesting poor stock quality influenced early field potential.

The biological basis behind aging in eukaryotic species is defined by a wide range of factors, from diet to environmental conditions and metabolic predisposition. The genetic factors associated with aging are of particular interest because they are highly conserved among all eukaryotes. Determining longevity genes that are broadly conserved among eukaryotic species is a critical step in understanding aging, and is one of the primary goals of this project. The Kaeberlein lab has analyzed the replicative lifespan (RLS) of more than 4700 single-gene deletion mutants of Saccharomyces cerevisiae, a eukaryotic model organism. RLS experiments measure the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Mother and daughter cells are visibly differentiated under a light microscope and physically separated using a fiber-optic needle. The number of daughter cells dissected are quantitated and recorded. The total number of cell divisions are then summed for statistical analysis. The result of the investigation yielded 238 gene deletion mutants that exhibited a statistically significant lifespan extension. Longevity genes were found to be clustered within specific parts of an organism's biology. Ribosomal genes and genes related to metabolic responses to nutrients were particularly significant. One particularly significant gene is the LOS1 gene, which functions as a tRNA exporter from the nucleus. Deletion of the LOS1 gene resulted in a dramatic enhancement of yeast RLS, opening the possibilities of lifespan research regarding the subcellular localization of tRNA. Also significant was the discovery that a majority of the longevity genes were concentrated among highly evolutionarily conserved parts of the genome. Ultimately, the success of the yeast RLS project in identifying novel longevity genes and establishing a database of conserved longevity genes in eukaryotes points to the efficacy of such methodology.

Aging is a phenomenon that brings about the onset of disease and eventually death. One of the hallmarks of aging is the decline in proteome maintenance that can lead to the accumulation of misfolded proteins. In extreme cases, protein misfolding can induce such devastating neurodegenerative diseases such as Huntington’s disease and Alzheimer’s for which very limited treatment is currently available. It has been observed previously that dietary restriction is a method that increases lifespan and delays the loss of function caused by age-associated pathologies. Specifically, complete food removal leads to robust increase in longevity in the roundworm Caenhorbabditis elegans, a popular model organism for aging research. Remarkably, when pre-reproductive adult worms are subjected to starvation, they enter a condition known as adult reproductive diapause (ARD). Upon reintroduction to food after prolonged starvation, ARD worms experience a pronounced morphological recovery and have normal lifespan. To understand how diapaused animals preserve this rejuvenation potential and normal lifespan we aim to analyze how proteome quality is maintained during the diapause. We hypothesize that proteome quality is either well maintained during the diapause or is restored back to youthful state upon exit from the diapause. To discriminate between these possibilities, we use C. elegans models of proteotoxicity wherein toxic protein species produced in muscle cells induce paralysis with age. By analyzing the accumulation of insoluble protein aggregates and motility during and after the diapause we will gain a better understanding of the physiological aspects of the ARD state. These insights will be useful in developing novel approaches to counter age-associated pathologies.

On June 16th, 2015 business mogul Donald J. Trump delivered a presidential announcement speech, announcing his candidacy for president of the United States, launching his ascension to the White House. Through a rhetorical analysis of Donald Trump’s Presidential Announcement Speech utilizing Kenneth Burke’s framework of dramatism, Trump’s speech is shown to mimic the effective rhetorical strategy of the United States Christian theological prosperity gospel movement. The speech establishes a negative status quo, provides testimony on a successful break from the status quo, and concludes with a plan for redemption, showing listeners how they, too, can break from the status quo utilizing an exemplary model. Trump serves as the exemplary model, claiming to know a path toward greatness, articulated with his campaign’s slogan, “Make America Great Again”. However, Trump’s imperfections, including his relationships with the elite and break from expected niceties in rhetoric, required mortification and scapegoating for justification to legitimately be seen as the exemplary model. The implications of Trump’s rhetorical strategy may inform future political candidates’ rhetoric and provide insight into President Trump’s framing of policy issues.

