Cancers are fueled by mutations which impart new capabilities to incipient cancer cells. Spontaneous mutations are, however, more likely to be deleterious than beneficial based on random chance alone. Therefore, inducing more mutations in an heavily mutated population could lead to an “error catastrophe” once the mutation level rises past a certain threshold. Accumulating too many of these debilitating mutations consequently leads to a reduction in the overall population fitness. Our hypothesis is that mutagenic versions of nucleoside analogs (modified versions of the building blocks of DNA) can induce more mutations to reach this ‘error’ threshold and beyond, and that this can selectively kill cancer cells. To evaluate the effectiveness of this strategy, termed therapeutic mutagenesis, we conducted cell culture experiments on various human colon cancer cell lines: HCT116 (mismatch repair deficient colorectal cancer) cells, SWS620 (non-MMR-deficient colorectal cancer), and a control normal human fibroblast line. We screened a library of candidate nucleoside analogues (e.g. O4-Ethyl-Thymidine) for mutagenicity and viability at physiologically relevant concentrations. I then quantified the short-term viability of the treated cell lines with a clonogenic survival assay. I used the highly accurate Duplex genetic sequencing methodology to monitor the changes in a cell line’s overall mutation load after treatment. After validating the concept of therapeutic mutagenesis in cancer, our next steps will be to determine whether possible animal and clinical trials would be possible in the future. Our approach may open up new therapeutic possibilities for patients in the most advanced stages of cancer, especially in the context of complementing increasingly common immunotherapies.

Bietti crystalline dystrophy (BCD) is an autosomal recessive eye disease caused by mutations in the human CYP4V2 gene. To understand the biochemistry and physiology associated with BCD, our lab has developed a “knockout” of the orthologous mouse gene, Cyp4v3. The gene targeting vector that was used to disrupt the Cyp4v3 gene incorporated a LacZ gene reporter and a neomycin antibiotic resistance gene to replace the majority (exons 2-10) of the Cyp4v3 gene. To differentiate endogenous mouse sequence from the targeting vector we designed primers to amplify by polymerase chain reaction (PCR) portions of the Cyp4v3 and LacZ genes, respectively. The experiments conducted for the LacZ primers failed to yield any PCR product when tested on samples of genomic DNA from Cyp4v3 heterozygous mice. However, we did get PCR products of the expected size from the same DNA samples for both Cyp4v3 a loading control (fatty acid binding protein-FABP). Because of the technical difficulties encountered with the LacZ primers, we designed new primers targeting the neomycin resistance gene sequence. When these new primers were tested, we were able to amplify the expected targets for all 3 genes when testing genomic DNA from a Cyp4v3 heterozygous mouse. We have developed a robust and reproducible method to genotype mice that are either Cyp4v3 +/+ (wild type), +/- (heterozygotes) or -/- (homozygous knockouts). Ongoing studies in the lab with these knockout mice are focused on characterizing the age-related progression of BCD ocular pathologies, including crystalline deposit chemical composition and defects in lipid metabolism.

People in agriculture commonly use pesticides to prevent insect damage on crops. One common pesticide is called acetamiprid (ACP). While this pesticide has low toxicity to mammals, there have been cases of acute toxicity from overexposure to this pesticide. In this study, we focused on pesticide handlers, people who apply pesticide to crops in the fields, from the Yakima valley and analyzed their urine samples to measure their exposure to the pesticide ACP. The primary goal of the study was to quantify urine biomarkers to ACP exposure, including the parent compound ACP, and two metabolites: acetamiprid-N-desmethyl (ACPDM) and 6-chloronicotinic acid (6-CNA). We collected urine samples from 10 pesticide handlers in the 24 hours after applying ACP in an orchard. Each person was given a liter sized sampling vial to collect each void. We collected 34 human samples in total. I identified and quantified the biomarkers in the human urine samples collected. I did this by processing the samples using Solid Phase Extraction (SPE) and then quantifying them by using high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). We expected to see small amounts of each biomarker in each sample, but found relatively high concentrations of the metabolite ACPDM. The results of this study will help to improve our ability to measure urinary markers of exposure, better estimate exposure and risk to pesticide handlers, understand the kinetics of ACP in the human body, and think of means to minimize exposure to prevent acute toxicity.

Our lab is using the nitrogen-vacancy (NV) center, which is a specific impurity in diamond, to measure magnetic fields on a microscopic scale. The ability to measure these fields unobtrusively and in real time will be a valuable tool for studying biological systems tagged with magnetic nanoparticles. The NV centers fluoresce when exposed to green light, but in the presence of a microwave field of a specific frequency this fluorescence decreases. The precise frequency at which this occurs is dependent on the local magnetic field strength and direction. Thus, by sweeping this microwave field over a range of frequencies, we can measure the magnetic field at the NV centers. Here we use computer software, HFSS, to design a resonator to produce the necessary microwave field. Critically, the resonator needs to produce a uniform microwave field over the imaging area on a diamond chip for a wide frequency bandwidth. It also needs to integrate with other aspects of the overall design; in particular it should be transparent at the center to allow us to image through it. Designs are fabricated at the University of Washington MakerSpace, and a comparison between the simulations and these fabricated devices will be presented.

