Sensory hair cells in our inner ear are necessary to detect sound, and hair cell death is the primary cause of hearing loss. A certain class of antibiotics, including neomycin, induces hair cell death. Understanding the mechanisms involved in preventing hair cell death may yield pharmacological advancements to protect sensory hair cells when these antibiotics are administered. Zebrafish are often used to study hair cells because fish have readily accessible hair cells on their exterior that are analogous to sensory hair cells within our inner ear. This project investigated the effects of heat shock on neomycin-induced hair cell death. Heat shock can trigger several signaling pathways known to protect cells against cellular insults. We hypothesized that applying heat shock prior to exposing the hair cells to neomycin would inhibit damage by increasing cellular defense mechanisms as seen in other organisms. The data were obtained by placing 5-day-old zebrafish in a 38 degree Celsius water bath for one hour (heat shock), followed by an hour break for recovery, and then a 30-minute incubation period in neomycin. We quantified the degree of damage to the hair cells 5 hours after neomycin treatment by scoring (ranking) hair cell clusters based on cluster size and counting the number of hair cells located in specific areas. Our results suggest that heat exposure partially inhibits neomycin-induced hair cell damage. Further research may yield insight toward pharmacological advances that prevent antibiotic-induced hair cell damage.

The hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcription factor that plays a crucial role in an animal’s response to changing oxygen availability. When oxygen is plentiful, HIF-1 is ubiquitinated and targeted for degradation by the von Hippel Lindau protein (VHL-1), which inactivates HIF-1. Recent studies have shown that HIF-1 stabilization through mutation of vhl-1 increases longevity in the model organism Caenorhabditis elegans. This is contrary to humans, where VHL-1 mutations cause a disease characterized by angiomas, renal carcinomas and various other tumors as a direct result of aberrant HIF-1 activation. Since nematodes are post-mitotic organisms in adulthood, constitutive activation of HIF-1 does not cause tumor formation and is able to increase lifespan. In order to better understand how HIF-1 increases longevity and to relate these findings to mammals, our project aims to find the specific genes and tissues downstream of HIF-1 that benefit worm longevity without the consequences of VHL disease. To identify these genes, we conducted an RNAi screen for genes that are necessary for HIF-1-mediated longevity. Using the results of this screen, we are creating transgenic worms that overexpress these genes under specific nematode promoters. To test for the tissue specificity of HIF-1-mediated longevity, we are stabilizing HIF-1 in different tissues using tissue specific promoters. Our initial data are promising; we have found two genes downstream of HIF-1 that when overexpressed increase nematode longevity. At least one of these genes, flavin-monooxygenase-2, is a well-conserved protein known to be involved in xenobiotic metabolism. Additionally, we have found that expressing stabilized HIF-1 in neurons is sufficient to increase nematode lifespan. Our continuing studies will identify additional target genes and tissues important for HIF-1 mediated longevity and will attempt to elucidate the mechanism by which they increase lifespan.

Adenoviruses (Ads) are the most exhaustively researched and well-understood viral platforms for gene therapy and oncolytic virotherapy, with potential applications including the treatment of numerous inborn and acquired genetic diseases, including cancer. However, when injected into the bloodstream, Ad vectors can bind several blood coagulation factors that facilitate the transduction of hepatocytes, sequestering the virus in the liver. Although both coagulation factors VII and X bind the Ad5 hexon with very high affinity, only FX appears to play a role in mediating Ad hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to the virus and its poor capacity at supporting cell entry, we conducted structural analyses on the Ad5-FVII complex. Using high-resolution cryo-electron microscopy followed by molecular dynamics flexible fitting (MDFF) simulations, we have determined that the FVII GLA-domain sits within a surface-exposed hexon trimer depression in a different orientation than found for FX, althouth several of the same hexon residues that mediate FX binding also mediate FVII binding to hexon. Furthermore, we found that two proximal FVII molecules on the viral surface may interact via their serine protease (SP) domains, burying potential heparin sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. Several viruses with point mutations in critical residues of the hexon protein were engineered and grown. Infection assays revealed cell transduction efficacy that varied independently of the strength of the interaction between the hexon and FVII. MDFF simulations revealed an altered interface between hexon and FVII that could affect the orientation of FVII in the pocket and, thus, the efficacy of dimer formation. Since the occluded HSPG domains are crucial for cell entry, our results suggest that differing binding orientations for FX and FVII GLA domains lead to disparate quaternary structures, which ultimately determine the cell-transduction efficacy of the complex.

Despite much investigation, there has been relatively little progress in the development of novel analgesic compounds to treat debilitating chronic pain conditions that affect millions of people. Inherent problems in behavioral screens to identify analgesic compounds include the use of expensive and time consuming low throughput rodent based assay as well as the use of behaviors that only measure the presence or absence of acute behavioral responses to noxious stimuli, whereas assays that measure an animal’s ability to choose between an innocuous and noxious environment overcome these setbacks. In this study, we aim to develop a high throughput two-choice thermal discrimination assay utilizing zebrafish (Danio rerio) larvae that can be used to screen for novel analgesic compounds. In order to do this, we will profile the ability of zebrafish larvae to discriminate between two temperatures. Individually arrayed 5 days post fertilization larvae will be placed in wells containing two distinct thermal zones and assayed to determine if they display a thermal preference between a testing temperature and a normal rearing temperature (28.5^oC). The testing temperature will range in one degree increments from noxious cold (10^oC) to noxious heat (38^oC). We will also test the limits of thermal preference by testing the ability of larvae to discriminate between two noxious temperatures, for example 36 vs 38^oC. To conclude if thermal preference is malleable based on the thermal environment to which larvae are acclimated, we will determine if prior incubation at a selected temperature other than 28.5^oC will alter the thermal preference of zebrafish larvae. Finally to determine if our assay may be appropriate for the use in a screen for novel analgesic compounds, we will investigate whether or not known noxious or analgesic compounds alter the thermal preference of zebrafish larvae.

Thermostability is the capacity of an organism to survive and thrive at high temperatures and is a characteristic with many biotechnological applications, such as food processing. We are seeking to understand the mechanisms of thermostability in viruses by using an RNA virus, phi-6 Cystovirus. We are evolving the phi-6 virus to become thermostable by exposing it to a target temperature of 61°C over many generations (wild type viruses grow at 25°C). In order to see if exposure to different intermediate temperatures affects the genetic pathway to thermostability, we will expose the phi-6 virus to different temperature regimes prior to reaching the target temperature. The temperature treatments are sudden, where the virus is exposed to the target temperature throughout the entire course of the experiment, moderate, where the virus is exposed to the target temperature halfway through the experiment, and gradual, where the virus is exposed to the target temperature on the final day of the experiment. The genome of phi-6 has already been characterized, which enables us to compare the genome of the evolved, thermostable phi-6 viruses to the ancestor and allows us to study the genetic basis of thermostability. In future studies, we hope to use these thermostable viruses to learn about costs and benefits of maintaining thermostability.

Most procedures for DNA transformation of chloroplasts involve the use of circular DNA as vectors and green leaves as the target tissue. Although such procedures are successful with some plants, they are inefficient and do not work with other plants, especially monocots. In maize, plastid DNA (ptDNA) is comprised largely of linear molecules. Furthermore, ptDNA is rapidly degraded as the cells become green. In consequence, the introduction of transgenes into maize chloroplasts has been problematic. In order to create vectors for monocot transformation, the plastid chromosome is most efficiently targeted in its natural form. Previous work suggests that the plastid chromosome is found primarily in a complex, branched linear form with defined ends. We chose one of these ends, “End 1,” which is near an origin of replication, as our target for transgene integration. We constructed vectors with the “End 1” gene sequences and a mannose-metabolizing transgene, PMI, as the selectable marker. Plastid transformation was performed by particle bombardment using gold microcarriers coated with either the circular or linear vector. The successful introduction of a transgene using the “End 1” DNA sequence indicates that the transgene integrates either by end joining of the linear vector to the linear ptDNA or by homologous strand invasion. Thus, using a linear vector with an end sequence produces optimized results when engineering monocot plastid transformants.

Eukaryotic genomes contain many copies of ribosomal DNA (rDNA) encoding the RNA components of ribosomes, ranging from 150 tandem repeated copies in the budding yeast Saccharomyces cerevisiae to approximately 700 copies total in diploid human cells. It is not known what dictates rDNA copy number, but we hope to gain insight by identifying genetic regulators of rDNA copy number. Recent work in the Brewer/Raghuraman lab using S. cerevisiae reveals connections between rDNA copy number and cellular processes, such as genome replication and replicative aging. These studies suggest that too few rDNA copies cannot satisfy ribosome demand and too many copies can interfere with DNA replication. Thus, certain genetic pathways must function to maintain such balance. Our preliminary survey finds that some strains with specific single gene deletions have varying rDNA copy numbers, suggesting genetic control of the flexibility of rDNA copy number. Our goal is to identify genetic regulators of rDNA copy number in a systematic genome-wide screen. I have developed a high throughput screen to look for strains with variation in rDNA copy number by measuring the size of chromosome XII, the chromosome that carries the rDNA locus. I will use this method to screen a yeast library of approximately 5000 non-essential single gene deletions. After finding and analyzing mutant strains with chromosome XII size changes, we will have a more comprehensive understanding of the cellular mechanism that controls rDNA repeat number. As rDNA repeat number is linked to replicative lifespan, we hope to uncover the common genetic pathways that regulate rDNA copy number and longevity.

