Recent advances in genome sequencing have allowed for the study of metagenomes, genetic material recovered directly from environmental samples. This has produced an enormous number of enzyme sequences without efficient methods for characterization. Since it is unknown whether a characterized enzyme is the best catalyst for a specific reaction, uncharacterized, natural enzymes in the metagenome may be better candidates for catalyzing the specific reaction. Thus, I am developing methods to search for and computationally characterize metagenomic enzymes with the same function as enzymes of therapeutic and commercial interest. By utilizing the Rosetta Software Suite to produce highly accurate models of enzyme structures, this method will evaluate an enzyme sequence’s potential for catalysis directly from a gene sequence. In order to validate my computational techniques, I will experimentally determine catalytic rates of enzyme candidates. As the method for an efficient metagenome search is still unknown, I am working to determine proper parameters for a homology search by using visual and statistical analysis of enzyme structure models in order to produce a diverse set of genes for experimental characterization.

Gluten intolerance disorders such as Celiac sprue elicit a painful immunological response in the intestines of patients who consume gluten. Gluten is a polypeptide found in wheat, barley, and rye, and contains repeating Pro-Gln-Leu-Pro (PQLP) motifs. Currently the only treatment for gluten intolerance is elimination of gluten from the diet. Our goal is to design an enzyme therapeutic that can be taken as a drug therapeutic in pill form. The engineered enzyme will break down the immunogenic PQLP motifs before gluten reaches the small intestine and can elicit an inflammatory response. Kumamolisin-As, a protein isolated from Alicyclobacillus sendaiensis, demonstrates promising proteolysis on PQ residues, and works in the low pH of the stomach. Using the protein-engineering program “FoldIt,” we designed novel mutations to Kumamolisin to improve its activity on PQLP. Our best version of the enzyme, dubbed KumaMax, degrades gluten over 700 times faster at pH 4 than SC-PEP, an enzyme currently in clinical trials. After further protein characterization, we hope to start clinical trials on KumaMax with the future goal of creating an effective treatment for gluten intolerance.

X-linked severe combined immunodeficiency (X-SCID) is caused by a mutation in the interleukin 2 receptor gamma (IL2Rγ) gene, which is required for lymphocyte development. Affected patients lack functional lymphocytes and die unless cured through bone marrow transplant or gene therapy. However, gene therapy treatment was stopped due to risk of leukemia resulting from insertional mutagenesis, and this prompted the pursuit of alternate treatments, such as gene repair. Gene repair requires the use of two essential components, a homing endonuclease and a repair template, that harness homology directed repair mechanism, which repairs double stranded DNA breaks in cells. Homing endonucleases are enzymes used to target and cut DNA near a mutation in the genome, while a repair template is the DNA sequence used to correct the mutation. My project involved performing experiments to optimize specific components of gene repair using the Sce-SCID mouse model that contained a recognition sequence for the homing endonuclease, I-SceI proximal to the IL2Rγ mutation. I used adeno-associated viral vectors (AAV) to deliver I-SceI and a repair template to hematopoietic stem cells (HSC) isolated from Sce-SCID mice. I tested the hypothesis that delivery of AAV encoding I-SceI homing endonuclease to Sce-SCID HSC would result in recognition and cutting of the Sce locus. Cutting of the Sce locus is essential to gene repair because it initiates the repair of DNA double stranded breaks through homology directed repair with the repair template. Previous experiments in the Rawlings lab demonstrated cutting of the Sce locus with stable I-SceI expression, but my experiments involved transient AAV delivery. Transient delivery is critical for the safety profile of gene repair. Once I-SceI cutting was established with transient delivery, I performed experiments that invovled both I-SceI and repair template in an attempt to correct the SCID phenotype of the Sce-SCID mouse.