The security level of the interactions between human users and cyber systems is becoming more and more important in many areas. The traditional way of identifying a user is to utilize alphanumerical passwords, which are easy to realize but hard to memorize and vulnerable to various kinds of attack. To enhance security performance for user authentication, we take the advantage of the haptic interaction of new touchscreen devices that have recently been introduced. Previous studies show that how a person interacts with a haptic interface is unique and can be used for identification and authentication purposes. We applied this technique using the Microsoft Surface Pro 3, which has a pen, the N-trig digitizer. Having its API (ways for developers to talk to another site or services) made it easier to transfer data to the haptic password system. By developing an app on a writable windows tablet, we successfully captured ink data and pointer properties such as pressure, twist and orientation. We built a haptic password system and invited subjects to take specific tests. After data analysis, we found that this system is secure with good performance and forgery attack resistance. This system provides a complex but intuitive way of creating signature passwords. This haptic interaction technique can be applied to those pressure sensitive devices, such as tablets and signature touch screens. This could provide a much safer way of authorizing credit card transactions and lead to reduced transaction fraud.
 

The psychology literature delves into understanding the role of men's self perception of masculinity in help-seeking behavior as well as help-seeking behavior in men with cancer. Help-seeking behavior is behavior that explains the utilization of healthcare of a population or group. What is the role of self perceptions of masculinity in help-seeking behavior of men with prostate cancer? This literature review focuses on understanding this role in men specifically with prostate cancer. The resources used for the review are MedLine, PsycINFO, PsycArticles, PubMed, and references within articles. The majority of the literature used were qualitative studies due to the availability in the literature. The literature suggests that men believe that prostate cancer affects their masculinity in both physical and emotional ways such as their physical aspects like strength, ability to provide for and protect their families, reaction to physical pain through peer pressure and social norms, and being viewed as unmanly. There are men that efficiently seek help from healthcare professionals for this disease to preserve their masculinity. Men also inefficiently seek help due to the potential embarrassment of those qualities being diminished by the disease. The limitations to writing the review included small sample sizes, support group participants, and participants that were already diagnosed. The review is also limited because there was no research on men that focused on how masculinity may have contributed to their help-seeking in the initial stages. Healthcare services, such as interventions or programs, should focus on the following indicators when promoting help-seeking behavior to men with prostate cancer: stoicism, privacy, family providing and protection, and being viewed as unmanly due to the physical effects on a man's body. If the healthcare system can overcome these barriers, it will be able to reach men more efficiently and prevent further advancement of diseases like prostate cancer. 

 Cyclic diadenosine monophosphate (c-di-AMP) is an essential nucleotide for bacterial virulence. C-di-AMP has been extensively studied in Listeria monocytogenes (Lm), but is also produced by other pathogens including E. coli, C. trachomatis, and M. tuberculosis. Although c-di-AMP is a signaling molecule in bacteria, host cells detect it as a pathogen-associated molecular pattern. During infection, the host receptor STING senses c-di-AMP and stimulates expression of interferon-beta, an anti-viral molecule. To date, STING is the only known sensor of c-di-AMP, and its promotion of an anti-viral versus anti-bacterial response has been somewhat perplexing. However, our lab has recently discovered another host c-di-AMP sensor, the murine aldo-keto reductase1C13 (AKR1C13) or “RECON”, that induces an alternative, anti-bacterial cellular response to infection. Based on the importance of bacterial clearance and the ubiquity of AKRs across species, we began to investigate whether other murine and human AKRs respond similarly to c-di-AMP. Sequence alignment of AKR genes and data on tissue-specific expression of these genes returned six candidates: the murine AKR1C12 as a potential paralogue, and human AKRs 1C1, 1C2, 1C3, and 1C4 as potential homologues. Cloning techniques allowed me to insert these genes into an E. coli expression vector that I used to purify recombinant protein via affinity chromatography. The binding affinity with c-di-AMP of each protein was tested with radio-ligand binding assays to determine dissociation constants (K_d) and we additionally used enzyme activity assays to quantify inhibition of protein activity. AKRs that bind c-di-AMP will be further characterized in vivo in mammalian cell lines in which expression has been knocked down via lentiviral transduction. Further work in cell lines may elucidate novel pathways that are activated downstream of c-di-AMP detection. Understanding the pathways that naturally clear infections is important to potential medical advances in bacterial infection management and prevention.