Hot flashes affect 75% of menopausal women, leaving them with recurring flushing, sweating, skin blotching, and occasional anxiety, palpitations, and sleep disturbances. 20% of those affected describe the symptoms as “intolerable,” and current treatment methods carry too many side effects or are insufficiently effective. We propose the drug nalfurafine to prevent these symptoms. Nalfurafine has the distinct clinical advantage of being taken orally and targeting κ-opiod receptors outside of the blood-brain barrier, leading to minimal side effects. κ-opiod receptors decrease the activity of Kiss1 neurons, and we believe kappa agonists such as nalfurafine inhibit the Kiss1 neuron activity we hold responsible for hot flashes. To test this hypothesis, we injected nalfurafine in mice and collected blood and brain samples, expecting reduced levels of specific hormones and proteins associated with Kiss1 neuronal activity. Analysis of data collected in this study is ongoing. If our hypothesis is incorrect, we will have a new understanding of how κ-opiod receptors regulate Kiss1 neuronal activity. However, if we find evidence that nalfurafine inhibits Kiss1 neuronal activity, it may alleviate hot flashes in women, thereby providing rationale for clinical studies and potentially leading to a therapy that would improve quality of life for millions of women.

Obesity is a major cause of health-related problems in the developed world and recently studies have demonstrated a link between environmental factors and obesity. 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) is one toxic chemical used as a flame-retardant in consumer products. BDE-47 contaminants are commonly found in dust, air, and soil and their accumulation in the food chain contributes to significant levels found in humans, particularly children. Recent studies have linked BDE-47 to a range of toxic impacts, including disruption of insulin signaling. The objective of this study is to investigate the in vivo effects of BDE-47 in hepatic metabolism. We hypothesize that the drug will cause increased cell injury, inflammation and accumulation of fat within the liver (hepatic steatosis) and will have significant effects on individuals predisposed to disruption in insulin signaling. In order to disrupt insulin signaling in vivo, we have generated mouse models with liver-specific deletion of Tsc1 and Pten, negative regulators of mTORC1 and Akt respectively. Both of these complexes play critical roles in the cell cycle and insulin metabolism within the liver. Cohorts of Tsc1-/-, Pten-/-, and control mice were exposed to BDE-47 or placebo for six weeks within their first 10-12 weeks of life. During exposure, the mice were tested for insulin sensitivity and glucose tolerance. Liver tissue was also tested for several markers of hepatic insulin signaling post-mortem. Differences in impacts on these genetic mutants could indicate if the BDE-47 targets the Akt or mTOR cascades differently and if those predisposed to aberrations in insulin signaling may be more sensitive to the toxic effects of BDE-47. This study will determine if a significant interaction between the environmental influence of BDE-47 and the genetic deregulation of insulin signaling occurs, which could have deleterious implications for the use of these compounds in consumer products in the future.

The nuclear transport pathway performs the fundamental function of moving cargo between the cytoplasm and nucleus in all eukaryotes. Nuclear transport is carried out through a highly conserved mechanism across all eukaryotes. Yet, in Drosophila, several components of the nuclear transport apparatus, including importins, evolve rapidly under positive selection. Genetic conflict with selfish elements has been suggested as a possible cause for this pattern of rapid evolution. Here, we present a comprehensive phylogenomic analysis of importin gene evolution in Drosophila. Importins are adapter molecules that directly mediate the transport of cargo into the nucleus. Our analysis reveals a recurrent pattern of gain and loss of importin paralogs across independent lineages of Drosophila. Interestingly, we discovered that almost all new copies of importins have acquired a testes-specific expression pattern since their birth through gene duplication. This pattern of repeated gains of testes-specific copies of importins suggests a function in suppressing genetic conflicts such as segregation distortion in the male germline. Segregation distorters such as SD in Drosophila melanogaster act by impairing nuclear transport in the testes. We are currently performing functional tests for the hypothesis that an increased dosage of these testes-specific importins may serve a role in suppressing segregation distortion in the male germline by restoring nuclear transport during spermatogenesis.

 Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction is one promising pro-longevity intervention, though its mechanism is poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species, including yeast, flies, mice, and primates. However, this extension is highly dependent on genetic background. To investigate this, we are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We have screened over 1500 genes, 61 of which are short-lived and 67 of which are long-lived. We are currently following up on our collected data by measuring the full lifespans using the RNAi clones that significantly shortened or extended lifespans in our screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

The hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcription factor that plays a crucial role in an animal’s response to changing oxygen availability. When oxygen is plentiful, HIF-1 is ubiquitinated and targeted for degradation by the von Hippel Lindau protein (VHL-1). Recent studies have shown that HIF-1 stabilization through mutation of vhl-1 increases longevity in the model organism Caenorhabditis elegans. This is contrary to humans, where VHL-1 mutations cause a disease characterized by angiomas, renal carcinomas and various other tumors as a direct result of aberrant HIF-1 activation. Since nematodes are post-mitotic organisms in adulthood, constitutive activation of HIF-1 does not cause tumor formation and is able to increase lifespan. In order to better understand the how HIF-1 increases longevity and to relate these findings to mammals, our project aims to find the specific genes and tissues downstream of HIF-1 necessary for its effect on worm longevity. Our goal is to find genes downstream of HIF-1 that benefit longevity without the consequences of VHL disease. To identify these genes, we conducted an RNAi screen for genes that are necessary for HIF-1-mediated longevity. Using the results of this screen, we are creating transgenic worms that overexpress these genes under specific nematode promoters. To test for the tissue specificity of HIF-1-mediated longevity, we are stabilizing HIF-1 in different tissues using tissue specific promoters. Our initial data are promising; we have found two genes downstream of HIF-1 that when overexpressed, increase nematode longevity. At least one of these genes, flavin-monooxygenase-2, is a well-conserved protein known to be involved in xenobiotic metabolism. Additionally, we have found that expressing stabilized HIF-1 in neurons is sufficient to increase nematode lifespan. Our continuing studies will identify additional target genes and tissues important for HIF-1 mediated longevity and will attempt to elucidate the mechanism for their action.

Leigh syndrome is a clinically defined collection of diseases comprised of multiple genetic defects related to mitochondrial function. This syndrome results in early death in children typically due to respiratory failure as a consequence of progressive neuropathy, and currently no viable treatment options exist. Leigh syndrome can result from mutations in several different mitochondrial genes, one of which encodes the NDUFS4 subunit of complex I in the mitochondrial electron transport chain. As the NDUFS4 knockout mice phenocopy human patients with Leigh syndrome remarkably well, this model was utilized for the purpose of this study. Dietary restriction (DR) was identified as a possible treatment for mitochondrial deficiencies through a yeast genetic screen, which found that mitochondrial gene mutants generally received a robust lifespan benefit from DR. Thus, the primary focus of our research is to determine the efficacy of rapamycin (a DR mimetic) as an intervention in the NDUFS4 -/- mitochondrial disease mouse model. Rapamycin functions as a DR mimetic through its inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway, which is responsible for modulating a variety of metabolic and cellular processes. Our model suggests that the increased NADH/NAD ratio resulting from a complex I deficiency in these knockout mice is sensed as energy abundance and results in systemic metabolic derailment. We propose that inhibiting mTOR signaling addresses this defect by altering intracellular energy sensing and modifying metabolic status. In order to test the efficacy of rapamycin treatment, NDUFS4 -/- mice were treated with daily injections of rapamycin or vehicle. Lifespan data for each treatment and additional assays for health related parameters were conducted to determine the response to the treatment regime. Investigating Leigh syndrome in this context will allow us to better understand the underlying mechanisms of pathogenesis, and directly test novel interventions aimed at attenuating this disease.

Protein phosphorylation is a common regulatory mechanism carried out by the cell, as adding a phosphate group to a particular protein can switch its activity on or off.  Signaling pathways employ this control method to instigate series of phosphorylation events that elicit a particular cell response.  Our research involves extracting and characterizing the phosphoproteome, or the cell’s entire set of expressed phosphorylated proteins, in both yeast and mammalian cell models.  To generate lists of phosphoproteins present in these cell models, we use mass spectrometry, an analytic technique that produces characteristic spectra for individual phosphopeptides that are then matched to a library of known protein spectra through the use of computer algorithms.  In our preliminary results, we have generated large-scale data sets identifying thousands of phosphorylated proteins present in each species and cell type.  The goal of the first project, the yeast study, is to determine the degree of evolutionary conservation in phosphorylation events across 19 yeast species to learn about the basic principles of phosphoregulation.  The conservation of phosphorylation can also give insight into phosphorylation events in other species evolutionarily related to yeast, such as humans.  The second project, the mammalian cell study, examines the phosphoproteome of different breast cancer cell types in order to identify common phosphorylated proteins, from which we can deduce a molecular signature for breast cancer.  This signature can be used in future studies to design a universal protein inhibitor drug as an alternative anti-cancer therapy.  Additionally, by identifying the phosphoproteins exclusive to each cell type, we can uniquely characterize specific types of breast cancer.  Knowledge of the proteins active in one type of breast cancer may facilitate development of inhibitor drugs as a form of personalized medicine.

