Climate change is a crucial factor that affects plant physiology, population dynamics and geographic ranges. Little is known about the effects of changing climate on conifer morphology, whether their key morphological structures have the ability to change in response to environmental differences and what the impact of these changes mean for future ecological population dynamics. Are morphological and physiological traits locally adapted within a species or do they acclimatize to specific environmental conditions? Two studies were conducted to test 1) whether morphological structures of conifers differ between source populations when transplanted across an elevation gradient and 2) whether photosynthetic levels under a light treatment differ between source populations. Tsuga mertensiana seedlings from three study sites, varying from high to low elevation on Mt. Rainier, were each transplanted across and beyond the species' elevation range. For the first study, measurements of shoot to root ratio, specific leaf area (SLA) (m²/g) and leaf mass vs. stem mass were calculated. We expect our results to show that as elevation increases, SLA and shoot to root ratio decrease, however overall values are smaller for trees at higher elevation. This indicates that within a single species, trees from distinctive ecotypes are inherently different morphologically, even though all seedlings have the ability to acclimate to changes in elevation. The second study manipulates light as a single variable and addresses whether the driving force of morphological and physiological characteristics are due to light levels at different elevations or whether T. mertensiana seedlings at different ecotypes are genetically different physiologically.

Levels of atmospheric carbon dioxide have been steadily increasing for decades, sparking the interest of many. In response, researchers have been studying the various components of the carbon cycle, including bodies of water as they act as both significant sinks and sources of carbon dioxide. This study examines the correlation between various water quality indicators and CO₂ flux within the Cedar-Sammamish watershed. At each site, CO₂ flux was measured and logged using an IRGA (Infrared Gas Analyzer) connected to a Plexiglas chamber dome floating on the surface of the water. Dissolved oxygen (DO) levels and pH were measured using Hach DO and pH meters, and water quality was measured using a field spectrometer.  Meteorological data and visual observations were also recorded.  Data was then analyzed for trends.  Preliminary data indicated a positive correlation between nitrate levels and carbon dioxide efflux.  Subsequent data has not necessarily followed this trend, indicating that other factors may be at work.  Understanding the various trends and possible predictors of CO₂ flux is important, as it will help create better models for global carbon cycling.

This study examines the effects of administering hydrogen sulfide (H2S) to ancient plant species to examine the organisms’ response to the stress. While sulfur is an essential macronutrient required by several organisms for growth and productivity, there are many toxic variants of the element that have detrimental effects and produce physiological stress. It is speculated that the accumulation of H2S, a lethal gas, may have existed as a major contributing factor in past mass extinction events, where the environment was fairly anoxic with fluctuating temperatures. The potential of this toxic variant to exist as an environmental stressor suggests that certain organisms may have adapted to survive these periods of mass extinctions. It is hypothesized that due to the abundant presence of hydrogen sulfide in the past, ancient land plants may have an adaptive advantage that allowed them to survive and thrive. In the study, species of bryophytes, specifically Hypnum and Dicranum are exposed to specific concentrations of aqueous H2S over a 7 day period and measured for their photosynthetic capacity at timed intervals using a FluorCam. Studying the effects of this toxic gas on ancient plants is imperative to our understanding of sulfur’s varying biological roles, and provides insight on the evolutionary phenotypic variations amongst plants and stress responses in order to survive mass extinctions. Current results indicate that bryophytes are able to tolerate significant quantities of the toxic substance and show resilience through increased photosynthetic capacity over a period of exposure, indicating a genetic and phenotypic legacy response. The results also imply that H2S may have the potential to act as a plant regulator due to its diffusive properties and influence in minute quantities.

Communication is prevalent and necessary in all social animals. It determines outcomes of competition for mates and food resources, and also allows for social organization. A wide range of communication strategies are used across the animal kingdom, but for primates - one of the most social groups in the animal kingdom -there are three main strategies for communication: auditory, visual, and olfactory. For my study, I focused on the question of how group size affects amount of communication within a group. I hypothesized that with larger group size comes a need for more direct communication, in order to maintain cohesion in a group with many individuals. Therefore, I predicted that a larger group would result in more complex vocalization usage in a primate which relies largely on vocalizations to communicate. Using the genus Eulemur as a study system, I investigated if vocalization complexity is related to social group size. Eulemur rubiventer and Eulemur rufifrons are an ideal system for this work, because they share similar habitats and predation risk, and are closely related, but differ in their social group size. I studied the species for a month in the primary forest of Ranomafana National Park, Madagascar, following and watching individual animals and continuously documenting their behaviors and vocalizations. I also used a microphone to record vocalizations to get better data on the different types of calls. I found that the species with larger groups (E. rufifrons) called 88% more often than the small grouped animals (E. rubiventer), and consistently used 4 more calls than the smaller grouped species. This preliminary evidence suggests that increased vocalization frequency and use of different calls are associated with higher sociality. I postulate that this increase in vocalization usage complexity is necessary to maintain cohesion in a large social group.

Antibiotic resistance is a challenging problem in the realm of human health today. As a fundamental evolutionary phenomenon, it provides an excellent system to test adaptive evolutionary theories in a short amount of time. Thus, I explored the rise of antibiotic resistance in two evolution experiments with Escherichia coli. In the first, I asked how differences in genetic background affect the rate of adaptation to a new environment. To address this question, I generated 19 unique antibiotic resistant mutants by evolving E. coli in an environment with low drug concentration, and then exposed thousands of populations of each isolate to a high concentration environment measuring the proportion of populations that survived. Since survival in the high-drug environment requires acquisition of a new beneficial mutation, I hypothesized that high mutant survivorship indicated an isolate with more mutational options available in the high drug environment (we call this “potentiation”). Isolates with low survivorship would have fewer available mutational options (we call this “doom”). Interestingly enough, some observed differences may be due to a phenomenon known as sign genetic epistasis – when mutations have different effects in combination than individually. Preliminary data suggests a few strong cases for potentiation. I also investigated the effect of an initial resistance mutation on the consequent adaptations in a no drug concentration environment. To study this, I generated 23 unique mutants of E. coli and tracked how fitness and drug resistance changed over time in 2,200 populations. Both of these experiments help illuminate the role of historical contingency in evolution, the idea that an initial mutation may dictate an organism’s future mutational trajectory (available mutational options). Therefore, understanding both the effect of initial mutations and the underlying mechanisms of mutation acquisition is necessary in order to combat the emergence of resistant bacteria, a current human health concern.

In the San Juan Islands, drift macrophytes from shallow waters represent a significant spatial subsidy provided to the subtidal zone. This organic matter is a potential food source for deep dwelling herbivores like the red urchin Strongylocentrotus franciscanus. Little is known about how urchins capture and use drift material. A racetrack flume was used to examine the capture of drift macrophytes of various species and size by S. franciscanus. No species-specific selectivity was found, but urchins caught 100% of small macrophyte pieces. A significant decrease in capture efficiency was noted as drift algal size was increased, probably due to the urchins’ inability to fight current drag acting on pieces with larger surface area. This suggests that drift macrophytes could be the main food source for deep dwelling urchins where available, potentially explaining how herbivores subsist at dysphotic depths. 

The mammalian cortex is composed of excitatory and inhibitory interneurons which regulate the firing of action potentials in the brain. Excitatory neurons increase the likelihood of an action potential in other cells while inhibitory neurons decrease this probability. Excitatory transmission develops first, resulting in electrical activity in the form of waves, which are important in the formation of neuronal circuit formation until the development of inhibition shuts down this spontaneous activity. While excitatory and inhibitory neurons are born in different locations in the brain, those that are born on the same day of development migrate and find each other in the same cortical layer. Excitatory neurons originate in the dorsal ventricles and migrate radially towards the plial surface. Inhibitory cells originate from the medial ganglionic eminence and migrate tangentially to the cortex where they then migrate radially to find their correct cortical layer. This project explores the idea that spontaneous electrical activity helps drive this organization. Tbr1 is a transcription factor shown to be important in neuronal layer finding. By using a Tbr1 KO (knockout) mouse, we show the disorganization of the neocortex compared to normal mice by quantifying the fraction of radially oriented interneurons versus non-radially oriented interneurons. Further, we examine the implication that waves and asynchronous electrical activity play a role in layer finding of excitatory and inhibitory neurons, as demonstrated by the differences in activity between Tbr1 knockout mice and controls.

Soil moisture is a variable that is difficult to quantify due to its large spatial heterogeneity, its lack of extensive ground-based measurement networks, and its strong dependence on many meteorological and biological factors. However, records of soil moisture are essential for developing more accurate climate models and providing constraints on processes controlling plant productivity and heat fluxes. My research aims to further develop these records by determining whether maximum temperatures can be related to the amount of soil moisture in a particular region. Previous studies have shown that plant transpiration accounts for more than 80% of evapotranspiration, the sum of evaporation and plant transpiration from the Earth's land and ocean surface to the atmosphere. We hypothesize that the variability of maximum temperatures in vegetated regions is determined by the amount of soil water available there. By analyzing latent heat, temperature, and soil moisture data collected from various flux measurement towers around the United States, we found that days with relatively low latent heat flux values occurring at high maximum temperatures have statistically drier soils compared to days where high latent heat flux values occur at high maximum temperatures. This relationship seems consistent only when the site under analysis is located in a wet environment that is rarely water limited.