The process of aging brings about susceptibility to disease and death, but its mechanisms are still poorly understood. Dietary restriction is a potent approach to slow down the aging process, which has shown efficacy in multiple species, including the roundworm, Caenorhabditis elegans, a popular model organism for aging studies. Specifically, pre-reproductive adult C. elegans subjected to starvation enter a state of adult reproductive diapause (ARD), which preserves their lifespan and reproductive potential. Intriguingly, diapaused animals show signs of age-related deterioration upon prolonged starvation, but exhibit dramatic morphological improvements and have normal lifespans following return to feeding. However, the mechanisms regulating this lifespan preservation and apparent rejuvenation are not known. In this project, we aim to identify the genes responsible for the recovery during diapause exit. For that purpose, we used an RNA interference (RNAi)-based screen to inactivate individual genes during exit from diapause and analyzed the resulting phenotypes. Our current results reveal many regulators of metabolism and cellular homeostasis as crucial mediators of post-diapause recovery. Further studies based on these findings will discover the critical steps required for post-diapause tissue rejuvenation and preservation of longevity.

It is known that dietary restriction is a positive intervention in aging. Its efficiency was demonstrated in multiple species suggesting that dietary restriction acts through conserved mechanisms that are not well understood. Specifically, in Caenorhabditis elegans (C. elegans), dietary restriction can extend lifespan well beyond that of their fed counterparts. Remarkably, it has been found that upon starvation young adult C. elegans enter a state of adult reproductive diapause (ARD) that allows a full lifespan preservation. We aim to characterize the mechanism by which lifespan can be preserved and extended in the diapaused animals. To this end we will investigate whether ARD animals experience typical age-related changes during prolonged starvation and whether they are reset upon diapause exit. Through fluorescent microscopy, we intend to distinctively characterize phenotypic differences of the cytoskeleton and nuclear morphology between ARD individuals and their fed counterparts. We expect these age-related markers either remain intact throughout the diapause state, or repaired upon diapause exit. This will further allow a better understanding of aging as wells the mechanisms of lifespan and healthspan preservation.

Aging can be defined as the time-related deterioration of physiological functions that are essential to the fertility and survival of a living organism. In the Kaeberlein Lab, we study how cells change with age and how these changes contribute to age-associated diseases in order to help people live healthier lives. The eukaryote Saccharomyces cerevisiae (yeast) is used as a model for cellular aging. Due to its conservation of cellular functions through evolution, modeling aging using yeast can provide insights into how mammalian cells age. Through replicative lifespan (RLS) analysis, we are able to determine the effects of interventions on aging by measuring the number of divisions that a single yeast cell goes through before it stops dividing. We are testing various nutraceuticals in order to identify novel pharmacological interventions that increase lifespan. In a screen of twenty nutraceuticals, we found that Pterocarpus marsupium (PME) extract significantly extended lifespan. P. marsupium is a deciduous tree native to the Indian regions. It is well-known to Ayurvedic medicine thanks to its purported curative and lenitive properties. The flowers are said to be effective in lowering fever; its heartwood may possess purifying and detoxifying effects; its wood is used to treat chest and body pain as well as indigestion. Additional studies will test whether the RLS-extending effects of PME can be attributed to pterostilbene, a compound found in PME that is structurally related to resveratrol. We tested concentrations of pterostilbene ranging from 1-50 µM to identify its effect on replicative lifespan. These studies will allow for further development of PME and pterostilbene for use as an intervention to promote healthy longevity.