Neutralizing antibodies, which prevent entry of the HIV virus into a host cell by binding to the HIV envelope and neutralizing the biological effects of the virus, is an important focus of HIV vaccine design. It is hypothesized that these neutralizing antibodies may protect against initial infection from HIV if they are elicited by vaccination and are thus present at the time of virus exposure. The high rate of HIV mutation results in an enormous number of HIV variants circulating in the human population. Thus, an effective neutralizing antibody-based vaccine must be broad and potent. In order to understand how to create factors that elicit broadly neutralizing antibodies (bNAbs), it is important to identify and map regions of the viral envelope important for recognition by bNAbs. The best way to map the molecular basis is to identify two closely related variants that are or are not susceptible to neutralization by a certain virus and identify the regions that differ between the two variants. PGT128- is a HIV-1 monoclonal antibody that has been identified as having a broad neutralizing activity that would be desirable for a vaccine to elicit. Our lab previously found that longitudinal viruses from a HIV-1 infected patient, QA255, across 560 days post infection (dpi) display increasing ability to be neutralized by PGT128. PGT128 recognition has recently been found to depend on residue N332 of the V3 envelope region of HIV. As this residue is intact in QA255 viruses over time, there are additional determinants of sensitivity to PGT128. We found that chimeric sequences containing 560 dpi DNA in the V1-C3 region of the HIV envelope sequence show more sensitivity to PGT128 compared to chimeric sequences from earlier time points. This result suggests that amino acids in the V1-C3 region are important for PGT128 sensitivity.

To be infected with human immunodeficiency virus (HIV) is to face a lifelong battle. Even with the combined force of multiple antiretroviral drugs to target different phases of the HIV life cycle, one cannot eliminate human immunodeficiency virus (HIV) from the body. This is due to persistence of virus reservoirs, compartments, or sanctuaries. Reservoirs are tissue sites characterized by collection of virus over time: viral populations show low divergence from the founder virus and high diversity between each other. Compartments are tissues with limited gene flow: they are pockets of virus with limited entry and exit. Sanctuaries are tissues that resist penetration of antiretroviral drugs: they can set up the conditions for reservoirs or compartments. To understand how and where these sites of persistent infection endure in the body even during antiretroviral therapy (ART), we are conducting a systematic characterization of possible sites in the infected host. We are studying viral populations present in biopsy-derived gut-associated lymphoid tissues (GALT) and other autopsy-derived tissues of patients who died while on antiretroviral therapy (ART). Viral populations are sampled from tissues using molecular lab techniques (including polymerase chain reaction and Sanger sequencing) and analyzed using phylogenetic trees and computational programs that measure diversity, divergence, and clustering. Our study addresses two main aims: (1) to find the tissue sites in the host critical in maintaining latent HIV infection during suppressive ART and (2) to find tissue sites critical in supporting continued viral replication and the emergence of pathogenic phenotypes during ART by characterizing sites as reservoirs, compartments, or drug sanctuaries. Hopefully our research will help to direct future methods of HIV treatment by allowing researchers and clinicians to better understand how and where virus persists in the body.

We are studying the roles of the p120-catenin protein in zebrafish development. p120-Catenin has a critical role in adhesion and migration by associating with E-cadherins and small, Rho family GTPases. The regulatory domain of p120-catenin contains 8 tyrosine and 8 serine or threonine residues that can be phosphorylated. To date, the function of these phosphorylation sites has been controversial. In cell cultures there is evidence that phosphorylation of 6 serine sites cause adhesions to recover quicker as well as form stronger connections. Some studies, however, claim that phosphorylation of these serine residues has no effect on adhesion. Tyrosine phosphorylation has been associated with nascent adhesions suggesting its role in adhesion formation, while its presence in carcinoma cell lines implies a role in cell motility. By selectively blocking phosphorylation at these 16 residues we hope determine the roles for the phosphorylation of serine and tyrosine residues in adhesion and migration of cells during embryogenesis. We mutated mouse p120-catenins at tyrosine residue 228 to phenylalanine, and at serine residues 252 and 268/288 to alanine via site-directed PCR mutagenesis. We inserted the DNA into PCS2+ vectors containing the fluorescent protein mcherry, and translated the mutant DNA into mRNA. We injected one-cell zebrafish embryos with an antisense morpholino RNA, complimentary to a splice-site region of endogenous zebrafish p120-catenin. Injections of this morpholino effectively knockdown the expression of newly translated p120-catenin mRNAs, and inhibit normal somite formation and axis extension during development. Co-injection of a wild-type mouse p120-catenin mRNA with the morpholino showed nearly normal development. Preliminary data suggests that co-injection of Y228F p120-catenin mRNA with the antisense morpholino RNA also produces nearly normal phenotypes. We will be testing the localization and function of the other mutated p120-catenins. This research could have important implications in development, treatment of some cancer types, and wound healing.
 

Organisms can develop normally and thrive in dynamic environments. The interplay between the environment and the biomechanics of morphogenesis can help us understand how development operates in a changing world. Effects of salinity on blastula expansion in the sand dollar Dendraster excentricus were used to investigate interactions among the environment, development, and mechanics. Salinity fluctuations, which are common in Dendraster's habitat, affect embryonic cell size. The hyaline layer, an extracellular matrix layer closely associated with the outside of the blastula, is hypothesized to resist blastula expansion. Four models describing how the hyaline layer responds to blastula expansion were compared. In three models the hyaline resists expansion as either an elastic, plastic, or viscous layer. In these models, salinity-driven changes in cell size were predicted to cause changes in the ratio of blastocoel volume to cell volume. In a fourth model, the hyaline is a perfectly accommodating layer that allows the embryo to expand freely as cells divide in a single layer. In this model, salinity changes were predicted not to affect the blastocoel-to-cell-volume ratio. To test these hypotheses, Dendraster embryos were placed in either 25‰ or 32‰ seawater wells and switched to wells of either the same or the other salinity. In a second experiment, Dendraster embryos were raised in 32‰ seawater until the 16-cell stage, at which point a subset of embryos were moved to 25‰ seawater for one cleavage stage and then moved back to 32‰ for the duration of the experiment. In both experiments blastocoel-to-cell-volume ratios were not affected by the salinity treatment. These results suggest that the hyaline layer does not resist blastula expansion in Dendraster, thereby maintaining a constant blastocoel-to-cell-volume ratio.

Only a few organisms possess the capacity to regenerate limbs and appendages from development into adulthood. Danio rerio, or zebrafish, are recognized as a powerful model for investigating regenerative processes. For example, following amputation of the tail fin, zebrafish rapidly restore lost bone, joints, nerves, and blood vessels within a few weeks. My research is focused on determining neuronal processes that contribute to the ability of zebrafish to regenerate their fins. My experiments have consisted of amputating the tail fin, and tracking fin growth and bone mineralization. To inhibit neuronal function, I developed an inducible model neuronal impairment by injecting the fins with Botulinum toxin type B. Data is analyzed through pictorial documentation, bone staining, image analysis, and microCT scanning. Preliminary experiments have pointed to the involvement of neuronal signals in regenerative growth and bone mineralization processes. Fish given a high Botulinum toxin dose had stagnated and significantly decreased fin regeneration compared to control fish administered sodium chloride. Fish given a low Botulinum toxin dose had normal fin growth, but significantly decreased bone mineralization activity. We hypothesize that this neurotoxin is preventing neuronal signals that promote fin regenerative growth as well as mineralization in regenerating bone tissue. Experiments are underway to develop standard procedures for amputation and Botulinum studies, and to better understand the mechanisms by which this occurs. Further experiments can be done to ensure that the neuronal pathway is the main contributor to bone regeneration or to develop ways to test other physical mechanisms that may also contribute. Provocatively, many organisms (including humans) possess the ability to regenerate limbs during embryonic stages; however, they lose this potential after birth. If the underlying mechanisms enabling fin regeneration can be discovered in Danio rerio, this will provide new strategies for regenerative medicine in humans.

Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy (SMEI), is a rare genetic form of epilepsy, caused by a mutation in the neuronal voltage-gated sodium channel, NaV1.1, encoded by the SCN1A gene. This form of epilepsy is characterized by febrile seizures at infancy that often change with age into afebrile seizures, which include generalized tonic-clonic and myoclonic seizures. In most cases, DS is a debilitating disorder that renders patients with decreased cognitive function and impaired development. To study this disorder, a mouse model of DS, with heterozygous deletion of the SCN1A gene (Scn1a +/-), was developed, demonstrating a phenocopy of the DS condition, with similar seizure patterns and atypical behaviors. In vitro work in the model demonstrates selective loss of sodium current and excitability in hippocampal GABAergic interneurons. However, it remains unknown if similar changes in neocortical GABAergic interneuron excitability occur. We hypothesize that fast spiking neocortical interneurons are responsible for control of cortical excitability via GABAergic inhibition and that reduced excitability of fast spiking interneurons associated with heterozygous loss of NaV1.1 contributes to hyperexcitability and seizures. To explore this hypothesis, we will compare neocortical interneuron excitability measured from single action potential parameters and repetitive action potential firing patterns in unaffected wild type mice and the DS mouse model, using pre-existing single-cell recordings. Excitability will be determined from the peak amplitude, width at half height, threshold for single action potentials, and from total number of action potentials in induced neuronal spike trains. If this hypothesis is correct, we expect that there will be atypical action potentials and firing patterns showing reduced excitability. This experiment will provide more insight into DS epilepsy and better therapeutic targets for the disorder.