Spontaneous neural activity (SA) during brain development has been implicated in processes ranging from regulation of gene expression to development of neural networks that ready organisms for post-natal life. Though SA is a well-known phenomenon, the mechanisms and developmental processes mediating its behavior are not well understood. This research aims to elucidate some of these processes. Preliminary findings have focused on defining dopamine’s (DA) and serotonin’s (5-HT) role in spontaneous activity in the developing mouse brainstem. It has been shown using transgenic mice expressing markers for proteins that interact with DA or 5-HT that SA propagates through regions containing these markers. I am now expanding this work by performing calcium imaging and confocal microscopy along with whole cell patch clamp in these transgenic mice to characterize SA in regions where the markers are highly concentrated. In addition, I have been working with a colleague from the Phillip’s lab, using voltammetry to try and record the extracellular release of DA. These experiments will establish whether 5-HT cells act as drivers for SA and if such activity can trigger DA release. The implications of this research are multifaceted. Primarily, it will expand on the anatomy and physiology of the developing dopaminergic and serotonergic pathways in the mouse brain. Second, its findings may shed light on pathogenesis of certain disorders involving these pathways by contributing to the understanding of how SA mediates their development.

Colicins are a family of antimicrobial toxins that are produced by E. coli. The colicin gene is carried on a small circular DNA molecule, called a plasmid, along with a gene encoding an immunity protein. The immunity protein binds and neutralizes the antimicrobial colicin in a highly specific manner. Bacteria lacking the plasmid (and thus lacking immunity) are killed by the colicin. The immunity and the colicin are mutually dependent on one another, creating a system that seems unable to evolve. If either changes then they will no longer fit together, which in this system would result in the death of the cell by its own colicin. However, evidence shows that there are many different pairs of colicin and immunity proteins that share common ancestors, suggesting that diversification has occurred. How is this diversity possible if the system seems evolutionarily trapped? Our research looks at the idea of promiscuous protein intermediates. Under this hypothesis, the immunity changes first so that it can bind both its original colicin as well as another distinct colicin protein, we then attempted to recreate this possible evolution pathway in lab by evolving our own colicin-immunity pair. Using directed evolution we first evolved a “broadened” immunity protein then a matching colicin through error-prone PCR, which created a diverse collection of mutated colicins. Then we screened for matching colicin-immunity pairs. In pursuit of our novel protein, we generated and screened over 6000 colicins and have successfully isolated two potential novel colicin proteins.Through sequencing analysis, we know that the two potential novel colicin genes each have two nonsynonymous mutations.

The plant genus Thalictrum (meadow-rues) contains numerous shifts in ploidy number, ranging from diploid (2x) in the earliest diverging species to 24x in recently derived species. Polyploidy can result from a whole genome duplication event within an individual (autopolyploidy) or following a hybridization event (allopolyploidy). The rates and evolutionary consequences of these two mechanisms of polyploidy are currently under debate, and I am therefore attempting to estimate their relative roles within this genus using a phylogenetic approach. The current Thalictrum phylogeny relies on chloroplast DNA and nuclear ribosomal regions, which tend to underestimate the role of hybridization due to maternal inheritance and concerted evolution patterns, respectively. I am using single-copy nuclear genes to uncover potential hybridization signals in Thalictrum’s evolutionary history. This information will be used to compare the relative numbers of autopolyploids and allopolyploids in the genus. I hypothesize that allopolyploidy played a larger role in shaping the diversity of this genus, due to its potential for differential survival and reproduction as a consequence of immediate, novel gene expression in hybrid progeny. Furthermore, this analysis will estimate the hybrid origins of particular alleles within a species. In the future, this information will be applied to questions on the evolution of genes involved in flower development after whole genome duplications.

Stickseed (Hackelia) is a genus of plants comprised of about forty species distributed primarily across the dry forests and mountains of interior western North America. This study seeks to build an evolutionary tree of Hackelia using DNA sequences from both the chloroplast and nuclear genomes. Preliminary analyses suggest possible conclusions regarding their geographic history and the evolution of their fruit characteristics. While it is currently unclear where the genus started, it appears there were two major migrations; one started in Washington and moved southward into Mexico, while another started in the Midwest and traveled southwest into California, Mexico, and Peru. Consequently, California is a diversity hotspot for this genus. Hackelia, like the rest of its family, has a fruit that divides into four nutlets at maturity. However, Hackelia is unique from other genera; it possesses elaborate prickles on its nutlets. These prickles can be described using two physical traits: whether the prickles along the border of the nutlet (marginal prickles) fuse to form a cup-like border, and if there additional prickles between the marginal prickles (intramarginal prickles). Our results suggest that ancestral species had nutlets with a cup-like border of marginal prickles and obvious intramarginal prickles. Most species of the genus have since lost the cup-like border, resulting in free marginal prickles. On the other hand, while most species have retained their intramarginal prickles, at least three lineages spread across the tree have lost them. This study will help us understand how plants have diversified in western North America, as well as how physical traits can be evolutionarily variable or conservative. 

Pollination syndrome refers to suites of floral traits that influence biotic or abiotic vectors transporting pollen from one flower to another. These traits typically include flower color, position, shape, size, scent and reward. Scent is a major component of pollination syndrome, as the volatile organic compounds (VOCs) that compose “scent” often attract pollinators or deter predators. The genus Thalictrum in the Ranunculaceae (buttercup family) includes both insect- and wind-pollinated species, enabling comparisons of the role of scent in floral diversification among recently diverged taxa with distinct pollination syndromes. To characterize floral scent, we identified and compared the bouquet of VOCs emitted by insect- and wind-pollination species, as well as by floral vs. vegetative tissue. Scent was collected from tissue samples by traps in a filtered air system, then eluted and analyzed with gas chromatography mass spectrometry. Quantitation and emission rate were calculated to generate floral bouquets. Electroantennograms were performed on selected compounds to examine pollinator response. Focusing on eleven species representative of the two pollination syndromes and spanning the Thalictrum phylogeny, we identified ten key compounds of which five are deterrents against herbivory, two are pollinator attractants, and three have unknown functions. Given that these scent profiles consist primarily of defensive rather than attractive compounds, we hypothesize that repeated transitions from insect- to wind-pollination in Thalictrum may be due to the breakdown of an ancestrally weak insect pollination syndrome. Moreover, scent bouquets appear to follow phylogenetic patterns more so than pollination mode. The variation in distribution of VOCs across species in a phylogenetic framework allows us to test evolutionary patterns of compound co-option during evolution.

Pollination is often facilitated by insects and animals guided to flowers by a mixture of volatile compounds: the components of floral scent. As an attractive cue for pollinators, scent emission may be highly regulated so that its release coincides with the activity of its preferred pollinator. This relationship is particuarly evident in the release of benzenoid volatiles by Petunia, through which it attracts its pollinator, the nocturnal hawk moth (Sphingidae). Though Petunia release volatile benzenoid compounds nocturnally through a process mediated by the circadian clock, the molecular mechanisms responsible remain uncharacterised. We hypothesise that the transcription of the ODORANT1 (ODO1) gene in Petunia, which encodes a key transcription factor involved in regulation of volatile benzenoid synthesis, is regulated by an ortholog of the highly conserved Arabidopsis core-clock genes - CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) - in Petunia. To investigate this potential association, we have characterised the Petunia ortholog of LHY (PhLHY) and will look to analyse its influence on volatile benzenoid production. We aim to understand the interaction between the PhLHY protein and ODO1, and will investigate the intracellular localisation of PhLHY as well as the tissue specific expression of ODO1. We anticipate that PhLHY will influence the rhythmic oscillation of the circadian clock, as well as influence the synthesis and emission of floral volatiles by directly regulating ODO1. In elucidating the mechanisms by which the circadian clock control scent emission, we will likely be able to generate floral varieties that display significant chronological shifts in their daily scent emmision regimes. 

 The first floral mutations ever documented, double flowers have been prized for centuries for their beauty. The double mutant variants ‘Shoaf’s Double’ and ‘Cameo’ are found in Thalictrum thalictroides, a member of the buttercup family. The purpose of this research is to determine the genetic mutation underlying this desirable phenotype. We predicted that a mutation in a C-class floral organ identity gene would cause the homeotic transformation (conversion of one organ type to another) of stamens and carpels to petalloid sepals in this species. C-class genes are also involved in floral determinacy, the termination of floral organ growth, which is why double flowers appear to have a second flower nested inside the first. As expected, the expression of the C-class gene ThtAG1 was greatly reduced in our mutants. Finding no changes in the coding region from wild type, we further investigated the entire genomic locus, where we found evidence of the action of a transposable element in both mutants. In ‘Shoaf’s Double’ a Mutator Like Element (MuLE) has inserted itself into a regulatory intron of TthAG1, disrupting its function. In ‘Cameo’ a ‘footprint’ at the exact same place in the sequence indicates that the same element may have jumped out, deleting a section of the intron. We hypothesize that the double phenotype in ‘Cameo’ is likely due to the accumulation of mutations before the MuLE jumped out. Since the MuLE’s transposase is expressed in ‘Shoaf’s Double’ (but not in wildtype), these results illustrate the rare case of an autonomous transposable element underlying a regulatory mutation that results in a visible phenotype. We hope that our work will provide insight into the genetic basis for the double phenotype of popular commercial flowers such as roses, camellias and carnations.