The Food and Drug Administration (FDA) approved drug rapamycin lengthens lifespan and rescues rodents from age-related diseases. Previous studies have shown that a 3-months regimen of rapamycin for old mice of the C57BL/6 strain changes the composition of gut bacteria that includes increased bacterial species such as lactobacillus, bacteroides and segmented filamentous bacteria (SFB). In mouse models with a mitochondrial disease, there was a decreased amount of bacteria and an extremely short lifespan. Injection of rapamycin increased the amount of bacteria and prolonged lifespan, showing an association between the quantity of bacteria and lifespan. We are interested in the effect that this experiment would have on a different strain of mice, Balb/c, which had not been previously tested for the rapamycin effect on lifespan nor the change in the composition of gut bacteria. First, we conducted a 90-day injection study on Balb/c mice, in which the mice were injected with different frequencies of rapamycin via an intraperitoneal (IP) injection. After collecting fecal samples at three different time stages – at the start of the 90-day injection period, immediately after the 90-day period, and 3 months after the 90-day period, we ran a qPCR (quantitative polymerase chain reaction) for each of the fecal samples collected, which quantifies the relative amount of microorganisms in the sample. We also monitored the lifespan of those rapamycin-treated Balb/c mice. This allowed us to see if the lifespan extension and gut microbiome change observed in the original model is reproducible in a different mouse strain. We hope that our findings help to support a future translational research strategy that applies the effect of rapamycin on specific types of bacteria to produce an intervention on gut bacteria to slow aging in pet animals and humans.

Alzheimer’s disease (AD) and cancer are two of the most devastating public health challenges in modern society costing 361 billion dollars and 685,240 lives each year. As these two classes of pathologies progress they both generate protein stress inside cells. Tumor cells are often characterized by greatly increased protein production while most neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease are classified as a “proteinopathies”; characterized by the accumulation of the misfolded proteins. Some recent reports suggest that the Parkinson’s disease related protein α- synuclein influences cancer growth in mice and cell culture models. The goal of our project is to uncover the cellular pathways that are shared between neurodegeneration and oncogenesis. We will study this using the C. elegans genetic model system. I have generated a transgenic line that ubiquitously expresses a human form of the AD-related protein, Amyloid-βeta, and observed that it develops similar tumors to cancer-model worm lines. We are also generating transgenic worm lines that ubiquitously express the other canonical neurodegenerative proteins α-synuclein and Tau to see if they also form tumors. These transgenic lines will allow me to knock down different cellular pathways using RNA interference (RNAi) which silences or inhibits the gene expression using double stranded RNA. First we will look chaperone functioning because this class of proteins is necessary for the cells to deal with protein stress. Research in our lab has already shown that RNAi of some chaperones decreases the occurrence of tumors in a worm cancer line. Next, we will generate worm lines with fluorescent reporters of the tumor altering genes and reporters of cancer-related cell-signaling pathways. Thus, we will begin to uncover the ways in which AD-related protein stress impacts cell processes that promote cancer. This could lead to new gene targets for future therapies.

Macrophages are specialized cells of the immune system that direct the pro-inflammatory response and its resolution. Matrix Metalloproteinase 28 (MMP28) is an extracellular matrix proteinase expressed in alveolar macrophages during lung inflammation and is involved in macrophage recruitment and polarization. Our aim was to explore the regulation of Mmp28 expression in macrophages. We hypothesized that LPS (lipopolysaccharide, a component of bacterial cell membranes which simulates bacterial infection) stimulation would increase Mmp28 expression via a TLR4-dependent (Toll-Like Receptor 4) pathway involving Type I Interferon (IFN) signaling. We also hypothesized that Polyinosinic-polycytidylic acid (Poly(I:C), a synthetic form of double-stranded RNA which simulates viral infection) would increase Mmp28 by a TLR3-dependent pathway. To test these hypotheses, we generated bone-marrow derived macrophages (BMDM) from wild-type mice. We exposed these cells to purified recombinant IFNα/β (two forms of IFN involved in antiviral responses), LPS, Poly(I:C), or control media; 4-6 h after treatment, we isolated RNA from the BMDM, synthesized cDNA, and used qPCR to measure mRNA levels of several inflammatory genes. We also included BMDMs from knockout mice for several proteins thought to be part of the Mmp28 regulatory pathway. We confirmed that LPS and Poly(I:C) increased Mmp28 expression in BMDM. By comparing knockout mice for two adaptor molecules shared by TLR4, we found that LPS-stimulated Mmp28 expression was retained in MyD88-/- but decreased in TRIF-/- macrophages. Since both poly(I:C) and the TRIF-dependent response to LPS induce a type I IFN response, we tested if Mmp28 expression was dependent on type I IFN by using IFNAR-/- (IFN Receptor) macrophages. We found that both TLR4 and TLR3 ligands require IFNAR for Mmp28 expression. These data link type I IFN responses to regulation of MMP28 and development of experimental COPD and emphysema models of lung disease, and further our understanding of how MMP28 may regulate macrophage responses.