Schizophrenia is a debilitating neuropsychiatric disorder with a worldwide prevalence of about 1%. Although the heritability of schizophrenia is high, there are many sporadic cases; that is, affected individuals with no family history of mental illness. We are sequencing persons with schizophrenia in the absence of family history of mental illness (probands), their unaffected parents, and whenever possible, an unaffected older sibling, in order to identify de novo mutations that may be responsible for the disorder. Our hypothesis is that de novo events will be observed in many different genes, and that the functions of the impacted genes will differ between probands and their unaffected siblings. From exome sequence, we filter variants to include only those predicted to be both de novo and damaging to the protein product. Next, we validate all candidate variants passing these filters by Sanger sequencing, using diagnostic PCR primers to amplify genomic DNA of probands, siblings, and parents. Results to date suggest that approximately 50% of probands and siblings carry at least one damaging de novo mutation. The analysis of pathways in which the genes converge is in progress. Next, we will target these candidate genes and determine their complete variant profiles in 5000 cases and 5000 controls. These results can help us identify critical pathways that are affected in schizophrenia.

The primate immune system has evolved to counter viral attacks. Yet, after several cross species transmission events of SIV, HIV has infected about 60 million people since the beginning of its epidemic. This is in part due to the ongoing evolutionary arms race between antiviral proteins and viral antagonists. SAMHD1 is an antiviral protein that is degraded by the Vpr protein expressed in HIV and SIV. In this evolutionary arms race, SAMHD1 evolves to escape degradation, while Vpr evolves to bind to these new variants of SAMHD1. African Green Monkeys (AGM) are divided into phenotypically distinct and geographically isolated subspecies, each infected with their own species-specific SIVagm, making them ideal to study. A cloned SAMHD1 gene, Tan4, from AGM has been found to be resistant to Vpr from a number of different viruses, except for one called the tan1 Vpr. Sequencing Tan4 revealed that one residue of its nuclear localization signal was mutated. Since nuclear localization of SAMHD1 is required for degradation by Vpr, we hypothesized Tan4 evolved to be cytoplasmic in order to resist degradation. Using immunoflouresence, our results confirmed Tan4 to be in the cytoplasm. Moreover, we found that tan1 Vpr and Vpr from another AGM subspecies, vervet, share similar gene sequences, but only tan1 can degrade Tan4. Currently, I am constructing chimeras between the two Vpr proteins to find the residues in tan1 responsible for degradation of Tan4. Likewise, two Vpr proteins of the sabeus monkey share similar gene sequences yet cannot degrade the same SAMHD1. My ongoing work explores the mechanisms used by the tan1 and sabeus Vpr proteins to evolve in order to successfully degrade SAMHD1 despite its attempts at escape. This will elucidate a better understanding of the ongoing evolutionary arms race for both primate host and virus survival.

Cancer genomic analyses performed by our team have allowed us to identify frequently mutated genes in Small Cell Lung Cancer (SCLC). Among these genes, MLL2 was found to undergo loss of function mutations suggesting it could act as a tumor suppressor. MLL2 is a histone methyl transferase that transfers a methyl group to a histone on a DNA strand. This process regulates chromatin state and transcription. More specifically, MLL2 methylates the lysine 4 of histone H3 leading to the activation of gene expression. Interestingly, MLL2 was also recently found mutated in squamous cell lung carcinoma, non-Hodgkin lymphoma, medulloblastoma and cell renal carcinoma pointing out that it could be a novel major tumor suppressor. To investigate this hypothesis, both in vivo (mouse models) and in vitro (cell based assays) will be used. The mouse model consists of a conditional, tissue specific inactivation of two prototype tumor suppressors namely p53 and Rb, which are found highly mutated in SCLC. The role of MLL2 will be studied in this mouse model by generating triple mutants (Rb, p53, and MLL2) mice. The survival of control SCLC mice (p53 and Rb inactivated) will be compared to the survival of the triple mutant (MLL2, Rb, and p53) mice. This critical experiment should enlighten us on the functional role of MLL2 during SCLC development. Molecular and histopathological analyses will also be performed. Additionally, in vitro approaches with human and mouse cells will be used to identify and understand the biological processes and pathways regulated by MLL2. Altogether, it should help us decipher the role of MLL2 in SCLC and it might ultimately lead to the identification of novel treatments and/or preventive options for SCLC.

Breast cancer tumors are classified within different subtypes based on the expression of three types of cell surface receptor proteins.  These proteins connect the extracellular environment with cellular activities via signaling pathways.  To elicit cell responses, signaling pathways employ protein phosphorylation as a regulatory mechanism, as adding a phosphate group to a particular protein can switch its activity on or off.  However, normal signaling can go awry if mutations in signaling proteins lead the cell to believe its components are always phosphorylated and active.  Consequently, the entire signaling pathway functions abnormally, which can lead to uncontrolled cell growth characteristic of cancer.  Given its potential role as a molecular mechanism of disease, our research focuses on obtaining a global view of the phosphoproteome, or the cell’s entire set of expressed, phosphorylated proteins, of 10 breast cancer cell lines.  To generate lists of phosphoproteins present in these cell lines, we are concurrently extracting and analyzing cell line phosphopeptide samples via mass spectrometry, an analytic technique that produces characteristic spectra for individual phosphopeptides that are then matched to a library of known protein spectra through the use of computer algorithms.  In our preliminary results, we have generated large-scale data sets identifying thousands of phosphorylated proteins present in two cell lines and generated side-by-side comparisons of the proteins expressed in each line.   By identifying common phosphorylated proteins across all of the cell lines, our goal is to deduce a molecular signature for breast cancer.  This signature can be used in future studies to design a universal protein inhibitor drug as an alternative anti-cancer therapy.   By identifying the phosphoproteins exclusive to each cell type, we can uniquely characterize specific proteins active in one type of breast cancer which may facilitate development of inhibitor drugs as a form of personalized medicine.

Perineuronal nets (PNNs) are a matrix of proteins that surround nerve cells and reinforce the most often used synaptic connections between neurons, essentially hard-wiring neural connections made as a child into permanent pathways that respond to specific stimuli. By dissolving these PNNs, we recreate the plasticity characteristic of a young developing brain, granting us the ability to rewire the brain with respect to any stimulus. In this study, we are looking at PNNs within the vestibular system – the sensory system that, under natural conditions, is primarily responsible for maintaining images on the fovea during movement. Hair cells within the semicircular canals of the vestibular system register head accelerations and then send reflex signals through vestibular afferent nerve endings to extra-ocular eye muscles that drive eye movements. This vestibular ocular reflex aids the maintenance of stability of images on the retina. As a therapy for vestibular loss, our lab has developed a vestibular prosthesis that electrically stimulates the afferent nerve endings of the vestibular system, thereby simulating natural vestibular response to movement. This electrical stimulation, however, spreads to untargeted as well as targeted vestibular afferents and cause inaccurate eye movements as a result. By dissolving the PNNs in the vestibular nuclei and then allowing them to regrow during a period of adaptation, we hope to suppress inappropriate eye movements in response to our prosthesis’ electrical stimulation. If our lab can prove that electrically elicited eye-movements become more accurate after the dissolving and guided re-growing of PNNs, we can show that it is possible to restore plasticity to the brain and shape its neural connections through controlled electrical stimulation to the target system. By extension, such results will ascertain if it is possible to use this method to re-wire a physiological system to accept prosthetic stimulation in place of a non-functioning system.

In typical ascidian development, a tailed larva hatches, swims, becomes competent, and then responds to settlement cues, that initiates metamorphosis. Metamorphosis triggers apoptosis, which is required for tail loss. At this time, the notochord is lost as the tail reabsorbs and the juvenile settles to a substrate. In the ascidian family Molgulidae, species have evolved tailless larval development at least four times independently from a tailed ancestor. We hypothesized that a heterochrony in metamorphosis is responsible for the evolution of tail loss in the tailless ascidian larvae. We show that there is early expression of metamorphosis genes in Molgulidae, including genes involved in the Mannose Binding Lectin (MBL) Complement pathway of innate immunity. We hypothesize that the maturation of the immune system early in tailless larvae could be a progression to competency and trigger to metamorphosis. Jeffery (2002) showed programmed cell death happens in cells that form a tail early in development. We further hypothesize that a heterochrony in metamorphosis promotes the early activation of programmed cell death in the tailless ascidian larvae. Using next generation sequencing techniques, we sequenced transcriptomes of a tailed and tailless species and their hybrid through development. We examined genes that have been shown to be expressed during metamorphosis in other ascidians and saw earlier expression of genes critical for metamorphosis in both species. Our ultimate goal is to elucidate the ascidian molecular trigger for metamorphosis and uncover whether heterochrony of metamorphosis leads to tailless ascidians.