In the model organism Arabidopsis thaliana, the B-class genes of flower development are responsible for proper formation of petals and stamens in a flower. Studies in Thalictrum thalictroides, a representative of the sister lineage to all other eudicots provides insight into the ancestral function of the B-class genes. Unlike Arabidopsis, the flowers of T. thalictroides lack petals, and yet the sepals are large and colored similar to petals. Here, we investigate the function of B-class proteins in T. thalictroides through the study of the horticultural mutant known as ‘Betty Blake.’ The ‘Betty Blake’ mutant phenotype consists of many non-petaloid (green) sepals and carpels but no stamens. We have previously shown reduced expression of most B class genes in ‘Betty Blake’, yet we were not able to find any meaningful mutations in these loci. Therefore, we hypothesize that an upstream regulator of the B-class genes may be mutated in ‘Betty Blake’. UNUSUAL FLORAL ORGAN (UFO) is a known upstream regulator of the B-class genes and is therefore a logical candidate. We hypothesize that the UFO mutation is the cause of the B class phenotype in ‘Betty Blake.’ The investigation of B-class function in this non-core eudicot has provided us with insight into the evolution of gene regulatory networks affecting ectopic petaloidy and stamen identity that we will continue to pursue in future experiments.

Neurons are specialized cells that receive and relay information throughout an organism, and the compartment that inputs the signal has multi-complex structures known as dendrites. The form of the dendrite is tightly regulated to neuron function; therefore, any abnormality that alters the structure of the dendritic arbor can lead to severe defects in neuronal function. However, the mechanisms of how dendrites develop and maintain their arborization are poorly understood. One mutant allele, Acsl (Acyl-CoA synthetase long-chain), leads to a complete loss of terminal dendrites, which carries a two amino acid change that renders the gene product non-functional. Interestingly, Acsl is also associated with human X-linked mental retardation (XMR). However, to date, little is known about the signaling pathway that may be involved in this unique function of Acsl in neurons. As such, we conducted a modifier screen, which serves to identify functional modulators with genetic interaction of Acsl in maintenance of terminal dendrites. Here we report our results of funcional signaling (and/or) regulatory genes from this screen. We also conducted comparative GC/MS of the lipid profiles in both whole larval body and brains to identify the specific lipid species being affected by acsl mutation. In return, this will reveal to us more about the function and signaling pathway of Acsl in maintenance of terminal dendrites. Results from this project will lead to a greater understanding of dendrite abnormalities in neuronal development and of neurodegenerative disorders.

Waves of synchronous spontaneous electrical activity propagate throughout the cerebral cortex of the brain during early stages of development. These waves consist of simultaneous action potential firing across large cell populations, with correlated increases in cellular calcium levels. Specific types of calcium signals mediate different developmental processes in the brain. While calcium signaling is known to regulate activity-dependent developmental programs that help to form essential connections between neurons, the particular function of spontaneous waves in cortical development is unclear. As many types of neurons participate in these waves in the cortex, it is possible that waves serve developmental roles that differ among neuronal subtypes. In this study we investigate the roles of spontaneous waves in the development of one neuronal subtype – inhibitory interneurons. At the time of wave expression, the last interneurons are migrating into the cortical plate and finding their appropriate layer within the cortex. To label inhibitory interneurons we use red fluorescent protein (RFP) under control of the dlx5/6 promoter region to label these cells, and a green calcium indicator to measure single cell activity of these interneurons. By imaging the red and green wavelengths and then analyzing the data, I have found two types of cortical interneurons: (1) Those that participate with other neurons in spontaneous waves; this type of activity is insensitive to blockers of L-type calcium channels, and (2) Those that do not participate in waves but do generate asynchronous activity between waves; this asynchronous activity is blocked by L-type calcium channel blockers. I am now testing the hypothesis that interneurons switch from asynchronous activity to participating in wave activity when they stop migrating and find their appropriate positions within the cortex. Such a finding would indicate a role for waves in determining the layer location of interneurons in the cortex.

Songbirds are a model organism of interest to neurobiologists as they are one of a few groups of organisms that display sensorimotor vocal learning, akin to that of humans. Once learned, birdsong is highly stereotyped and while slightly variable, has not been thought to vary systematically from instance to instance. However, biological processes separate from learning mediate the qualities of a bird’s song. It is well known that the amount of singing varies throughout the day with the light cycle, but is not well understood how the acoustics of birdsong vary with light. Recent work has shown that pitch and loudness vary in a consistent manner throughout the day, rising through the morning and early afternoon and falling through the afternoon and evening. Whether these changes in song depend on an internal circadian rhythm or external light cues remains unknown. My project focuses on determining whether finch song is endogenously driven by an internal clock. I collected song data from zebra finches housed under typical light patterns (14 hours of light, 10 hours of dark) and constant light conditions. The fundamental frequency of a sample syllable was analyzed for each bout of singing and then plotted across the span of weeks. Preliminary results suggest that birds under constant light conditions take a few days to unentrain from the light cycle, then sing throughout each 24-hour day, with FF fluctuation around a baseline frequency. I hypothesize that if birdsong acoustics vary according to a circadian cycle, FF fluctuations that occur during constant light conditions are part of a regular pattern that is regulated by an internal clock. Finding that finch song is circadian has implications for understanding how a clock might connect to song system nuclei and demonstrating how complex motor behavior and learning can be influenced by time of day.

As intertidal communities face predicted increases in temperature and hydrodynamic disturbances due to climate change, organisms may find it necessary to reallocate energy among normal physiological processes for survival. Specifically, mussels must distribute energy among processes such as attachment, shell growth, metabolism, and reproduction, all of which may be influenced by seasonality and other natural environmental fluctuations (temperature, pCO2, etc.). Examining energy distributions is the first step in predicting responses to environmental change. In the present study, we investigated the energetic cost of byssus production for the intertidal mussel Mytilus trossulus. After performing an initial pilot study to establish methods for manipulating byssus production by controlling how often the mussels had to replenish byssal threads (ie. daily, weekly, never), we exposed collected mussels to each treatment in triplicate for a four-week period. We then assessed the energetic cost associated with byssus production in relation to energy allocated to other processes such as growth and development. For all measures (length, width, height, shell mass, etc.), forcing the mussel to produce more byssus resulted in a decrease in growth rate; this was significant for length and shell mass. For this study, activities for making longer shells were first to be forfeited as mussels were manipulated into manufacturing greater amounts of byssus during a season where developing reproductive structures were of highest priority. This research identifies byssus production as a major energetic constraint in mussels, which play an important role both economically through aquaculture as well as in intertidal marine ecosystems. As these globally marketed shellfish are placing more amounts of energy towards byssus production, less energy is available for growth. Coupled with modeling predictions, this information could be useful for aquaculture practices as well as understanding physical changes mussels undergo in response predicted climate change.

Circadian rhythms are daily biological processes regulated by circadian clocks, endogenous 24-hr biological clocks. Our master circadian clock, located within the suprachiasmatic nucleus (SCN) in the brain, modulates the activity of circadian oscillators in peripheral organs, including the adrenal gland. While the circadian rhythm in glucocorticoid hormone production correlates with adrenal clock gene expression, the molecular mechanisms of adrenal circadian clock regulation require further investigation. Since the transcription factor phosphorylated cAMP response element binding (pCREB) protein is involved in the regulation of the clock gene PER1 in the SCN, we hypothesize that similar pathways to those found in the SCN control clock gene expression and circadian hormone production in the adrenal. Previous results from our laboratory display that the phase of PER1 circadian expression, in cultured adrenal tissue, can be shifted by drug treatments that increase cyclic adenosine monophosphate (cAMP) levels. Since increases in cAMP lead to CREB phosphorylation, we propose that pCREB ultimately regulates adrenal expression of PER1 in vivo. We measured adrenal pCREB levels in rats housed under constant laboratory conditions and sacrificed 11 hours after starting their subjective day (circadian time 11, CT11) or 11 hours after starting their subjective night (CT23), equivalent time points of peak and trough cAMP levels in the SCN respectively. Immunohistochemical detection of pCREB showed significantly higher number of cells expressing pCREB in the adrenal medulla at CT11 than at CT23. Elevated expression of pCREB before the onset of nocturnal activity in rats is consistent with the predicted peak of glucocorticoid levels. Currently we are investigating the presence of pCREB in the adrenal cortex. Exploring the relationship between pCREB and PER1 expression in the adrenal, and the role of pCREB in phase shifting the adrenal clock, may help elucidating how the master circadian clock relays circadian time to the adrenal gland.

The acrosome is a sperm-specific organelle which is best known for its release of digestive enzymes to degrade the egg’s outer membrane during fertilization. Interestingly, it is required for successful fertilization in Drosophila despite the absence of gamete membrane fusion. Here, we show that sperm lacking Pskl, a predicted membrane protein, display irregularly small and round acrosomes, are unable to breakdown the sperm’s plasma membrane after entry into the egg, and prevent the completion of fertilization. Although Pskl's role in acrosome biogenesis is unknown, comparative analysis indicates the pskl gene gains certain groups of exons over evolutionary time. We propose that Pskl has a modular structure and are testing which modules are necessary for subcellular localization and protein function. To determine the minimal requirements for targeting Pskl to the secretory pathway and specifically the acrosomal membrane, we constructed fluorescently tagged deletion derivatives. The results of these studies will be informative for understanding the protein’s complex evolutionary origin and its role in Drosophila-specific features of fertilization.