Most adults have some degree of atherosclerosis. In a subset of these individuals, atherosclerosis progresses more rapidly. Increased activity of proteases (enzymes that digest proteins) in atherosclerotic plaques is a plausible cause both of atherosclerosis progression and of atherosclerotic plaque rupture. Plaque rupture is often a terminal event that results in blockage of arteries, heart attacks and strokes. Urokinase plasminogen activator (uPA, a protease that is present in plaques) functions by converting plasminogen (a zymogen, or enzyme precursor) into its active form, plasmin. The uPA/plasminogen pathway facilitates macrophage migration, enhances the release of inflammatory mediators, and activates other proteolytic enzymes, all of which may contribute to atherosclerosis progression and plaque rupture. We hypothesize that inhibition of uPA will decrease atherosclerosis progression and prevent plaque rupture. To begin to test our hypothesis, we use a mouse model of uPA-accelerated atherosclerosis (mice that are genetically predisposed to develop atherosclerosis and express high levels of uPA). We inject an antibody (mU1) that binds to and inhibits uPA, or a control antibody, and we measure atherosclerosis in aortic roots and pinned aortas. We anticipate finding less atherosclerosis in mice treated with mU1. We also expect to find fewer macrophages in atherosclerotic plaques of mice treated with mU1. If we find that inhibition of uPA decreases atherosclerosis progression and that it is also well tolerated by mice, we will later set up an experiment in older mice to determine whether anti-uPA treatment with mU1 will prevent plaque rupture. If uPA activity contributes to atherosclerosis progression, and if inhibiting uPA decreases atherosclerosis progression and prevents plaque rupture, drugs could be developed that inhibit uPA. These drugs could be used to treat humans who are at high risk for atherosclerosis progression and plaque rupture.

Diffuse Large B-Cell Lymphoma (DLBCL) is one of the most common types of non-Hodgkin's Lymphoma with a 50.4% five-year survival rate.  Previous work has shown that some patients with aggressive DLBCL have increased expression of the transcription factor NF-kß, which can alter normal growth and function of B-lymphocytes.  Interestingly, a protein upstream of NF-kß in the B-Cell Receptor Pathway called PKCß is found to be highly activated in many DLBCL cases, suggesting that PKCß overexpression may be altering the lifecycle of those with DLBCL.  Therefore, PKCß may be a significant new target for therapy.  We designed a transgenic mouse expressing a dominant active form of PKCß expressed only in B-cells (Rosa26 DAPKCß XCD19-Cre).  With this model, we measured differences in B-cell  germinal center development and in antibody responses between WT and DA PKCß-Cre mice immunized with T-dependent antigen NP-CGG.  We hypothesized that DA PKCß-Cre mice may have an increased response compared to WT because of its dominant active form of PKCß, which leads to increased NF-kß and hyper-activation of the responding B-cells.  Thus, we used ELISA and ELISPOT assays to quantify the expressed antibodies and immunohistochemistry to analyze the architecture of the spleen.  Immunohistochemistry has shown that the immunized DA PKCß-Cre mice have slightly larger germinal centers than WT mice, although not statistically significant.  Immunized DA PKCß-Cre mice show no difference in serum antibody levels by ELISA, but have slightly increased numbers of anitbody producing cells by ELISPOT.  Overall, the data suggests that over expression of PKCß in B-cells results in a hyper-proliferative phenotype, but does not strongly alter the antibody response to the T-dependent antigen NP-CGG.  Since we saw a difference in mice immunized with Pertussis, which stimulates the Toll-like Receptor (TLR) Pathway, we will look at different responses of TLR ligands in vivo and in vitro.