 The diatoms are a prolific and productive group of phytoplankton belonging to the phylum Heterokontophyta. They are responsible for ~30% of oceanic primary production and their high photosynthetic capacity depends on their abilities to synthesize chlorophyll. The penultimate step in the biosynthesis of chlorophyll is catalyzed by the enzyme ‘light-dependent protochlorophyllide oxidoreductase’. To understand the evolutionary history of the gene (por) encoding this enzyme (POR) we have constructed an algal por gene tree using diatom por genes as well as those from sister taxa and distantly related taxa. This process involved the cultivation of various strains of algae, DNA extraction, PCR, gene sequencing, data mining from public databases, and bioinformatic analysis. We found two distinct por genes (por1 and por2) within the diatoms and a single haptophyte alga. We propose three hypotheses as to the origin of dual por genes in diatoms: (1) a por gene duplication event occurred before the divergence of the Heterokonts and Haptophytes; (2) a por gene was laterally transferred to a common ancestor of these two algal taxa; or (3) reciprocal lateral gene transfers occurred between these two algal taxa. Whereas the first two hypotheses are more parsimonious, we are unable to determine which scenario is correct with the current dataset. Nonetheless, these data provide evidence for a shared common ancestor between the heterokonts and haptophytes, a hypothesis that is currently hotly debated. Furthermore, the presence of these two por genes in various algal taxa presents a rich opportunity to study the evolutionary forces that either modify or maintain cellular gene function after gene duplication or lateral gene transfer. Thus, the results of this study will soon be complemented with research regarding the enzymatic function and regulation of the two por genes in diatoms.

The goal of our research is to understand mechanisms of the earthworm immune system responsible for distinguishing symbionts from potential pathogens. The Hygiene hypothesis, proposed by David Strachan and published in British Medical Journal in 1989, fundamentally expanded our view on microorganisms and natural development of immune system. The hypothesis states that early childhood exposure to microorganisms is essential for acquiring healthy immunity. Studies in mice showing that host-specific bacteria in the gut are required for full maturation and function of the immune system support this hypothesis. Although numerous symbioses of beneficial bacteria with animals have been studied, the role of symbionts in the development of host immunity remains uncertain. Additional models are needed to study the roles and regulation of the immune responses to host-specific beneficial bacteria. The earthworm offers a tractable system in which to study immune system development and responses to both beneficial symbionts and potential pathogens. Eisenia fetida, a common composting worm, has an effective immune system that eliminates bacteria from the coelom. E. fetida also harbors specific bacterial symbionts in the excretory organs (nephridia) that process coelomic fluid, thus potentially exposing the bacteria to immune elements in the fluid. We hypothesize that the immune system adapts to the symbiotic bacteria in order to avoid eliminating them, or cause unnecessary inflammation in the nephridial tissue. Therefore, we investigated the contribution of the bacterial symbiont Verminephrobacter eiseniae to the development of immune system responses in E. fetida. Coelomocytes were extracted from symbiont-containing earthworms and symbiont-free earthworms, and were examined for differences in their binding and engulfment responses to V. eiseniae and non-symbionts. We believe further studies of the role of immunity in symbiont recruitment mechanisms in the earthworm would help us understand more about the co-evolution of beneficial microbiota and the host organisms in other species. 

A recent discovery in reproductive endocrinology, the kisspeptin system has been found to play a pivotal role in the regulation of the brain-pituitary-gonadal axis. Kisspeptin signaling in the brain is vital to the onset of puberty, control and regulation of sex steroid production, and regulation of fertility and reproductive function in mammals. However, the importance of the kisspeptin system in other vertebrates, such as fish, is not as clear. The goal of the current study was to clone the kisspeptin genes (kiss1, kiss2, and the receptor kiss1ra) for the first time in the marine teleost Anoplopoma fimbria (sablefish), and explore their potential roles in gonadal development. Previously, expression of the kiss2 gene was demonstrated in the gonads of sablefish. Partial kiss mRNA sequences were obtained by PCR and rapid amplification of cDNA ends (RACE) cloning techniques. Semi-quantitative PCR assays developed for each gene allowed us to measure kiss1, kiss2 and kiss1ra mRNA levels in a variety of adult tissues. We found the highest abundance of these mRNAs in the gonads, brain, and pituitary. In particular, kiss2 mRNA was abundant in the ovary and appeared to be the most highly expressed of the kiss genes. We are currently analyzing the expression of the kiss genes in gonads of fish undergoing sexual differentiation. Preliminary data suggest that there is sexually dimorphic expression of all three genes early in life. The ultimate goal of this project is to determine if gonadal kiss gene expression plays an important role in sexual differentiation and/or gametogenesis in fish.

Recent studies demonstrate that certain reproductive factors such as parity (defined as the number of completed pregnancies) are disparately associated with various pathologic subtypes of breast cancer. One factor, fetal microchimerism - the term for persistent child-origin cells in a woman’s circulation and tissues long after delivery - is generally deficient in women with unselected breast cancer and suggests a protective role. Here, we are testing the hypothesis that high-grade triple negative breast cancer (positive association with parity) will be distinctly associated with fetal microchimerism compared to low-grade luminal breast cancer (inversely associated with parity). Buffy coat specimens (n=450) were obtained from breast cancer patients and control participants aged 18-44 from the REACH project (Fred Hutchinson Cancer Research Center, PI Christopher Li). Using quantitative polymerase chain reaction, DNA extracts from these specimens were tested for the presence and concentration of Y chromosome sequence DYS14, a marker of male fetal microchimersim. In an interim analysis from the first 87 specimens, 33% (9/27) of women who developed luminal breast cancer tested positively for the presence of DYS14, as did 33% (10/30) of healthy controls. 47% (14/30) of women who developed triple negative breast cancer tested positively for the presence of DYS14. Preliminary results demonstrate a higher prevalence of fetal microchimerism in patients with triple negative breast cancer compared to control or luminal participants consistent with the original hypothesis. Additional samples will be tested. Fetal microchimerism testing may represent a novel non-invasive method to identify women with poor prognosis breast cancers.

American students regularly get advised to “follow your passion” in choosing a career. However, people's passions are often shaped by what is considered appropriate for their gender. Could encouraging students to follow their passion inadvertently perpetuate gendered career choices? The present work examines how the cultural ideal to follow your passion may maintain gender imbalance in stereotypically masculine or feminine careers. In the current study, some participants were randomly assigned to write about what it means to “follow one’s passion” before writing about their career plans. We hypothesized that women who wrote about following their passions would report more feminine career plans compared to women who did not. Similarly, we predicted that men who wrote about following their passions would report more masculine career plans compared to men who did not. The American cultural ideal to “follow one’s passion” may help reinforce the gender makeup of career fields, contributing to the underrepresentation of women in certain fields like science.

Auxin is the plant hormone, guiding phototropism, gravitropism, cell elongation, cell division, cell determination, and a host of other processes. Plant cells respond to auxin by turning on certain genes. When auxin enters the cell, it binds to a receptor protein; this receptor then binds to a repressor protein which typically prevents expression of auxin genes. This interaction triggers repressor breakdown and gene expression. We are investigating auxin-induced interactions between the receptor and repressor proteins (IAA’s). In our model plant, Arabidopsis, there are 29 IAA repressor proteins and 6 receptor proteins. Some combinations of repressors and receptors are known to result in differential degradation of the repressor; other interactions do not seem to lead to degradation. Our data suggests that receptors AFB4 and AFB5 cannot degrade repressors. This could be because AFB4/5 do not interact with the repressors (hypothesis 1) or because their interaction with the repressors does not trigger degradation (hypothesis 2). We are testing these hypotheses by examining repressor/receptor interactions in Arabidopsis proteins expressed in budding yeast cells. To address hypothesis 1, we are using a yeast two- hybrid assay to investigate the strength of interaction between several IAA repressors and the AFB4/5 receptors in the presence of auxin. To address hypothesis 2, we will determine whether IAA: AFB interactions identified at the yeast two-hybrid assay lead to repressor degradation. We have tagged the IAAs with yellow florescent protein and will co-express them with AFB4/5 in yeast. Flow cytometry will be used to measure changes in florescence levels upon addition of auxin. The next step will be to confirm these findings in plants. Understanding the variation in auxin repressor/receptor interactions brings us a step closer to understanding how auxin drives so many different plant behaviors and responses.