FLOWERING LOCUS T (FT) is a highly conserved protein in plants which promotes flowering by influencing the differentiation of shoot apex meristem cells from vegetative to floral identity. CONSTANS (CO) is a protein involved in sensing photoperiodic cues and promoting FT expression. Arabidopsis thaliana express a single evening peak of FT when placed under long-day and constant temperature conditions. Under natural conditions, temperature fluctuates throughout the course of a day. In order to determine if experimental conditions of constant temperature accurately describe flowering time mechanisms in nature, wild type Arabidopsis thaliana plants were grown outside during the long days around the summer solstice of 2013. Real Time qPCR analysis of FT mRNA expression levels in 14-day-old plants over the course of 3 days revealed a morning peak of FT absent under long-day constant temperature conditions – in addition to the evening peak of FT present under long-day, constant temperature conditions. To determine what factors might induce this morning peak, Arabidopsis thaliana were grown in two sets of environmental chambers. Both sets simulated average outdoor temperature conditions over the week of original tissue collection. One set simulated average outdoor light intensity conditions over the same period, while the other used constant light intensity. Real Time qPCR analysis of plants grown under these conditions revealed that the simulation of outdoor temperature conditions is sufficient in producing the morning peak of FT. Different mutants of genes associated with flowering time were then grown under simulated temperature conditions to determine what genes might be involved in the induction of the morning peak of FT. By looking at the different expression profiles of FT and CO, we seek to determine what genes are involved in the induction of this morning FT peak.

The distinctive pigment pattern of the adult zebrafish Danio rerio is formed by stripes made up of black pigment cells called melanophores. Melanophores are the homologue of human melanocytes, which can give rise to deadly melanomas; understanding the signals that regulate the differentiation and proliferation of these cells may be key in treating cancer. Previous work in our lab demonstrated that thyroid hormone (TH) plays an important role in limiting the number of melanophores that appear during development. I wanted to know if TH was also required to maintain appropriate numbers of melanophores after the adult pigment pattern had formed. TH is produced by thyroid follicles, and if the thyroid follicles are ablated, fish cannot produce TH. I used a line of transgenic zebrafish to ablate the thyroid at adult stages to determine if this had any effect on melanophore numbers. Indeed, after several months with no TH these thyroid ablated adults showed an increase in the number of melanophores, demonstrating that TH does play a role in inhibiting proliferation of adult melanophores.

During the Late Cretaceous (100 to 66 million years ago), most mammals were relatively small (< 200 g) and occupied very few ecological niches. Didelphodon vorax was the largest mammal of that time period (~1.7 kg). Thus, study of Didelphodon vorax is key to understanding the range of mammal evolution during the Late Cretaceous. Although a significant amount of research has been conducted on this species, it has exclusively focused on cranial elements, specifically dentition and rare skull fragments. From these elements, researchers have hypothesized Didelphodon had a semiaquatic habit and bulbous premolars for a shell-crushing diet. However, a major limitation of these hypotheses is that they have not been tested with postcranial data. I describe parts of a disarticulated but associated partial skull and skeleton of Didelphodon vorax. The fossils were excavated from a locality called Marshoupial in the Late Cretaceous-age Hell Creek Formation in McCone County, Montana. Specifically, I compared the morphology and dimensions of the hands and feet to those of extant marsupials (e.g., Didelphis virginiana). I have determined that the hand and foot elements preserved at the Marshoupial locality include two distal metacarpals, one distal metatarsal, one complete proximal phalanx (digit III or IV), one distal phalanx, one proximal phalanx (digit II or V), and three intermediate phalanges. From these elements, I infer the locomotion, habitat, and body size of Didelphodon vorax to better understand the ecology of this species. In preliminary observations, Didelphodon vorax is larger than Didelphis virginiana (1.9–6.0 kg) and the shape of its distal phalanx (ungual flexor protruding anteriorly, broad profile in dorsal view) resembles that of aquatic extant mammals. If this interpretation of Didelphodon vorax is confirmed, it would suggest some Late Cretaceous mammals were more ecologically diverse than previously thought.

The fossils of the Cabbage Patch beds of western Montana record over five million years of mammalian evolution leading up to and across the Oligocene-Miocene boundary (23 million years ago [Ma]). This boundary marks the advent of many modern mammalian families. This evolutionary event may have been driven by the climatic and environmental changes of the time. Prior studies of these deposits have identified three faunal divisions occurring chronologically within the beds. To test whether faunal change was correlated with climatic or environmental change in our study area, we quantified the number of mammalian families and their relative abundance in the two oldest divisions of the Cabbage Patch beds (28-25 Ma), and compared this record with the record of habitat change and fossil preservation biases. A preliminary analysis of 495 microfossil specimens from five well-sampled different rock layers in these two divisions shows no change in the number of families throughout the time span; however, temporal shifts in relative abundances of families likely reflect differences in habitat and fossil preservation. Despite potential preservational biases, turnover in mammalian taxa occurs between the older and the younger layers. For example, mouse-deer, interpreted as forest specialists, are very abundant in the oldest layers but absent in the younger layers suggesting their local extinction. Conversely, gophers, native to more open, drier habitats, occur in the younger layers but are absent from the older ones. Together, these data provide circumstantial evidence of changing environments in our study area. These preliminary findings increase our understanding of habitat change and preservation bias in the two oldest divisions of the Cabbage Patch beds. We are currently improving the taxonomic resolution (from family to genus level) of our dataset to provide a more detailed view of changes in regional mammalian diversity during this critical interval in mammalian evolution.

The physiological mechanism of light-induced leaf expansion may begin with the capture of blue light by the photoreceptor phototropin. This process is responsible for growth of the plant which influences photosynthetic ability and overall productivity. Our goal was to determine whether phototropin has an affect on expansion and if so, what phototropin's role in growth is. In this study, we grew Arabidopsis thaliana WT (wild type), phot1, phot2, and phot1/2 mutants in pots in a growth chamber under continuous light. Each plant's fifth leaf was photographed every day and measured with ImageJ to obtain growth rates. This experiment was run three times. The results show that phot1 mutants do not significantly differ from WT, phot2 mutants had a similar growth rate to WT but continued growing at a fast rate for a day longer, and phot1/2 had a much slower growth rate than WT. A second experiment was done using the same growth conditions, but using only WT, phot2, and phot1/2. On the sixth day after its appearance, the fifth leaf was removed from each plant and floated in a 5 millimolar solution of KCl for 24 hours. These leaves were photographed before and after to obtain their growth rates and the results were consistent with the first experiment which used intact leaves. The second experiment demonstrates that there is a phot-mediated mechanism contained within the leaf which influences growth. Knowing the cellular mechanism regulating leaf growth rate may help to understand how environmental stresses such as drought reduce plant productivity. Our next step will be to determine the mechanism which is causing the difference between the rates. First, we will look at anatomy of the leaves to determine if cell expansion or division is most affected by phototropin.

Placental mammals (Eutheria) are one of the most ecologically diverse and dominant groups of terrestrial vertebrates alive today. However, prior to the Cretaceous-Paleogene (K-Pg) mass extinction event 66 million years ago (m.y.), two groups of mammals known as multituberculates and metatherians (group including marsupials) were far more successful in terms of number of species (species richness) and relative abundance. Substantial paleontological research has been conducted on the recovery of mammals immediately after the K-Pg mass extinction and their major diversification 5-10 million years later, but relatively little is known about the transition from recovery to diversification in the intervening interval. Recent research suggests that ca. 65 to 64.8 m.y. the placental radiation began, but whether this is an artifact of sampling or a meaningful evolutionary trend remains somewhat unclear. Here, I add to our knowledge of this important interval in mammalian evolution by describing and analyzing a new sample of fossils from the upper Tullock Formation of northeastern Montana, USA. The data set consists of over 70 isolated fossil teeth from the Horsethief Canyon 1 Locality, nearly doubling the size of the previously known sample. I identified each specimen to at least family level based on morphology and dimensions. Using the resulting identifications, I quantified the species richness and relative abundance of the major mammal groups: Multituberculata, Metatheria, “archaic ungulates,” Primates, “insectivorans,” Triisodontidae, Creodonta, Taeniodonta, and Pantodonta. Species richness was greatest for “archaic ungulates,” with more than 10 species, and lowest for metatherians, with 0 species. Primates had a relative abundance of ~55%, “archaic ungulates” ~27%, Multituberculates ~16%, and “insectivorans” ~1.6%. These results corroborate previous research implicating this time period as the beginning of placental radiation and takeover, and help illuminate patterns of decline and diversification during this critical chapter in mammalian history.

The ability to learn and perform complex motor skills varies with the time of day. One complex motor skill that oscillates daily is song produced by songbirds. The study of song is particularly useful for understanding mechanisms driving the learning of vocalizations and the cellular and morphological changes that accompany changes in song production rate and quality. In one such songbird, Gambel’s White-crowned Sparrow (Zonotrichia leucophrys gambelii), the production of song and the underlying neural circuit that controls this song change dramatically between breeding and nonbreeding seasons. We sought to determine whether song also changed on a daily time scale. We analyzed song production rate, spectral features, and stereotypy of more than 1500 songs produced over the course of two consecutive days by sparrows maintained in breeding conditions. We identified daily patterns in song rate, frequency, entropy, power, and energy of breeding song within both individual birds and pooled bird data. The demonstration of daily oscillations in song rate and spectral features song supports both descriptions of ‘the dawn chorus’ and growing evidence on the modulation of the song control circuit by the internal circadian clock. Having shown daily changes in song production in white-crowned sparrows, we can now begin to study the relationship between song production, cellular changes like neuronal birth and death that occur within the song circuit, and the endogenous circadian clock.