English holly (Ilex aquifolium) is an increasingly prominent invader of western Washington forests, yet little data exists on its effects on western Washington native flora. A population study of holly in a 15.9 acre study area in St. Edward State Park suggests that holly reduces the abundance and diversity of native vegetation. We hypothesized that these negative effects are caused by shading and/or litter fall from holly canopy. We studied native vegetation cover in four treatments (holly shade, holly litter, holly shade + litter, neither) to determine the importance of these two factors. We also contrasted photosynthetic photon flux densities (PPFD) under holly canopy with PPFD under native tree canopy and in full sun, and compared PPFD under holly canopy with existing data on compensation points for native plant species occurring in the park. If holly reduces native vegetation through shading, we expect to see reduced native vegetation abundance in our shade treatments and to find that PPFD levels underneath holly canopy are lower than required for some or all native plant species. If holly leaf litter is suppressing growth, we expect to see reduced native vegetation abundance in our litter treatments and to find that holly leaf litter is deep enough to negatively affect seed germination. Preliminary results suggest a significantly lower total vegetation cover underneath holly canopies compared with total cover outside of holly canopies, and we have recorded leaf litter depth as much as 17 cm underneath holly canopies. This study will provide quantitative data which can aid forest managers in assessing the threat holly poses to western Washington forest biodiversity. Results and implications will be discussed.

What makes one country happier than another? For centuries mankind has searched for an answer to what brings bliss, producing a great variety of answers. Over the past two years I have been on my own search; first collecting data and readings from varying scholars and fields of education, I then presented my findings at last year’s URS. A major division between western medicine and eastern wisdom split my presentation, and left me wanting a more visceral experience. Fortunately, my advisers Dr. David Barash and Dr. Judith Lipton took on a book to tackle my primary question, and allowed me to watch their house in the Central American sunspot, Costa Rica. Additionally, Costa Rica had been named the world’s happiest country by several organizations. Both my advisers and I had some qualms on how these organizations (World Happiness Database, Yale Environmental Performance, Happy Planet Index) addressed such subjective variables, and thus my project split. For the summer, I was to explore the country, conduct informal interviews with locals, tourists, and collect field notes to form answers towards some broad questions. Are Costa Rican’s the world’s happiest? What mechanics create such joy? Finally, what can we learn from them? Presently, I am conducting further investigations on how these organizations rank happiness. Possible methodological faults are addressed by comparing mental health and social inequalities to their rankings. In my attempt to put boundaries on an abstract emotion, quantitative and qualitative in nature, I have distilled cloudy answers to what makes us happy. I view these as primal ingredients towards a universal ‘elixir’. This ‘happiness variable’ takes flexibility, openness, purpose, & awareness to create. I believe we all have the capacity to be happy and hope to present some tools discovered while researching to help construct understanding and accessibility to this enriching experience.

Human embryonic stem cells (hESCs) are pluripotent, or able to differentiate into all cell types of a developing embryo. They can be used to engineer human tissue, but are prone to immune rejection when used in medical treatment. Similar to hESCs but derived from the reprogramming of adult somatic cells, human induced pluripotent stem cells (hiPSCs) were developed that have the potential for use in regenerative therapy with less risk of rejection. However, hiPSC research faces its own challenges, such as inefficient reprogramming rates and variation in observed pluripotency. Previous research has shown that methylation patterns of hESCs can be distinguished from hiPSCs at both the DNA and histone level, suggesting less pluripotency in hiPSCs than in hESCs. While exhibiting greater overall methylation on DNA but less on histones, many of the hypermethylated DNA regions of hESCs are adjacent to these differentially methylated histones. The location of the hypermethylated regions suggests that erasing specific histone methylation marks may improve hiPSC reprogramming rates by creating a more open chromatin landscape, and may create a cascade effect that allows for additional methylation of the genome. This experiment investigates the correlation between histone methylation and hiPSC reprogramming efficiency and accuracy. We reprogrammed human fibroblast cells with lentiviruses expressing various demethylases and transcription factors in order to compare resultant hiPSC methylomes to traditional hiPSC and hESC lines. Opening of the chromatin is expected to improve reprogramming rates and allow for proper methylation of the genome, creating an hiPSC closer in phenotype to hESCs and more useful for clinical therapies.