Research over the years has shown that effective oral hygiene with brushing and flossing can help reduce bleeding, gingivitis and plaque buildup. When bacterial plaque is left alone or removed improperly, it can lead to further dental disease such as caries. During orthodontic treatment, fixed appliances such as brackets are bonded to the teeth and archwires connect the brackets. These appliances make it especially hard for orthodontic patients to brush and floss effectively on their own, and failure to do so leads to an increase in plaque and gingivitis seen in the orthodontic population. A pilot study at the University of Washington’s Center for Pediatric Dentistry (CPD) will assess development of white spot lesions during orthodontic treatments. White spot lesions are the first sign of a caries lesion on enamel that can be detected with the naked eye. Pediatric orthodontic patients from CPD will be enrolled in this pilot study. Study participants will be randomly assigned to one of two oral hygiene regimens. Group A will use the WaterPik Water Flosser with orthodontic tip (dental water jet irrigation system) in conjunction with daily oral hygiene and Group B will use floss with a threader in conjunction with daily oral hygiene. Both groups will manually brush their teeth with fluoridated toothpaste twice daily as part of daily oral hygiene maintenance. The White Spot Lesion Index will be used to evaluate white spots. The primary objective of this study is to evaluate prevalence of white spots in fixed orthodontic cases following these assigned oral hygiene regimens. Secondary objectives include assessment of safety and of plaque and gingival status for both groups. Intraoral examinations will be performed; and visible plaque index and bleeding on Marginal Probing scored at each follow-up evaluation. Study participants will be evaluated at every orthodontic appointment for a minimum of 18 months.

The host seeking behavior of disease-vector mosquitoes may be impacted by olfactory and visual learning. By enabling mosquitoes to better find bloodhosts, this behavior may allow mosquitoes to remain longer in the environment, transmitting diseases to more organisms. Recent work has shown that mosquitoes respond to Pavlovian conditioning that pairs visual and olfactory cues with an unconditioned stimulus. However, the function and relative importance of each sensory system has not yet been explored. We investigated both subjects using adult and larval Aedes aegypti. Firstly, we conducted appetitive conditioning experiments on Aedes adults. We found that adults were able to learn associations between novel odors and blood-feeding: they recruited to the conditioned odor consistently and significantly more than the odorless location choice. In contrast, naïve animals showed no innate preference to these odors. Secondly, we conducted aversive conditioning experiments on Aedes larvae. We compared the learning capabilities of wild-type specimens and specimens molecularly (RNAi) or chemically altered (receptor antagonists) to decrease response to dopamine, a critical neuromodulator involved in the aversive learning pathway. Our assays included a conditioned stimulus of indole and multiple unconditioned aversive stimuli (e.g., salt, bitter tastants). We found that wild-type Aedes exhibited a consistent behavioral change after conditioning, whereas transgenic or pharmacologically altered Aedes larvae did not. Finally, we analyzed threat-sensitive larval learning. We tested larval mortality in treatments containing a natural predator (a dragonfly nymph). We found improved survivorship in trained control-injected larvae versus dopamine antagonist injected larva. This is consistent with our data that lowering dopamine sensitivity alters an important pathway in the Aedes larvae olfactory systems to learn and recognize threats. These experiments suggest that both appetitive and aversive learning may be important to mosquito behavior. Our data also supports the conclusion that learning in mosquitoes is at least in part mediated by dopamine.

Ceftaroline fosamil, otherwise known by the name ceftaroline (trade name Teflaro) is a cephalosporin antibiotic indicated for the use against MRSA skin and soft tissue infection and bacterial pneumonia. Though approved by the FDA, the drug is thought to be safe and thought to be well tolerated, however, our team has found it to cause neutropenia in patients. Neutropenia is defined as the abnormally low number of neutrophils, which seems to be present in some patients during and after taking ceftaroline. It is our objective in this study to determine the incidence of neutropenia among patients using ceftaroline. The drug has been approved for more than a year but the post marketing experience has been limited. We have seen two cases of agranulocytosis, which has led us to conduct this study. The study will be conducted in the Tri Cities, which is comprised of Kennewick, Pasco, and Richland. The Tri Cities has a diverse population of 193,000 individuals and there are three major medical facilities: the Kadlec Regional Medical Center, Lourdes Medical Center and the Kennewick General Hospital. The study will determine the incidence of neutropenia among patients receiving ceftaroline. We also hope to describe the side effects of the drug, and track patients’ health over the course of a 7 month period by reviewing patients' charts and through phone interviews. All patients that are included in the study received ceftaroline from August 2011 to March 2012. The patients’ data will be secured from hospital records from the three local hospitals. Our hope is to find a link between usage of ceftaroline and incidence in neutropenia in patients.

Learning on a cellular level actively guides the stabilization or elimination of neuron connections, selecting the optimal pathways that are intrinsically and extrinsically stimulated. The strength and resilience of these neuron networks is what defines neuroplasticity, which can be inhibited by many factors, such as stress, ageing, or developmental disorders. Evidence shows rapid seasonal morphological changes in the High Vocal Center (HVC) and acropallium (RA) regions of the brain, defining regions for song-voice production in vertebrates. Testosterone (T) plays a cyclic role facilitating these changes and can act locally on the brain to prevent neuron loss and regression yet the T mechanistic pathway acting on neuron death and neurogenesis remains grossly uncertain. We address this issue by injecting eight cohorts of Gambler White-Crowned Sparrows (GWCS), that have been exposed to breeding and non-breeding conditions, with T and or/ a potent inhibitor of P13K (an intercellular molecule that indirectly regulates cell growth and apoptosis), Ly294002 (Ly). Information obtained will allow for the determination, if regulation of P13K is required in T mediated signal transduction. We evaluate the data by extracting the bird brain and staining the tissue with Nissl stain to examine HVC and RA regions. With the aid of camera imaging and a volumetric algorithm we then determine HVC and RA volumes with the expectation that Ly inhibits T-stimulated HVC and RA growth. Our results will provide insight to the complex molecular pathway that underlines adult neural plasticity contributing to specifying clinical treatments for neurodegenerative injuries.

Genome-Wide Association Studies (GWAS) determine the places in the genome where there are differences, Single Nucleotide Polymorphisms or SNPs, in genetic sequences of individuals who have a disease as compared to a control group. I used the Human Genome Epidemiology (HuGE) Navigator to analyze the literature on GWAS related to autoimmune disorders.  I then performed a meta-analysis on published literature that had combined multiple GWAS with previously published findings on candidate genes for multiple autoimmune diseases and that had also established shared risk genes. The meta-analysis assisted in extracting not only the statistical methods most often employed, but also biostatistical software used in their analyses.  Using these studies as models, I took sets of autoimmune disorder GWAS and combined that information with previously proposed candidate genes for which studies were lacking.  I statistically and systematically explored possible shared genetic locations to discover any novel and significant associations.  I expect that such a systematic review of existing studies will highlight as yet unseen patterns of shared risk genes when data from two or more autoimmune GWAS studies are compared to previously proposed candidate genes.  The ability to identify shared risk genes is increased by combining data obtained from GWAS of autoimmune diseases.  Although clinically distinct, autoimmune diseases may share common disease pathogenesis mechanisms that may be implicated only by using analysis of genetic factors between diseases.  By further elucidating shared genetic risk factors, this knowledge will contribute to our understanding of genetic epidemiology and will help to define and characterize environmental interventions with the potential to prevent the development of autoimmune diseases.

GPSM2 is required for orienting the mitotic spindle during asymmetric cell division during development. The hearing loss and brain malformations in Chudley-McCullough Syndrome (CMS) suggest that this autosomal recessive condition is due to mutations in GPSM2. The clinical phenotype of CMS includes partial agenesis of the corpus callosum, arachnoid cyst, cerebellar dysplasia, gray matter heterotopia, and frontal polymicrogyria. This combination of brain malformations is highly distinctive and not seen in any other genetic syndrome. Homozygosity mapping and sequencing of affected individuals revealed eight molecular variations in the G protein-signaling modulator 2 gene, GPSM2, that underlie CMS. To identify additional GPSM2 mutations, I utilized a molecular inversion probe (MIP) method enabling minimal DNA input, high output performance, and low-cost sequencing for less than $1 per gene per sample. Each MIP is a common 30 bp linker flanked by an extension arm and a ligation arm giving a total MIP length always equal to 70 bp. The unique arms of each MIP target a specific 112 bp genomic region for a gap-fill and circularization. Post-capture PCR amplification with primers corresponding to the common linker was used to adjoin sample-specific barcodes. Samples were then PCR amplified, pooled together, and ran on Illumina MiSeq. Currently, I am sequencing six confirmed CMS patients in addition to eight samples we obtained from patients presenting only with agenesis of the corpus collosum and hearing loss. These results are pending. By sequencing patients who present with classic CMS features, as well as those who present with limited CMS features, I hope to identify a genotype-phenotype correlation in Chudley-McCullough patients.

Influenza has long plagued the human population, and continues to cause sickness throughout the world. As an enveloped virus that utilizes the process of endocytosis to enter and infect cells, it must fuse its membrane with the host’s endosomal membrane in order to deliver its genetic material to the cell. This specific process is mediated by the viral glycoprotein hemagglutinin. Through my research, I hope to determine the underlying mechanisms of the HA-mediated fusion process. One aspect of the membrane fusion process that remains poorly understood involves the association between HA and the internal structural protein M1 or matrix protein. It has been hypothesized that these work together to keep the virus intact while the host membrane is remodeled. The M1 protein is located between the viral envelope and ribonuclear core, and is believed to be a bridge between these two layers. We hypothesize that the interactions between the cytoplasmic tail of HA and the M1 protein layer in the virus interior are disrupted in a low pH environment. To test this hypothesis, I have isolated and purifyed the influenza matrix protein from whole virus particles. Having obtained a pure matrix protein, I can further study the oligomerization process and start to determine the behavior of the M1 protein in solution. I hope to be able to use dynamic light scattering and gel filtration chromatography to observe the M1-M1 complexes. I will also examine how M1 interacts with membranes of different compositions using lipid flotation gradients and electron microscopy. By understanding the dynamics of M1 secondary structure and HA membrane fusion, my research could help to generate information for rational drug and vaccine targets against the influenza virus.