The Northwestern Crow (Corvus caurinus) and American Crow (Corvus brachyrhynchos) are difficult to distinguish using morphological or vocal characters. The Northwestern Crow is considered ecologically distinct, occupying only coastal and shoreline habitats from Central Washington, north through much of coastal Alaska. The more widely distributed American Crow occurs throughout greater North America, including Pacific coastal habitats north to Central Washington, potentially overlapping and hybridizing with Northwestern Crow in this region. Until now, no comprehensive genetic analysis has been performed to examine crows in this potential zone of overlap. This study examines Pacific Northwest Corvus species distributions with minimal spanning networks and phylogenetic analyses via the mitochondrial DNA ND2 gene. Mitochondrial DNA is inherited maternally and does not recombine giving a minimally distorted view of an individual's ancestry. Our data show that the Northwestern Crow occurs from Alaska to Southern British Columbia, and not in central Puget Sound or along the southern coast of Washington. The American Crow occurs throughout the continental U.S. and comes into contact with the Northwestern Crow in southern B.C. Further analysis is required to reveal the possible extent of this contact zone north of Vancouver and/or into northern Washington. A better understanding of this overlap zone will allow science to update natural history species distributions and further explore the effects of human impact on biogeography.

The Miocene epoch represents a hotbed of cetacean evolution; during the epoch, whales underwent a large radiation of morphological forms and ecological specialization. The superfamily Platanistoidea experienced high diversity, especially during the Miocene, with 47 extinct and extant species currently known to science. However, of these 47 species, the Ganges River Dolphin is the sole extant member of this once large clade. Here in Washington State, the University of Washington Burke Museum (UWBM) specimen 87105 is a probable representative of the Platanistoidea in the Pacific Northwest. The specimen consists of the posterior third of the skull, along with some non-diagnostic ribs and vertebrae. It is tentatively a member of the Squalodelphidae, and shares morphological similarities, namely thickened supraorbital bosses, with other members of the Platanistoidea, such as the Platanistids found in the Astoria formation in Oregon and in the Calvert Cliffs formation in Maryland. This project's goal is to describe UWBM 87105, and place it as either a member of the Squalodelphidae or the Platanistidae.  Research into the distinguishing synapomorphies of the Platanistoidea and its families is ongoing; from these synapomorphies, I am deriving a character matrix for UWBM 87105. UWBM 87105's place within the whale phylogeny will expand what we know about the cetaceans present in the Pacific Northwest during the Miocene. In addition, studying this specimen will shed light on the diversity of the Platanistoidea, diversity that is no longer present in the oceans today.

The Mesozoic Era (250–66 million years ago) was a significant interval in Earth’s history when the supercontinent Pangaea broke apart into the continents we know today, allowing continental biotas to evolve independently from one another. Throughout this era, vertebrates diversified into many of the major groups known today. Although there has been much research on the vertebrate fauna of this era from many parts of the world, the Mesozoic vertebrate fossil record of Africa is still poorly known. In 2008, a team of paleontologists from the University of California, Berkeley, the University of Washington, and the University of Utah collected large volumes of fossiliferous sediment (~1,000 lbs) from a Late Jurassic locality (Jema-5) near Addis Ababa in Ethiopia. Our team of undergraduates has begun to process, sort, and identify vertebrate microfossils from these fossiliferous sediment samples. We are in the preliminary stages of our project, but previous workers have recorded a diversity of cartilaginous and bony fish, such as hybodont sharks, guitarfish (Rhinobatos), lungfish (Asioceratodus tiguidensis), and primitive ray-finned fish (Lepidotes, Pycnodus) from nearby localities. Other aquatic vertebrates and terrestrial vertebrates have also been identified from this area, including pleurodiran and cryptodiran turtles, crocodyliforms, ornithiscian and theropod dinosaurs, and primitive mammals. We aim to (1) develop a faunal list from this locality; (2) add to our understanding of vertebrate diversity in Ethiopia during the Late Jurassic; (3) better constrain the age of the fossil locality within the latter half of the Mesozoic; and (4) shed light on vertebrate evolution from a critical area and time in Earth’s history.

During spermatogenesis in animals, the developing male germ cell undergoes dramatic changes in nuclear morphology. This transformation of round spermatid nuclei into condensed nuclei of mature sperm is accompanied by loss of histones and enrichment of several sperm-specific chromosomal proteins. To date, the function of only a few sperm-specific chromosomal proteins has been studied. We are using the fruit fly, Drosophila melanogaster, to investigate the function of a protein that is encoded by the CG14835 gene. The protein is interesting because it is small, highly enriched in positively charged amino acids and it contains a small region of similarity to other known sperm-specific chromosomal proteins. To test for the possible requirement of CG14835 for spermatogenesis and male fertility, we are generating mutations by imprecisely excising a transposable element that resides within the gene. Currently, we are molecularly analyzing independent excision lines to identify gene deletions. The phenotype of males deleted for the CG14835 should reveal whether the protein plays a role in sperm chromatin remodeling, sperm success during fertilization, or the maintenance of paternal chromosomes in the early embryo. Overall, these studies should contribute to our understanding of the possible roles that sperm-specific chromosomal proteins play in sperm function.

At the Cretaceous-Paleogene (K-Pg) boundary, approximately 66 million years ago, a major mass extinction event eliminated up to 75% of all species on the planet. Considerable debate surrounds the cause of this extinction. Was the extraterrestrial bolide impact at the K-Pg boundary the sole cause? Or were ecosystems already deteriorating prior to the impact? In non-marine ecosystems, dinosaurs (except birds), lizards, metatherian mammals, and sharks and rays (Chondrichthyes) suffered the greatest levels of extinction. Chondrichthyans have received little research attention relative to other taxonomic groups. Here, I investigated the fossil record of freshwater chondrichthyans through the Hell Creek Formation in eastern Montana. The formation is ~90 meters thick, divided into lower, upper and middle parts, and spans the last two million years of the Cretaceous. I examined 12 vertebrate microfossil assemblages through the section, identifying teeth, dermal denticles, and vertebrae of freshwater sharks and rays to the genus level. I used this dataset to estimate generic richness and relative abundance trends leading up to the K-Pg boundary. My richness results show that one genus of ray occurs through the entire section. From the lower to middle parts of the section, sharks increase from one to five genera and then drop back down to one genus in the upper part of the section. This pattern temporally correlates with a minor rise and fall, respectively, in sea level of the Western Interior Seaway. Alternatively, this pattern may reflect differences in sampling intensity and site availability through the Hell Creek Formation. I will test the latter hypothesis using rarefaction analysis, which compensates for differences in sampling intensity. Results will help me determine whether these trends are a true glimpse into the shifting biogeographic ranges of non-marine cartilaginous fish communities of the latest Cretaceous or an artifact of variable sampling through the section.

 Stomata play a crucial role in global carbon and water cycles, and disruption of stomatal function could impact our life and sustenance. In order for stomata to function correctly, proper density and distribution of stomata need to be tightly regulated. Several family members of cysteine rich secreted peptides called Epidermal Patterning Factors(EPFs) have been discovered by the Torii Lab and other groups as important signaling molecules controlling stomatal patterning and differentiation in Arabidopsis. Within the EPF family, EPF2 is secreted from stomatal precursor cells to suppress neighboring cells from differentiating into stomata by interacting with ERECTA-family receptor kinases (RKs) and a receptor-like protein, TMM. Thus EPF2 acts as a negative signal of stomatal development. In contrast, EPFL9 (Stomagen), was discovered to enhance stomata development, therefore acting as a positive signal of stomatal development. Conversely, suppression of EPFL9 gene expression by RNA interference leads to reduction in stomatal numbers. The question I addressed was whether EPFL9 requires ERECTA-family RKs and how ERECTA-family RKs process negative signals and positive signals simultaneously. I hypothesized that EPF2 and Stomagen activate and inhibit ERECTA-family receptors and that TMM influence the receptor activity. I addressed this hypothesis through characterizing the effects of their loss-of-function and gain-of-function mutant phenotypes and genetic interactions. For loss-of-function approach, I reduced EPFL9 expression in different genetic backgrounds with respect to different combinations of erecta-family RKs and tmm mutants to analyze the stomata density and index . For gain-of-function approach, I overexpressed EPFL9 in different genetic backgrounds to analyze the stomata density and index. Through the data, I am able to delineate the mechanism by which EPFL9 interacts with ERECTA-family RKs. This will further help to understand stomatal signal regulation and interaction by antagonistic peptide hormones in Arabidopsis.