High gradient mountain streams are characterized by drops known as steps, followed by depressions in the bed known as pools. This step-pool morphology governs habitat availability and sediment transport of many high gradient streams. A notable characteristic of most pools is the presence of clast-size based sorting on their bottoms. We seek to understand how this sorting forms, as it may provide more insight into the currently debated topic of how such pools are formed. We propose that there is a decrease in shear stress on the bed from the base of a step to the lip of a pool. We suspect the effects of this difference in shear stress are proportional to changes in stream gradient, as step height has been shown to increase with gradient. If our hypothesis is true, we suspect to see a range of sorting patterns from low gradient to high gradient mountain streams. We will survey a number of pools in streams of varying gradient to determine whether such a range can be documented. Preliminary field observations have indicated that such a range is present, although we seek to quantify that observation. For each pool, we will use a pebble count procedure accompanied by geometric measurements of the pool to quantify sorting patterns and evaluate the relationship between sorting and stream gradient. By understanding how sorting is affected by gradient, we can start to determine what aspects of stream hydraulics control pool formation.

This research explores the function and dynamics of gold nanoparticles functionalized with azobenzene- modified DNA. When exposed to UV light, the azobenzene groups photoisomerize, melting the DNA and dissociating aggregated nanoparticles. This process is reversible with introduction to blue light, initiating the isomerization that allows the DNA to rehybridize and the particles to form aggregates once again. Our research focuses on the dynamics of this transition, determining the extent of trans-cis isomerization of the azobenzene DNA coupled to gold nanoparticles, a process which is monitored through pronounced changes in light absorption between the trans and cis conformations. This information creates a baseline for future photoswitching calculations, accounting for differences in behavior between free azobenzene DNA and such DNA in the presence of gold nanoparticles. We also use the kinetics of the light-induced dissociation and reassociation to provide information about the composition of the DNA, with respect to location of the azobenzene group embedded in the DNA strand. This information can assist with detection and location of base-pair mismatches within the coupled DNA.

Magellanic penguin morphology is likely to influence the survival and reproductive success of an individual. Although Magellanic penguin colonies are not genetically differentiated, there may be significant differences in the structural size of penguins among colonies, as suggested by Bergmann’s and Allen’s rules that appendage size changes with latitude. We used data collected on 792 Magellanic penguins at 12 colonies in Argentina, Chile and Falkland/Malvinas Islands to compare weight, bill length, bill depth, flipper and foot. We expected penguins to be larger at southern colonies and have longer flippers and bills at northern colonies to shed heat. We expected variation among colonies as morphology should reflect adaptation to the local environment. We matched the years, months and approximate gender ratio of the measurements collected from the colonies with a randomly selected sample of penguins from Punta Tombo, Argentina, a northern colony. We performed an ANOVA on these data using Stata. When ANOVAs were significant, we performed pairwise comparisons (Tukey HSD) among the colonies. All traits varied among colonies except flipper length for females. Average female flipper length was 14.6 cm. suggesting that flipper surface area may be more important for foraging success and survival than for shedding heat on land or conserving heat in the water. Bill length decreased with latitude for males and females (males: R2 = 0 .73, females: R2 = 0.58). Exploring the interactions between latitude and morphology suggests selection is favoring penguins with longer bills at lower latitudes. Does bill size reflect diet changes with latitude or changes in heat and cold stress? Penguin morphology changed with location but flipper size did not which is not consistent with Allen’s rule. Flipper size may be so important in foraging success that selection favors a “standard" flipper size regardless of latitude and temperature.

Zebrafish have a defined striped pattern that results from pigment cell differentiation and migration during development, and this developmental process provides a good model for studying the process of boundary formation. Elucidating the mechanisms by which boundaries are formed is critical for understanding vertebrate development, and will give insight into what might cause them to malfunction during metastasis where cancer cells invade other tissue types, thus breaking the boundaries that have formed. While zebrafish have these defined patterns, a closely related species, the pearl danio, lacks stripes and instead has different pigment cell types intermingled. We hypothesize that the pearl danios have lost the mechanism for pigment cell boundary formation found in zebrafish and my goal is to identify genetic differences between the two species that could have caused this change pattern development. Preliminary RNA sequencing has identified candidate genes that appear to be differentially expressed. I am using in situ hybridization to compare the expression patterns of candidate genes between the two species. This is a technique where whole larval fish are incubated with RNA probes which are complimentary to the mRNAs of the genes I am screening for. Wherever the candidate genes are transcribed into mRNA, and thus expressed, the probe will hybridize. I am able to visualize this through labeling of the probe and staining with antibodies. I expect to find that some of the candidate genes will have different patterns of expression between the two species. This result will indicate that these genes are involved in the pigment pattern formation process, although it will not explain their specific function. However, once I know which genes are involved I will be able to manipulate their expression through knockdown or over-expression, which will provide further insight on the mechanisms of boundary formation in pigment patterns.