Barrett’s Esophagus (BE) is an inflammation of the lower esophagus. This inflammation can progress to cancer through a stage called High Grade Dysplasia (HGD). One approach to early detection of HGD is the use of light emitting “beacons “consisting of fluorescently labeled peptides targeted to HGD tissue biomarkers. Our laser based Scanning Fiber Endoscope (SFE) possesses the ability to capture live images of the esophagus during examinations and also excite and locate fluorescently tagged targets. Refining SFE fluorescence imaging for clinical use requires mathematical algorithms such as image stitching and distance compensation. Image stitching combines a series of partial views of a 3-D surface into a composite view of the entire surface. Distance compensation addresses the issue of fluorescence signal reduction as the distance between the light emitting beacon and endoscope increases. Phantoms are synthetic mimics of disease conditions used to refine and calibrate new medical devices. This research was directed toward the development of a low-cost, flexible, and durable esophagus phantom with color matching to provide a realistic model of BE. The color of inflamed BE tissue and healthy tissue were matched using quantitative combinations of commercial acrylic paints to create recipes for both tissue types. The resulting paint mixtures were applied to the interior of a latex cast cylinder mimicking the dimensions of an adult esophagus. The painted latex models were shown to a panel of gastroenterologists for approval. The end result was the development of a color realistic phantom onto which fluorescent targets could be affixed. The phantom was imaged using the SFE and demonstrates 2D stitching with distance compensation. (C. Yang, V. Hou, L. Nelson, E. Seibel. J. Biomed. Optics, Feb 2013) Future work extends similar techniques to develop brain tissue phantoms for the calibration of the SFE after integration onto the RAVEN surgical robot.

Aging in the budding yeast Saccharomyces cerevisiae can be measured by its replicative life span, which is defined by the finite number of mitotic divisions of mother cells. The Kaeberlein lab measures the replicative life span of S. cerevisiae through the microdissection of dividing mother cells. This laborious process separates daughter cells from their mothers, which quantifies the replicative life span. We are assessing how various environmental factors affect the replicative life span of the cells. Our research project involves paraquat, a commonly used pesticide that produces reactive oxygen species within cells. This substance is normally considered to be toxic, and as expected, we have observed that the replicative lifespan of yeast is decreased in the presence of high concentrations of paraquat. However, we have also found that low concentrations of paraquat increased the replicative life span of the yeast. This observation is an example of hormesis- a phenomenon where beneficial effects are obtained at low doses of an agent that is otherwise toxic when given at higher doses. These findings are significant because a similar effect has been published with the nematode worm, Caenorhabditis elegans. C. elegans and S. cerevisiae are evolutionarily separated by approximately one billion years, suggesting that there is a conserved mechanism of life span extension influenced by paraquat. We are currently characterizing the effect of paraquat and other environmental conditions that will help us to find conserved pathways that modulate longevity.

Paternal effect mutations provide experimental tools to identify the unique contributions that fathers provide to their offspring. We are studying the ddbt (deadbeat) gene of Drosophila melanogaster, which encodes a chromosomal protein that is incorporated into spermatid nuclei during spermatogenesis and is required for normal embryogenesis. Embryos fathered by ddbt mutant males have extensive chromosome damage and embryogenesis terminates prematurely. This study asks whether ddbt-induced damage is a consequence of improper mitotic chromosome segregation in the embryo, or if the damage is independent of chromosome segregation. To address these alternatives, ddbt males will be mated to females carrying the giant nuclei (gnu) mutation. This maternal effect mutation permits repeated rounds of DNA replication but prevents chromosome segregation in the embryo. If DNA replicates to the same extent in embryos produced by gnu mothers and ddbt fathers compared to those of control fathers, this would suggest that ddbt-induced breaks occur during chromosome segregation. The alternative is that the chromosomes of ddbt fathers have preexisting damage, in which case replication would arrest due to preexisting DNA breaks. We are examining nuclear size and morphology in experimental and control embryos using confocal microscopy and assay for chromosome damage using techniques that detect breaks in DNA. Investigation of ddbt promises to improve our understanding of the paternal contributions to embryonic development.

Diatoms are major players in Earth’s biogeochemical cycling, accounting for approximately 20% of global primary production. Their dominance in several aquatic ecosystems is a testament to adaptations that provide an advantage in nutrient acquisition and growth. Ocean fertilization experiments have demonstrated that iron is the primary limiting nutrient in many open ocean ecosystems. This metal is critical to the functioning of numerous important proteins, particularly in photoautotrophs such as diatoms. An excess of intracellular iron, however, can cause oxidative damage to the cell. This project investigates genes related to iron metabolism in the recently sequenced transcriptomes of 21 diatoms and select outgroup species. Of particular interest are genes coding for ferritin subunits. Ferritin is a multimeric protein that serves as an iron storage unit, safely sequestering iron atoms in an oxidized state and releasing them back into the cytoplasm when iron supply is low. Ferritin in diatoms was previously thought to exist only in pennate diatoms. Here we show that ferritin-coding genes are expressed in members of every major diatom group. 13 out of 21 diatom transcriptomes contain full-length sequences, forming a clear monophyletic clade most closely related to cyanobacterial ferritins. Additionally, 7 of the 21 transcriptomes surveyed reveal two distinct ferritin genes, possibly due to an ancient duplication event. The presence of ferritin helps to explain the success of some diatom species in iron-poor waters, and our expanded understanding of which diatoms possess ferritin genes will help to predict the responses of this important group to a dynamic ocean environment.

Seabird distribution and abundance can be used as indicators of the health of the coastal ecosystem. In the Gulf of Alaska over the last four decades there has been growing evidence of climate change and ocean acidification. The largest oil spill in the United States, the Exxon Valdez, occurred here. We used data from the Coastal Observation and Seabird Survey Team (COASST), a citizen science, beached bird survey program with 14 years of surveys from beaches in the Pacific Northwest, and eight years of data from Alaska. We seek to compare U.S. Fish and Wildlife Service (USFWS) data from historical surveys in the Gulf of Alaska (1970-1989), to the current beached bird data collected through the COASST program (2005-present). Historic data include monthly surveys of sites from the Kenai Peninsula to Yakutat Bay, and one offshore island, Middleton (n>250 surveys). Current COASST data cover this same range, include additional surveys from Kodiak Island, but exclude Middleton (n>1200 surveys). These data will be analyzed to examine the differences in monthly encounter rate (carcasses/km) averaged over this region, cumulative species diversity, and oiling rate (percent of recorded carcasses that are oiled). This comparison will provide insight into possible changes in the encounter rate index in the Gulf of Alaska over the past 40 years.

Lily blooming has recently been suggested to result from differential growth of the petals. The tissue expands along the edges, making them longer than the midrib. To accommodate for this excess length, the petal reverses its curvature, causing the bud to open. This is the growth driven mechanism for lily blooming. We inquired into the shape of the ripples resulting from edge growth and if we could observe direct evidence of the growth. We first investigated rippling, another consequence of edge growth. When the growth rate is high enough, lily petals ripple and their edges adopt their characteristic wavy shape. Using time-lapse imaging, we observed lily petals' edges rippling. We monitored the formation of several generations of ripples, with a fractal-like pattern of smaller waves superimposed onto larger ones. We also measured the temporal evolution of the ripples' wavelength, amplitude and arc length. We found that these quantities could be related using a geometric argument. Agreement between our prediction and observations is excellent. This indicates that in addition to biology, physics plays a significant role in determining the shape of lily petals. Second, we sought to quantify edge growth. We took quantitative measurements of the different growth rates, by tracking the positions of marks along the edge and along the midrib. We found that the growth rate is 35% larger along the edge than along the midrib, supporting the edge growth driven blooming model. We now want to measure the growth rates of different lily species, and investigate whether smaller growth rates can correlate to the absence of ripples. The purpose was to understand the 3D shape resulting from growth in a 2D sheet to supply knowledge leading to understanding plant development. It will also lead to applications in the packaging industry where flat shapes are made into 3D objects.

This action-research project describes a particular student-lead model created to foster community and awareness of global health across campus. We, the student leaders, who form the Global Health Undergraduate Leadership Committee (GHULC), have shaped this model to best respond to an ongoing challenge of engaging all undergraduates in awareness and community building around global health issues. The model for this approach involves bimonthly meetings with the committee and two advisors in addition to attending events planned by the GHULC and the Department of Global Health. Methods we have taken to increase undergraduate involvement in the minor include regular information sessions about the minor, monthly student-planned Community Connections events on critical issues, a joint student- and faculty-run journal club, peer advising, walking tours of Global Health organizations in Seattle, and a GHULC blog. Preliminary quantitative data suggests that there has already been an increase in the number of students enrolled in the minor this year as well as an increase in the academic diversity of participating students. Our next step will be to develop a qualitative assessment of the GHULC’s impact by administering a feedback form to all Global Health minor students regarding their perception of and involvement in the minor. We intend to gather this information on a yearly basis in order to improve the minor as well as the GHULC’s role with the goal of continuing to increase diversity, engagement, and mentorship.