Grackles are gregarious blackbirds (Icteridae) native to the New World. Our research focuses on the genetic structuring and biogeography of two sister species: the Boat-tailed Grackle (Quiscalus major), and the Great-tailed Grackle (Quiscalus mexicanus), the latter consisting of western and eastern clades. We constructed a molecular phylogeny using ND2 mitochondrial sequences that suggest the Great-tailed Grackle is paraphyletic with respect to the Boat-tailed Grackle. In other words, the Great-tailed Grackle species as currently recognized includes some, but not all, of the descendants of its most recent common ancestor. Phylogeographic analysis of the same genetic data suggests that eastern and western populations of Great-tailed Grackles are currently interbreeding freely, while Boat-tailed Grackles remain reproductively, morphologically, and ecologically isolated, despite their more recent split from the eastern Great-tailed Grackle clade.

Modern frogs and toads (Anura) are environmentally sensitive vertebrates that are often viewed as bellwether taxa for the modern biodiversity crisis. As such, one would expect that they did not fare well in the Cretaceous-Paleogene (K-Pg) mass extinction event (ca. 66 million years ago) that killed off the non-avian dinosaurs. However, relative to other taxa, little is known about Late Cretaceous and early Paleogene anurans or how they fared in the K-Pg event. I have documented temporal changes in anuran diversity leading up to and across the K-Pg boundary, based on the vertebrate microfossil assemblages in the Hell Creek and Tullock formations in Garfield County, Montana. These geologic formations span approximately the last two million years of the Cretaceous and the first one million years of the Paleogene. I have identified known anuran species and distinct morphotypes, which likely represent unnamed species, from more than 200 isolated fossil anuran jaws and hip bones. Initial results, based on maxillae, show an increase in anuran species richness from 9 morphospecies in the lower Hell Creek Formation to 17 morphospecies in the middle of the formation. Then, species richness drops before the K-Pg boundary down to 9 morphotypes in the upper Hell Creek Formation. Anuran fossils are relatively scarce after the K-Pg boundary: the lower middle Tullock Formation includes one species that locally survived the mass extinction and one apparent immigrant taxon. Later in the middle Tullock Formation, I document only one unnamed immigrant taxon. These results parallel pre-K-Pg declines in some vertebrate taxa (e.g., mammals) from this study area but contrast with the high survivorship of more fully aquatic lissamphibians (e.g., salamanders). I hypothesize that the high levels of extinction suffered by anurans at the K-Pg boundary may be due to the greater vulnerability of their more terrestrial life habit.  My research aims to bring a greater understanding to the timing and mechanisims of one of the most important events in the history of terrestrial life (the K-Pg mass extinction), and possibly give insite into the current biodiversity crisis effecting extant amphibians.

Diatoms are an important taxon of phytoplankton that contribute to both freshwater and marine ecosystem function. For example, marine diatoms are responsible for about 14% of global primary production. My research is focused on determining the ability of these algae to regulate the production of chlorophyll a- the core pigment component of photosynthetic complexes. The enzyme light-dependent NADPH protochlorophyllide oxidoreductase (POR) is a key regulator of chlorophyll synthesis. Whereas most organisms have just one POR enzyme, diatoms have two (POR1 and POR2). The goals of this project are to understand how these enzymes are metabolically different from one another in: (a.) their enzymology and (b.) their regulation at the transcriptional and posttranslational levels. To this end, plasmids were constructed encoding the cDNAs of the por genes, adding N-terminal histidine tags to assist in affinity purification. E. coli, transformed with these plasmids, was induced to express the proteins; harvested, and lysed. Recovered POR1 and POR2 proteins were purified from the lysate by affinity chromatography. Antibodies raised against these proteins were used in Western blot protein identification and quantitation of the targeted proteins. Preliminary experiments demonstrate good affinity of generated antibodies for POR proteins, and indicate that POR 2 may be post-transcriptionally truncated. Developing an understanding of chlorophyll metabolism in diatoms has not only ecological implication, but also could prove important in commercial aspects of biofuel development- increasingly important issues driven by changing ocean conditions and the critical need for generating alternative fuel sources. Determining the properties of these enzymes and their regulation is just one step towards understanding the successful productivity of diatoms. Further studies may be required to determine if there are additional regulatory pathways and enzymes that are critical in photosynthetic production.

Forensic entomology uses the life cycles of insects to determine the time of death (post mortem interval, PMI) of victims of homicide and abuse. Currently, PMI is not used on bodies that have been burned with the use of an accelerant because little is known about how the accelerants affect the life cycles of insects at the scene. The purpose of this study was to investigate whether flesh flies (Sarcophaga bullata) will colonize and complete a full life cycle on charred carrion that has been burned with an accelerant. Laboratory reared adult flesh flies were provided with various substrates; one not burned, one organically burned and one burned with the accelerant, gasoline. Early results of this experiment have shown normal progression of the fly life cycle with larvae deposited on the control carrion. No progression of the life cycle of the flies has been observed on either of the charred substrates. The results of these experiments can show how carrion burned with accelerants affects the colonization and life cycle of an important forensic indicator species, the flesh fly. This pilot study can lead to further studies regarding using PMI for charred remains. The broad scope of this study is to gain insight into the use of insect life cycles to determine the time of death of victims burned with accelerants.

The circadian system generates daily oscillations of behavior and physiology including the sleep-wake cycle, rhythms of mood and hormonal release. These oscillations represent a predictable source of variance for neural centers that generate specific motor programs such as walking or hand movements. How and where in the brain this internal 24-h information is integrated to achieve effective motor patterns remains unknown. I hypothesized that the mouse primary motor cortex (PMC), a region responsible for complex motor programs, would show a signature of electrical activity associated with wheel running. I also hypothesized that this wheel running-associated electrical activity pattern would change predictably across the circadian cycle. I implanted electrocorticographic (ECoG) electrodes onto the PMC of mice and recorded brain wave activity while mice ran on the wheel at different circadian times. My results indicate a broad-spectrum power increase in the frequency of PMC electrical signals associated with wheel running. Furthermore, the spectrum frequency associated with wheel running changes predictably throughout the circadian cycle. My results provide a new model system in the use of ECoG in mice to assess circadian components associated with complex behaviors. It sheds new light on neural motor-system control, and it may have immediate application for the design of brain-computer interface devices that can operate accurately throughout the 24-h day.

Circadian rhythms such as the sleep-wake cycle and rhythms of hormonal release are regulated by biological clocks within the brain. Because these clocks have a period of about 24 hours, they need to be synchronized (or entrained) to the solar day by 24-h cyclic environmental input. Although the light-dark cycle is typically the strongest synchronizer, several studies have shown that the restriction of food access to a few hours a day can act as a stronger synchronizer than light. Under these conditions the suprachiasmatic nucleus (SCN) of the hypothalamus, which contains the master circadian oscillator that regulates circadian rhythms, stops regulating the timing of rest and activity. This regulation is instead achieved by other circadian oscillators entrained by restricted food access, allowing rodents to anticipate a predictable daily mealtime. The location of these food-entrainable oscillators (FEOs) in the brain remains unknown. To identify the putative location of the FEO, we divided the rats under a normal 12:12 light-dark cycle into 3 groups: Group 1 had access to food 2 hours during the day, group 2 had access to food 2 hours during the night, and group 3 had access to food 2 hours during the day the night. I carried out in situ hybridizations targeted to the clock genes PER2 and BMAL1, whose circadian expression is essential to sustain a normal circadian rhythmicity. To quantify the mRNA levels for each gene, I then exposed the brain slices hybridized with radioactive probes specific to each gene to X-ray-sensitive films and measured the optical densities within the dorsomedial hypothalamus and the SCN. The results show that the temporal pattern of expression of Bmal1 and Per2 mRNAs within both the SCN and the DMH are affected by the feeding schedule in opposite ways, suggesting that the DMH may contain an FEO.

 Sleep is essential for survival, yet its function remains unknown. In our society, sleep deprivation and disruption of sleep architecture, namely the temporal structure of rapid eye movement (REM) and non-REM sleep stages—are common. Sleep deprivation impairs memory consolidation dependent on the hippocampus region of the brain. We hypothesize that disruption of sleep architecture in the absence of sleep deprivation impairs hippocampus-dependent memory. Our laboratory developed a rat model in which sleep architecture is predictably disrupted without the confounding effects of sleep deprivation, and without genetic, pharmacological or surgical manipulations. Exposure to an artificially short light-dark (LD) cycle (22 hours) produces two circadian rhythms within the same animal. In this “forced desynchrony” protocol, animals cycle between “aligned” days, during which the two oscillations are in phase and sleep architecture is similar to that of 24-hour LD cycle control animals, and “misaligned” days, during which oscillations are out of phase and sleep architecture is disrupted. Previous results indicated that rats trained in a contextual fear-conditioning hippocampus-dependent memory task during misaligned days show impaired memory consolidation. Because memory consolidation was measured as “freezing”, the animal’s immobility in the fearful context, we asked whether memory consolidation would also be impaired in a hippocampus-dependent task requiring active spatial navigation (the Morris water maze or MWM). We trained aligned, misaligned and control animals to swim to a hidden escape platform within a pool containing visual cues. After removing the platform, we tested memory consolidation by measuring latency to reach the original platform location along with other parameters. Our analysis showed that while circadian forced desynchrony associated with sleep-stage architecture disruption may impact memory acquisition in the MWM task, it does not appear to impair long-term memory consolidation. We conclude that normal sleep architecture may be critical for some hippocampus-dependent memory tasks but not others.