Aging is characterized by time-dependent deterioration of cellular functions and increased risk for disease and death. The onset of age-related diseases including cancer, Alzheimer's, and other neurological disorders decrease the healthspan of humans. Healthspan is defined as the period of life without infirmity. My research focuses on understanding the basic biology of aging and determinants of healthspan within the emerging interdisciplinary field of geroscience. I use the model system Saccharomyces cerevisiae, a unicellular fungus, to study aging at the cellular level. To analyze cellular aging, I focus on replicative lifespan (RLS), which quantifies the number of daughter cells produced by individual mother cells before they reach replicative senescence and cease to divide. Our replicative lifespan team is composed of a group of undergraduate, graduate, and post-doctoral scientists at the University of Washington who use light microscopy and manual micromanipulation to quantify the RLS of hundreds of yeast mother cells each week. There are four important steps to analyze RLS in yeast: assay setup, incubation to outgrow cells, dissection of daughter cells from mother cells, and quantification of the number of daughter cells produced prior to replicative senescence. Using the replicative lifespan assay, I investigate genetic, environmental, and pharmacological treatments to determine their impact on cellular aging. Research produced by our RLS dissection team has led to a better understanding of evolutionarily-conserved factors that modulate aging, as well as potential treatments for certain human mitochondrial diseases.

The large-scale configuration of the cosmos has long been a mystery to astronomers. My research project aims to shed light on this mystery. The overarching goal for my research project is to further understand the distribution of matter on 10⁵ –10⁷ light-year distances by comparing numerical simulations to actual observations of the universe. This comparison tests the current theoretical parameters of the physics governing the largest scales of the cosmos. I used simulated data from models made by Professor Matthew McQuinn and observations of quasar spectra from the Very Large Telescope (VLT). From the VLT data, I analyzed quasar spectra for absorption lines at a hydrogen electronic transition called the Lyman-alpha line. The IGM absorbs amply at this transition, creating absorption lines throughout the quasars’ spectra. I processed this data by programming Python scripts to compare flux probability distribution functions (PDFs) of the Lyman-alpha absorption in the observed quasar spectra to the simulated quasar spectra. Through this comparison, PDFs of observations fell within one standard deviation of the simulated PDFs. Although at higher redshifts (further back in cosmological time), there was considerably more variance. In order to further constrain the observational flux PDFs, I aim to eliminate systematic and statistical uncertainties that may make the observations spuriously differ from models. The salient errors include fitting precise continua (spectra without absorption) and removing metal (predominantly carbon-4) contamination from the Lyman-alpha forest. I used Principal Component Analysis to predict the continuum spectrum but found that a fit by eye was more effective. Through analysis of absorption probabilities outside the Lyman-alpha forest, I statistically removed carbon-4 from the data inside the Lyman-alpha forest. Ensuring the most precise flux PDF measurements are used is critical to the comparison of observations to simulations and to the constraints of parameters governing the universe.