GCS1 is a protein that plays a crucial role in angiosperm plant fertilization. Surprisingly, comparative studies show that GCS1-like proteins exist within a wide variety of eukaryotic species. In Drosophila, the protein Pskl is predicted to have a GCS1-like domain that affects male gametes, but its exact role in sperm formation and sperm function is not well characterized. Studying Pskl is imperative towards broadening our understanding of sexual reproduction in eukaryotes. Comparisons of Pskl to its homologs in angiosperms, algae, and protists may provide insight into mechanisms of fertilization that are evolutionarily conserved across diverse species. Furthermore, defining how the protein works could contribute to the development of reproductive therapeutics or drugs that inhibit the procreation of illness-causing organisms. The goal of this research is to understand how Pskl regulates fertility by defining its functional domains. Through gene deletions generated by imprecise excisions of a transposable element called MiMIC, analysis of both the molecular and phenotypic consequences can improve our understanding of sequences necessary for normal pskl expression. If male flies with the chromosomal derivative are sterile or have reduced fertility, then removed sequences flanking the MiMIC insertion site are likely crucial for successful fertilization. Alternatively, generated deletions may extend in either or both direction of the insertion site and impact viability, shedding light on whether pskl or its adjacent genes are essential for development. In this report, we describe the nature and consequences of the transposon excisions in pskl, both molecularly and phenotypically.

The Kiss1 gene encodes a neurotransmitter whose expression in the brain is essential for reproduction. Previous studies with in situ hybridization and immunocytochemistry have identified Kiss1 expression in several hypothalamic nuclei, including the arcuate. To determine whether Kiss1 expression is limited to the hypothalamus, we developed a transgenic mouse that expresses a marker gene (tdTomato) if and only if Kiss1 expression is turned on. tdTomato encodes a red marker protein that is constitutively expressed, labeling the cell forever as having once expressed Kiss1. We looked for tdTomato labeling in brain sections to identify areas that reveal expression of Kiss1 at some time during development. Surprisingly, we found tdTomato labeling outside of the hypothalamus. Possible causes include Kiss1 expression early in development or low-level Kiss1 expression in adulthood. Distinguishing between these possibilities has important implications for developing tools to genetically manipulate Kiss1 neurons in adult animals. We will test the hypothesis that Kiss1 is widely expressed at low levels in the adult brain. Kiss1 transcription will be mapped in two ways. First, brain sections will be examined for the presence of green fluorescent protein (GFP). GFP is expressed under the Kiss1 promoter, but it may not mark low levels of Kiss1 transcription. Therefore, Kiss1 expression in adulthood will also be tested by in situ hybridization. In situ hybridization radioactively marks Kiss1 mRNA, illuminating even low levels of transcription. Comparing in situ hybridization between wild-type and Kiss1 knockout mice may reveal low levels of Kiss1 mRNA outside the adult hypothalamus— and open new possibilities for a physiological role of Kiss1 signaling that extends beyond reproduction.

Aging is a process characterized by the degeneration of bodily components and functions. Scientists are limited in their ability to study this process in humans, in part because a human lifespan is too long to conveniently study. Instead, model organisms are studied extensively in the hope that discoveries made will be applicable to another organism of interest. Model organisms are usually short-lived, easy to manipulate, and easy to handle. One of the best examples of a model organism is the budding yeast Saccharomyces cerevisiae. S. cerevisiae lifespans are easy to measure and reproduce, and since they have a short lifespan we can study a large amount at one time. Aging in Saccharomyces can be measured in two different ways. A yeast cell can be kept in a non-dividing, nutrient limited environment and tested to see if it can successfully re-enter the cell cycle, when it cannot, its lifespan has ended. This method is used to measure the chronological lifespan, and the model can be applied to human cells that are no longer replicating (post-mitotic cells). There is another model we can use to understand a different aging process, called the replicative lifespan. The replicative lifespan refers to the number of times a mother cell can divide to produce daughter cells, and is a much better representation of cells that are still dividing (mitotically active cells) in other organisms. As undergraduates, we help study this process by physcially separating daughter cells from the mothers using fiberoptic needles (dissecting) and counting them at regular intervals. When the mother cell no longer produces daughter cells, the replicative lifespan has ended. The data collected can be compared to past and expected results in order to help us gauge how well the cells are dividing. By manipulating the environment that each cell line is grown in, we can gain insight towards specific drugs and conditions that mother cells divide best in, and apply this to other mitotically active cells in other organisms. Both measurements of lifespan can be manipulated in similar ways, and many labs have started to establish a bank of genes and environmental factors that can be manipulated in order to change the outcomes.

Ischemic preconditioning (IPC) refers to the neuroprotective phenomenon in which a brief ischemic exposure in the brain increases resistance to injurious effects of subsequent prolonged ischemia. Ischemia is the restriction of blood to tissues. Interferons (IFNs) are cytokines released during ischemic exposure to activate the Interferon alpha receptor (IFNAR)  complex and trigger the transcription of interferon stimulated genes (ISG). ISG gene products may contribute to IPC-mediated neuroprotection. However, IFN receptors are expressed on myeloid cells as well as neural cells; the cell type-specific contribution of IFN signaling in IPC is not established. Microglia are the resident immune cells in the brain and are central in the inflammatory responses to stroke. Data from our lab strongly suggest that microglia exposed to ischemia both in vitro or in vivo demonstrate a robust expression of ISGs. We hypothesize that expression of IFNAR1 and intact IFN signaling on microglia is required for IPC mediated neuroprotection. Thus, the administration of IFNβ prior to stroke will mimic IPC in the wild type (WT) but not in myeloid cell targeted IFNAR1 (Cre/loxP) knockdown mice. We obtained IFNAR1fl/fl strains through collaboration and purchased LysM-Cre. We then used selective breeding and PCR genomic strategies to generate mice that are combined for homozygous IFNAR1fl/fl and heterozygous for LysM-Cre and wild type (WT) genes. We are also currently optimizing our method to administer IFN β using a stereotactic injection system through the lateral ventricle of the brain. We have recently confirmed the appropriate positioning of the intracerebroventicular (ICV) injection and plan to carry out ICV injection of IFN β on the WT and IFNAR1fl/fl /LysM-Cre strains. After inducing stroke, we will quantify and compare the infarct volume and neurobehavioral outcomes. This study will demonstrate the cell-type specific contribution of microglia in IFN-mediated neuroprotection. If successful, our findings can be used to develop pharmaceutical therapies for stroke and gene therapy remedies.

Approximately 30% of patients who receive standard therapy for Acute Myeloid Leukemia survive the treatment, but do not enter complete remission. These patients are classified as resistant to the first course, and it must be decided if they should receive a second course of standard therapy or switch to a new therapy in a clinical trial. The complete remission rate produced by a clinical trial is by definition unknown, so the probability of a patient achieving a complete remission with a second course of standard therapy must be used. In this study, we aim to determine which factors can be used by physicians to predict if a patient will enter complete remission with a second course of standard therapy. Medical records from three separate clinical trials conducted by the Southwest Oncology Group were analyzed to find patients who received two courses of standard therapy. Specific information such as blast percentage and marrow cellularity before and after treatment was then recorded. A multivariate analysis of this data will be performed to determine which factors can predict response to the second course of standard therapy. This information has the potential to be used in making the decision to treat patients with a second course of standard therapy or to switch to a new therapy in a clinical trial.

Individual bees often show flower constancy, specializing on a floral resource while avoiding switches to newly available resources. The familiar plant species benefits by receiving high rates of visitation even when competing floral resources become available. However, the phenology of some plants is shifting more rapidly than others in response to climate change, resulting in a changed sequence of floral resources bees encounter. To determine whether this affects plant fitness, we asked how pollinator visitation to competing plant species depends on the order in which those species bloom. We used two plant species with overlapping phenology and a common pollinator, Hypochaeris radicata and Campanula rotundifolia. We allowed Bombus impatiens to forage in floral arrays representing three phenological progressions: (1) CR Early, with C. rotundifolia decreasing from 100% to 0% while H. radicata gradually increased from 0% to 100%; (2) HR Early, with H. radicata decreasing from 100% to 0% while C. rotundifolia gradually increased from 0% to 100%; and (3) a Control with both species at 50% throughout. We quantified visitation rate to both species, and seed production by H. radicata. Overall, the number of bee visits received by both species was significantly higher when that species was introduced first than when it was introduced after its competitor. In treatments where the proportion of the early species decreased over time, the visitation rate per flower to that species increased, for both species and across all stages of experimental treatments. Seed set for H. radicata showed a similar pattern to visitation. Our results corroborate other work suggesting that individual bees learn and become specialized on a single floral resource and are slow to switch to a newly emerging resource. As a result, the flower species that blooms first can benefit from higher bee visitation and thus, potentially higher reproductive success.