Organic photovoltaics (OPVs) are a promising solar energy technology due to the possibility of cost-competitive large-scale manufacture. Polymer based devices typically have a bulk heterojunction (BHJ) architecture. However, BHJ materials degrade rapidly under typical operating conditions, contributing to limited device lifetimes. The degradation due to photooxidation of a model OPV system of poly[9,9’-dioctylfluorene-alt-(bis(N,N’-(4-butylphenyl))-bis(N, N’-phenyl-1,4-phenylenediamine)] (PFB) and poly[9,9’-dioctylfluorene-alt-1,4-benzothaidiazole] (F8BT) was studied by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Photooxidation products as a function of increasing photon dose at 405 nm were identified in PFB:F8BT, PFB-only, and F8BT-only films. Degradation kinetics on these films were determined by fitting Gaussian peaks to the IR spectra and measuring changes in peak area as a function of photon dose. Preliminary results suggest that multiple carbonyl species are formed in both polymers under ambient conditions. Analysis of the data collected in this experiment will be used to potentially evaluate the chemical kinetics of the degradation process and the formation of undesired chemical species in order to find a way to extend the lifetime of the device. Gaining insight into what limits the lifetime of current solar cell materials will help to improve the durability of plastic solar cells, and thereby solar energy as a widespread technology.

In the past decade, there has been increasing interest in the potential of new thin-film solar technologies. Research in this field has focused largely on organic photovoltaics (OPVs) utilizing a bulk heterojunction (BHJ) design, consisting of blended films of conjugated polymer electron donors and fullerene electron acceptors.Use of semiconductor nanocrystals or “quantum dots” as a replacement acceptor material for the fullerene has been proposed as an alternative. In this research, charge transfer between lead sulfide nanocrystals and a conducting polymer, "poly(2,3-didecyl-quinoxaline-5,8-diyl-alt-N-octyldithieno[3,2-b:2′,3′-d]pyrrole) " or PDTPQx is investigated by time resolved spectroscopy. Previous work has found that incident light exciting the polymer results in transfer of an electron from the polymer to lead sulfide nanocrystals present in the blend. This electron transfer leaves a net positive charge on the polymer, which is accompanied by a characteristic spectroscopic signature. It is found in this research that selective excitation of the lead sulfide nanocrystals using an infrared laser also results in observable positive charge carriers on PDTPQx. This indicates that light absorption at the lead sulfide nanocrystals results in transfer of positive charges from the nanocrystals to the polymer. Confirmation of this additional charge transfer process has important implications for the potential this system in photovoltaics.

Solar cells, through their ability to convert energy from light directly into electricity, pose a promising alternative for supplying our world’s growing demand for clean affordable energy. Many factors governing the efficiency of these photovoltaic devices are still being explored and improved upon through methods aimed at boosting photo-voltages, improving energy-conversion efficiency, and finding materials accessible for large-scale production and implementation. Here, I investigate the potential of a new type of solar cell that utilizes electron-impurity Auger de-excitation. I will discuss the effect of Auger de-excitation observed in Mn doped CdS quantum dots and explore the potential for boosting the photo-voltage via the harvesting of hot electrons. I will explain the processes involved in both synthesizing Mn:CdS quantum dots and fabricating quantum dot-based solid-state devices. I will show preliminary results from photoluminescence quenching measurements used to quantify Auger de-excitation in fabricated solid-state devices as well as device properties and characterization measurements. In the future, full-cell PV devices will be constructed and tested against the efficiencies of today’s leading solar cells.