Aging remains as the biggest risk factor for developing the most prevalent diseases found in developing countries: cancer, cardiovascular disease, and neurodegeneration. Reduced insulin/IGF signaling (IIS) extends lifespan in diverse organisms, but the underlying molecular mechanisms that regulate this remain unclear. Recently, non-coding RNAs, i.e. those RNAs not translated into proteins, have become a hot topic, being identified as regulators of gene expression during aging. Circular RNAs (circRNAs) are one such form of non-coding RNAs. Although they are identified in a diverse number of species, the function of most circRNAs is unknown. CircRNAs are distinct from their linear counterparts because they are devoid of the terminal structures (e.g. 5’cap or a poly A tail) that frequently determine the fate of RNA transcripts thereby escaping normal RNA turnover. It has been established that circRNAs are abundant and differentially expressed; they can serve as either transcriptional regulators, and/or microRNA sponges. Furthermore, some circRNAs are produced at the expense of linear RNA, thereby affecting gene expression and thus tissue or organismal function. CircRNAs are also evolutionary conserved, which begs the question why they are so important. Recently, the Partridge lab conducted gene expression profiling of long-lived IIS mutants and identified differentially expressed circRNAs, which may influence IIS-mediated longevity and thus aging. The Partridge lab have generated in vivo Drosophila models, mutating individual circRNAs through genomic engineering (CRISPR/Cas9) and the Gal4/UAS system, to study their function. Using these novel circRNA mutants, I will contribute to discovering the in vivo function of circRNAs in aging by performing quantitative experiments. I will analyze circRNAs mutant lifespan and phenotypes associated to longevity e.g. development, fecundity, stress resistance, neurodegeneration. Genetic epistasis experiments will determine the role of the given circRNAs in insulin signaling. Finally, I will use molecular biology techniques to unravel the mechanism underlying any observed phenotype.

MSN2 is an environmental stress response transcription factor that is activated in yeast by multiple different stresses—e.g. glucose deprivation, heat, oxidative stress, misfolded proteins among many others—and in turn activates a collection of downstream transcriptional programs. Interestingly, while MSN2 becomes increasingly activated with age, deletion of MSN2 increases the replicative lifespan of the budding yeast. We show that in a glucose-rich environment, MSN2 misreads the environment and drives a pathological glucose starvation response that limits lifespan. Our experiments are facilitated by a novel budget microfluidic/microscopy method rather than the golden standard of microdissection. This innovative system analyzes data through time-lapse images to develop replicative lifespan curves rather than picking off daughter cells with a needle. Microfluidics drastically increases the throughput of experiments and allows for whole-lifespan monitoring of aging yeast cells. The system allows us to efficiently further the research of MSN2 and other transcription factors so that we can potentially translate this to other complex organisms that have similar types of transcription.

MicroRNAs (miRNA) are non-coding RNA (ncRNA) which post-transcriptionally regulate gene expression. In cancer, many miRNAs are overexpressed leading to the down-regulation of tumor suppressing genes. While the general processing steps of miRNA are known, the specific reason certain miRNAs are overexpressed in cancer remains unclear. Our particular miRNA of interest is miR-21, which inhibits genes encoding tumor suppressing proteins, and upregulation is associated with cancer. Specifically, we are interested in understanding how the pre-miRNA-21 processing step is regulated to generate functionally mature miRNA, and what proteins are involved in this process. Many RNA binding proteins are involved in miRNA biogenesis and influence transcription levels. This project explores the possible role of the RNA binding protein NOVA. NOVA is a neuron-specific RNA-binding protein exclusively expressed in the brain and neuronal tissues except for patients with Paraneoplastic opsoclonus-myoclonus ataxia, where it is found site of malignancy. NOVA is composed of 3 K-homology RNA binding domains selective for the sequence 5’ -YCAY-3’ (Y = pyrimidine). This KH domain recognition sequence is present in the hairpin loop in pre-miR-21. Additionally, in-vitro DICER processing of pre-miR21 is very slow relative to other miRNAs, however, if NOVA interacts with the pre-miR-21, this could stimulate its processing. We are trying to determine a possible mechanism of NOVA stimulated miR-21 expression through binding a conserved YCAY sequence. MiR-21 and NOVA have been isolated and purified, and their binding affinities are being evaluated using electro mobility shift assays. If binding is indicated, gene knockdowns will be preformed in cells to assess how NOVA affects miR-21 levels. It is difficult to design microRNA specific inhibitors, however, finding inhibitors for proteins is possible. If NOVA is found to lead to miR-21 up-regulation, a possible cancer treatment could be designed by targeting NOVA to lower levels of miR-21.