During zebrafish gastrulation, presumptive mesodermal cells internalize beneath the ectodermal cells and migrate toward the dorsal axis to form the notochord and the presomitic mesoderm. Our research focuses on how p120 catenin is involved in the pathways of cell adhesion and migration during zebrafish morphogenesis. p120 catenin is a regulatory molecule which has dual functions. The first is to stabilize cell-cell adhesion by binding to the intracellular and juxtamembrane region of cadherins at the cell membrane which form adherens junctions with other cells within a tissue, holding them together. The second role of p120 catenin is to function upstream of CDC42 and Rac1 GTPases in the cytosol to initiate cell motility by stimulating the formation of lamellipodia and filopodia. Both functions are greatly regulated by p120 catenin phosphorylation. The hypothesis is that tyrosine phosphorylation in p120 catenin mediates its binding to cadherins while serine/threonine phosphorylation leads to activation of CDC42 and Rac1 and thus, cell migration. Our general approach is to apply immunoprecipitation of embryo lysates to determine which proteins interact with p120 catenin when it is phosphorylated on tyrosine or serine/threonine residues. Next we will inject mutated forms of p120 catenin into the embryos and determine how that changes protein interactions. So far, we have been testing different mammalian commercial antibodies on normal embryo lysates to determine which ones work with zebrafish. This research could have important implications on treatments of some cancers and wound healing. 

The protein that I am interested in, TOPLESS (TPL), is in a class of co-repressors that is conserved across eukaryotes. A protein is classified as a co-repressor if it cannot bind to DNA itself, but is recruited by a DNA-binding protein (typically a transcription factor) to repress the expression of a specific gene. Although we know that in the model plant, Arabidopsis thaliana, the TPL mutant phenotype does not have a normally developed top half (giving the protein its name) it is not clear how TPL confers repression. Research on Tup1, the yeast homolog of TPL, suggests several possible mechanisms: TPL could physically block key interactions, recruit other repressive proteins, or cause changes in the DNA to prevent gene expression. My research aims to test the hypothesis that TPL recruits a partner protein to induce repression. To test this hypothesis I have been using a yeast two-hybrid assay. This assay works by splitting a transcription factor that activates a reporter gene into two pieces. One of the pieces is fused to TPL and the other is fused to a protein I believe interacts with TPL. If they interact, then together they will form a functional transcription factor and activate reporter gene expression. In this case, I have been using histidine as the reporter gene. I can determine if histidine is being produced by growing the yeast on an agar plate that lacks that key amino acid, so only the yeast that have interacting proteins can synthesize it on its own. The yeast containing proteins that interact form colonies, while yeast containing proteins that do not interact will fail to grow. This experiment will allow me to better understand an important mechanism that is not only present in plants, but is present in many other organisms.

Auxin is a major plant hormone, guiding phototropism, gravitropism, cell elongation, cell division, cell determination, and a host of other processes. One of the main mechanisms through which auxin acts is by triggering the expression of auxin-related genes. Under high auxin conditions auxin will act as molecular glue bringing together a co-receptor complex that consists of a repressor protein and a receptor protein. This triggers the ubiquitination and breakdown of the repressor protein allowing for the expression of auxin genes. Under low auxin conditions the repressor persists and auxin genes are blocked. In our model plant, Arabidopsis, there are 29 IAA (Indole Acetic Acid) repressor proteins and 6 receptor proteins. Previous works shows that degradation rates between IAA’s vary widely and that rate-motif sequences outside of the degron (the minimum region of the IAA required for degradation) accelerate or decelerate auxin-mediated degradation. The work presented here tested the hypothesis that the rate of repressor degradation is linked to the binding strength between the repressor and the receptor. A yeast-two-hybrid (Y2H) assay was used to test the binding strength between the TIR1 receptor and truncations and mutations of the IAA1 and IAA28 repressors. Combined with degradation rate data collected through flow cytometry, the Y2H data shows that mutations in the rate motif region of the IAA that change the degradation rate do not change the binding strength between the two proteins. This suggests that the rate motifs do not affect the strength of the IAA:TIR1 interaction and instead affect another aspect of the interaction.

The focus of this research is on p120 catenin, an essential protein in the development of vertebrate embryos. p120 catenin is associated with the regulation of cell migration during development, which includes cells migrating towards the dorsal axis. The mesodermal cells that migrate towards the dorsal axis will become the notochord and somites. We hypothesize that p120 catenin regulates this movement through its association with cadherins, and its activation of the actin cytoskeleton. p120 catenin binds the cytoplasmic tail of E-cadherin to stabilizes and strengthen adhesion and in the cytosol regulates small Rho GTPases to mediate actin dynamics. The regulatory domain of p120 catenin contains tyrosine and serine/threonine residues that are phosphorylated. We are modifying these sites to amino acid residues that lack the ability to be phosphorylated, phenylalanine or alanine, or are constitutively charged, glutamic acid. A splice site antisense morpholino injected into single celled zebrafish embryos knocks down the expression p120 catenin proteins, which can then be rescued by co-injection of wild-type p120 catenin mRNA. The modified p120 catenin mRNAs are co-injected with splice site RNA morpholino and the embryos’ development is then compared to those embryos that were rescued with wild-type p120 catenin. Thus we are studying the phosphorylation sites essential in the cell migration process. Data from the tyrosine 228 to non-charged phenylalanine mutation rescues the normal phenotype. This suggests phosphorylation of the tyrosine at the 228 site is not required for cell motility. Whether serines 268 and 288 are required for cellular motility is not yet clear. Other phosphorylation sites are currently being studied for their roles in cell adhesion and migration. This research is significant for developmental biology as well as cancer biology and wound healing.

Over the last four decades, marine community composition has changed in the San Juan Archipelago, showing a density decline in the scleractinian (hard skeleton) solitary cup coral Balanophyllia elegans. Determining predictors of B. elegans abundance, distribution, and size is important for ecological research in the eastern Pacific Ocean because changes observed over time could be an indication of realized impact from climate change and ocean acidification on calcifying organisms. Though reef corals are heavily studied, little is known about temperate, solitary corals. We studied B. elegans in the San Juan Islands in transect photos from 2008-2011 at 11 sites. At two of the sites, we analyzed photos to determine the distributions of size and abundance at 3-27 m depth. At all of the sites, we evaluated the effect of flow rate on coral size and population density by grouping the data from depths between 15-21m at the 11 sites into 4 categories of flow: low flow, medium flow, high flow, and very high flow. We used multiple statistical tests, including the Gaussian General Linear Model and the Poisson General Linear Model, with ANOVA to analyze the data. We found that the distributions of B. elegans vary by site, depth, and year. The results tell us that each site is unique. Even though some of these sites are separated by mere meters, characterized by the same flow rates, and evaluated on equivalent slopes, they appear to differ enough to create measurable variance. The patterns we found will need to be explained by researching these sites longer and adding additional site variables, such as pH. Fluctuations in pH, potentially caused by changes in rates of upwelling in the northern Pacific Ocean, could cause short term trends of increasing population size of calcifying organisms when pH is higher, but significantly reduced populations when pH is lowered by increased upwelling.

During mammalian central nervous system development, waves of spontaneous synchronous activity (SSA) are a widespread developmental phenomenon, serving to regulate neuronal migration, physiological maturation, and synaptic connectivity. In the neonatal mouse cortex, these waves of electrical activity involve a vast number of neurons firing bursts of action potentials simultaneously and producing transient increases in intracellular calcium, which can be imaged using calcium-fluorescent dyes. The Moody lab has previously determined that waves of SSA are initiated by a pacemaker in the ventral piriform cortex and are dependent on GABAergic transmission. Since developmental problems may arise when waves fail to occur during the proper developmental time period, it is crucial to determine exactly how waves are initiated. In order to gain insight into how waves are generated, I estimated key network and cellular parameters that define developing cortical networks using past experimental data. I then built a computational model of GABAergic neurons that simulates wave initiation and propagation, implementing the previously estimated parameters. By matching the simulated wave propagation frequency to those determined by experimental data, I estimated the parameter space of cell connectivity, a free parameter of the model. Finally, I am validating the model by gathering data that estimates synaptic connectivity via patch clamp experiments. This knowledge will ultimately help elucidate the pacemaking function of GABAergic cells that generate waves of SSA by characterizing intrinsic physiological properties and synaptic connectivity.

During neurological development, waves of spontaneous activity propagate through structures in the central nervous system including the hindbrain, retina, hippocampus, and midbrain. The presence of waves of activity is essential for proper maturation of these structures and aids in cell path-finding, the establishment of synaptic connections, and the expression of mature firing properties of neurons. These waves initiate at pacemaker sites and electrical stimulation of those sites can produce waves comparable to those initiated spontaneously. In our research, we compare the excitability of the hippocampus to that of a known pacemaker site in the ventral cortex and a non-pacemaker site in the dorsal cortex. To do this, we use blunt glass electrodes to electrically stimulate a mouse brain slice preparation at P3 or P4 and determine the threshold of electrical stimulation that is necessary to evoke a wave of activity at various sites in the cortex. We have found that the threshold of the dorsal cortex is higher than that of the ventral cortex—a known pacemaker region—or that of the hippocampus. The hippocampus is a common initiation site for seizure activity. So, the finding that the hippocampus and pacemaker sites are similarly excitable may aid in developing a better understanding of temporal lobe epilepsy.