Voltage-gated calcium channels are vital to electrical signaling in the human body, and their defects are associated with various neurological and cardiovascular disorders. Finding the 3-D molecular structure of these channels allows us to understand the physical basis for their function of selectively allowing calcium ions through the cell membrane. The Catterall lab uses X-ray crystallography, a technique in which a protein is grown into crystals and then diffracted with X-rays, to study the structure of Ca_vAb, a voltage-gated calcium channel from the bacterium Arcobacter butzleri. However, crystals of Ca_vAb are currently weak and give structures with relatively low resolution. This makes it difficult to see details in the protein’s structure and limits these structures' usefulness. To this end, I have introduced a zinc-binding site into Ca_vAb that increases the strength of the protein-protein interactions that cause it to crystallize. I anticipate that crystals of this altered form of Ca_vAb, when grown in the presence of zinc ions, will incorporate zinc into the introduced binding site, strengthening the crystal. Current results show some evidence for the inclusion of this zinc-binding site in crystals of Ca_vAb. The crystals show X-ray absorption and electron density within the binding site that indicate the presence of zinc. While further studies are needed to completely elucidate the effect of this zinc-binding site, current results are promising. In the future, we hope to take advantage of the anticipated crystal quality from this zinc-binding site for further structural studies on Ca_vAb. For instance, greater resolution will allow our lab to fully determine the mechanism of action by which calcium channel blocker drugs bind to a voltage-gated calcium channel. Furthermore, similar introduced metal-binding sites could be applied to the crystallization of other proteins that have so far evaded structure determination by crystallography.

In typically developing children, obstructive sleep apnea (OSA) is associated with neurobehavioral performance deficits including impaired behavioral regulation and working memory. Less is known about the effects of OSA on neurobehavioral performance in children living with a chronic health condition, such as Juvenile Idiopathic Arthritis (JIA). Our recent study showed a high prevalence of OSA in children with JIA that was associated with slower reaction times and increased impulsivity. The purpose of this study is to compare the frequency of OSA, and the associations between OSA and neurobehavioral performance (behavior regulation [emotional control, inhibition], metacognition [working memory, organization]) in 150 school-age children with and without JIA. We hypothesized that OSA will be increased in children with JIA, and OSA will be negatively associated with emotional control, inhibition, working memory, and organization in comparison to an age, sex match group of control children. Children and their parents underwent an overnight polysomnography (PSG) in a sleep research laboratory. Parents completed neurobehavioral performance measures including Behavioral Rating Inventory of Executive Function (BRIEF) and Child Behavioral Check List (CBCL). OSA frequency between JIA and control children was examined with Chi-square analysis. Group differences (JIA vs. control) in neurobehavioral performance measures were examined with analysis of variance (ANOVA). Bivariate correlations were done to examine the associations between OSA and neurobehavioral performance tests. Findings from this study will provide new knowledge about the effects of obstructive sleep apnea on neurobehavioral performance in children living with a chronic health condition.

Obstructive sleep apnea (OSA) is a major public health concern associated with poor health outcomes (health-related quality of life (HRQOL), cognitive deficits). An estimated 1-4% of typically developing children have OSA. Less is known about the prevalence of OSA and co-morbid effects of OSA in children with chronic health conditions such as juvenile idiopathic arthritis (JIA). JIA is an autoimmune disorder associated with joint inflammation, pain, fatigue, and disturbed sleep. A recent study reported a high prevalence of OSA in JIA, however the associations between OSA and health outcomes were not reported. The aims of this study were to: 1) compare the prevalence of OSA, HRQOL, and fatigue in JIA and control children; and 2) compare HRQOL and fatigue in JIA and controls with OSA to those without OSA. Participants included children 6-11 years, with JIA (n=68) and without JIA (n=75). Children underwent overnight polysomnography (PSG). Children and parents also completed the Pediatric Generic Core scale (HRQOL), Multidimensional Fatigue Scale; and parents completed the Pediatric Sleep Questionnaire (PSQ). The analysis for aim 1 examined group differences in demographic and clinical characteristics, PSG OSA indices, parent-reported OSA symptoms, HRQOL, and fatigue. For aim 2, we categorized JIA and control children who met the clinical criteria for OSA by PSG and by PSQ. Mean scores for HRQOL and fatigue were examined in children with OSA confirmed by PSG and PSQ, to children without OSA confirmed by PSG or PSQ. We found that children with JIA have increased OSA by PSG and parent report, lower HRQOL, and increased fatigue severity compared to controls. Results of this study will inform clinical practice by highlighting the need to routinely screen children with JIA for OSA, and the impact of OSA on important patient-reported health outcomes in JIA.