Surface plasmons of metal nanoparticles have been the source of much scientific study over the last decade due to their immediate and significant applications in biosensing, surface-enhanced Raman Specroscopy (SERS), and light absorption engineering in semiconductor nanodevices. Surface plasmons can be generated by high-energy electrons coupling with natural plasmonic resonances of a nanometal’s electron cloud. An abundant source of such relativistic electrons can be found in common electron energy-loss spectroscopy (EELS) experiment, in which surface plasmons generated by the experiment can further couple with plasmons of other nearby nanoparticles, giving rise to “hot spots” with very strong electric fields. Often these hot spots can magnify an incident electric field over 100-fold, giving rise to many of the applications mentioned previously. This work aims to produce a computational model that computes plasmon excitation due to a fast electron by using an existing Discrete Dipole Approximation (DDA) framework, and to compare theoretical results from metal nano-rods and metal nano-cubes with experimental data. Unlike experimental data, computational models are not limited by detector resolution; meaning that such a model will give significant insight into details of the EEL experiment. In this work we explore results that are higher-resolution than what is currently obtainable by modern techniques. The existence of such a model will allow researchers to obtain preliminary data before running time-consuming and expensive experiments, will allow enormous insight into the deeper physics of surface plasmons, and will give experimental results a theoretical foothold while also improving on their accuracy.

Under constant volume muscle contraction myofilament overlap increases and muscle expands radially. While myofilament overlap is considered a major factor in force generation, considerably less is known about the role of myofilament expansion accompanying shortening during contraction. Recent theoretical evidence suggests that the spacing between filaments will indeed be an important determinant of force generation at any level of filament overlap, however, there are scant data that show the extent to which filament overlap changes in intact muscles. To address this issue we performed work loops on intact Manduca sexta flight muscles while simultaneously measuring myofilament spacing, force, and length using time resolved small angle x-ray diffraction imaging. Muscles were stimulated at 25 Hz and held at a temperature of 35C while simultaneously measuring length and force. Five x-ray images were taken during each wingbeat cycle for 100 contractions. These images were averaged over all cycles, and lattice spacing was measured for each phase. During work loops with a 6% strain cycle, lattice expansion changes by 2.77 ± 0.839%. Thus we do not reject the constant volume assumption, which would predict a 2.4% change in radial dimension for a 6% longitudinal strain. Taken together these data suggest that lattice expansion during muscle shortening is a critical component of muscle force generation.

This project explores the innate immunity of the earthworm species Eisenia fetida during development in context of its symbiotic relationship with Verminephrobacter eiseniae, a known bacterial symbiont. Innate immunity, unlike adaptive, has largely unspecific properties against foreign particles yet many organisms that possess only innate immunity manage to allow select beneficial symbionts to thrive within their body while at the same time defending against pathogenic infection. In this study I begin to explore involvement of innate immunity in the relationship between E. fetida and its symbiont V. eiseniae. This project examines expression of a common innate response, oxidative stress, in early stages of V. eiseniae colonization of its host E. fetida. We hypothesize that innate immunity activity is spatially separated from colonization of bacterial symbionts. To test this hypothesis, I will stain for nitric oxide synthase (NOS), an enzyme commonly involved in oxidative stress, during the development of E. fetida in the presence of V. eiseniae. I will then proceed to compare the location of NOS to the location of V. eiseniae. If the hypothesis is correct, then oxidative stress response in the worm eliminates foreign organisms everywhere except where the symbionts reside. On the other hand, if oxidative stress is at the same location as the known symbionts at the same time during development, then the oxidative stress may eliminate other foreign organisms except the symbionts, which have developed a mechanism for overcoming the stress. The results of this research can reveal how innate immunity overcomes the dilemma of accommodating symbionts. In addition, if E. fetida is particularly easy to work with for this type of experimentation, then E. fetida could prove to be an ideal model organism for studying innate immunity. Furthermore, insights into innate immunity may hold the key to new innovative ways of combating disease.