Chronic graft-versus-host disease (cGVHD) is a frequent complication of bone marrow and hematopoietic stem cell transplants and one of the leading causes of death in patients who receive such treatments. cGVHD arises as a consequence of incompatibilities between the donor and host immune systems, including disparites between donor and host major histocompatibility antigens (mHA). Donor CD8 T cells, which are responsible for distinguishing self and non-self tissue, contribute to cGVHD development through recognition of mismatched mHA and MHC (major histocompatibility complex, a protein responsible for recognition of foreign antigens in the body) molecules. This results in subsequent destruction of host tissue and cGVHD onset. In the female-to-male transplant environment, an immune response to certain peptide fragments derived from the Y chromosomes, called H-Y antigens, correlates with an increased risk and severity of cGVHD. Less is known, however, about the nature of the T cell receptor (TCR) repertoire responsible for the reaction to H-Y antigens. This project examines the TCR repertoire of cells reactive to an H-Y antigen derived from the Y-chromosomal protein KDM5D, an antigen known to increase the risk of cGVHD. Cells were cultured in the presence of peptide, sorted to generate monoclonal cell lines, and Sanger sequenced in order to determine the exact nucleotide sequence responsible for TCR expression in order to catalog TCR sequences and look for patterns in the CD8 response to KDM5D. Five separate CD8 cell lines have been successfully established, of which one’s TCR genes have been sequenced. While still in progress, the work completed thus far on this project demonstrates the viability of the methods used; alternative strategies may yield more comprehensive results faster. Completion of this and similar projects promises to inform new efforts in the diagnosis and management of cGVHD.

Giardia lamblia is a protozoan parasite common in mammalian hosts and belongs to one of the earliest diverging eukaryotic groups. Subsequently, Giardia has a simplified and highly divergent genome that lacks classical actin associated proteins, yet has retained a conserved microtubule (MT) cytoskeleton. This unique cell biology could provide a window into ancient cellular processes, particularly cytoskeletal function and regulation. Giardia has evolved novel and elaborate MT-arrays that mediate important stages in a complex life cycle and assemble into structures critical to its virulence, motility, and cellular organization. Like most eukaryotes, Giardia possesses a superfamily of five tubulin paralogs (gene duplicates) that constitute the MT cytoskeleton (alpha, beta, delta, epsilon, and gamma-tubulin). Interestingly, the Giardia genome contains multiple paralogs of the alpha- (TUBA1, TUBA2) and beta- (TUBB1, TUBB2, and TUBB3) types. It remains unresolved why multiple, highly conserved TUB paralogs are maintained in the Giardia genome. To address this question, we localized tubulin proteins by expressing GFP-tagged tubulin under its native promoter in a pGFPa.pac expression vector. These constructs were transfected into cells and proteins were localized using 3D fluorescence microscopy. Microscopy of fixed cells revealed that TUBA and TUBB proteins localize similarly in interphase cells and are largely interchangeable (ventral disk, median body, flagella), yet display differential levels of expression. Namely, GFP::TUBB3 and GFP::TUBBA1 signal is much brighter than that of GFP::TUBB1, GFP::TUBB2, or GFP::TUBA2. Microscopy also demonstrated delta-tubulin is diffuse throughout the cytoplasm, epsilon-tubulin localizes faintly to the ventral disk, and gamma-tubulin appears to track the internal axonemes of the flagella and concentrate at the flagellar tips. This data could suggest Giardia utilizes alpha- and beta-tubulin paralogs in different stages of the life or cell cycle, which has not yet been demonstrated. This data will also inform the development of a robust live cell marker for Giardia’s MT-arrays.

Phylogeography is the study of the geographic and spatiotemporal distributions of populations, and it is an important tool for investigating the evolutionary history of species. Determining when and where species originated, merged, or moved is critical in understanding the factors that generate biodiversity. Novel Bayesian phylogeographic methods provide a useful tool for obtaining accurate estimates of these important population dynamic parameters. West Africa is a global biodiversity hotspot with a high number of endemic species, and Agama lizards are a diverse and prominent member of this distinctive ecoregion. To investigate the phylogeography of Agama lizards, I constructed phylogenetic trees from over 300 unique samples of Agama lizards from 20 countries using Bayesian phylogeographic inference. Molecular genetic data from a quickly evolving gene (16S rRNA) provided sufficient variation for estimating the divergences between distinct populations. Visualization of the phylogeographic history for the populations was conducted with Google Earth. This group of species is predicted to have originated in the central part of West Africa (i.e., Cameroon, Nigeria, and Equatorial Guinea). At least five distinctive species formed across the region from that one ancestral population, and one paraphyletic group requires further investigation. Outlining the history of species formation in subtropical West Africa, with an emphasis on when and where populations divided, moved, and reconnected, serves to show the benefits of phylogeographic studies as they can reveal patterns that are otherwise too difficult to detect using other approaches. These studies also serve as tools in providing information for the detection of distinct species.

As twilight falls on the University of Washington Bothell campus, the skies turn black with the arrival of 10-15,000 crows (Corvus brachyrhynchos) that use the restored North Creek Wetlands as their winter nocturnal roost.  The crows leave large amounts of nitrogenous waste under their roost, and the impact this has on the biodiversity of flora in the wetlands is unknown.  Using simple random sampling, we surveyed flora composition and collected soil samples at sites on- and off-roost across seasons (spring 2013, fall 2013, and winter 2014).  We analyzed soil from each site for moisture, pH, and levels of nitrates.  Across all seasons, significantly higher levels of nitrates and lower pH levels were found on the roost compared to non-roost locations; pH levels varied both with season and by location, but there was no significant interaction effect. Plant community composition was not significantly different on and off the roost, nor did it vary significantly across seasons.  Although the crows are fundamentally changing the chemical composition of the soil under their roost, they are not yet affecting plant biodiversity or abundance.  Birds, however, are vectors for invasive plant species, and these variables are predicted to change over the long term, which will have important management implications.  The North Creek Wetlands are an exemplar of ecological restoration, surrounded by rapidly-developing urban centers, so any future impacts the crows have on these wetlands could cause larger ripple effects over the remaining fragmented forests, streams, and wetlands of the Lake Washington watershed.

Testosterone plays a major role in aggressive and territorial behavior in many male vertebrates during the breeding season. Song sparrows, Melospiza melodia morphna, are not only aggressive in the breeding season but also in the non-breeding period, when their testes are regressed and plasma testosterone levels are low. Previous studies suggest that this territorial aggression is mediated in part by high levels of an androgen and estrogen precursor, dehydroepiandrosterone (DHEA), produced by the adrenals. In this study, we aim to determine whether DHEA exerts these effects via an androgen or estrogen receptor mediated mechanism, or both. Male song sparrows were captured, kept on a short day cycle (8L: 16D) typical of the non-breeding season, and implanted subcutaneously with either empty or DHEA-filled silastic implants for fourteen days. DHEA implanted birds had significantly higher circulating DHEA and testosterone levels and showed increased aggression in a laboratory-based aggression test. Brains were extracted and in situ hybridization was used to label androgen receptor (AR) and aromatase mRNA. Aromatase is the enzyme that catalyzes the transformation of androgens to estrogens. Using dark field microscopy, AR and aromatase mRNA expression in brain regions previously associated with aggressive and reproductive behaviors were quantified. In the ventromedial nucleus of hypothalamus (VMH), AR expression was significantly greater in DHEA birds compared to controls. However, in the preoptic area (POA), aromatase mRNA was significantly higher in DHEA birds over controls. In the medial preoptic area (mPOA), neither AR nor aromatase expression differed between DHEA and control birds. We conclude that DHEA may cause non-breeding aggression via both androgen and estrogen receptor mediated mechanisms. Deeper understanding of how exogenous DHEA affects the brain and behavior of song sparrows may provide useful insights for biomedical researchers interested in investigating the health benefits and side effects of DHEA supplements.

Tree growth rate is a major driver of tree population dynamics, forest structure, and forest function. As temperature and competition (e.g., for light) can both influence growth, a clear understanding of the relationship between climate, competitive environment, and tree growth will be critical for predicting how forest ecyosystems will respond to climate change. We hypothesized that growth rates would decrease toward higher elevations as mean annual temperatures and growing season length decrease. Additionally, we expected growth to be faster in canopy gaps (low competition) than in closed canopy areas (high competition). To examine this issue, we assessed the relationship between tree height and age (based on tree ring counts) for Abies amabilis, a dominant conifer species of the Pacific Northwest, across a large environmental gradient encompassing this species elevational range. Specifically, we collected 231 tree saplings from three different elevations (600m, 1000m, 1400m) in gaps and closed canopy areas in Mount Rainier National Park, USA. We found that growth rate declined as elevation increased, suggesting that growth might be constrained by temperature and growing season length. For example, the average age of a 1 meter tree increased by 30 years for every 400 meter increase in elevation (e.g., 28, 58, 88 years from low to high elevation). However, competition for light had little effect on growth rate, possibly because gaps in the forest canopy are short lived (10 - 25 years) relative to the lifespan of these saplings. Our results suggest that global climate change may accelerate growth rates, which could shift this species geographic range, accelerate forest regeneration, and alter forest structure. To further understand this issue we plan to 1) examine the period of time these trees experienced gap and closed canopy conditions and 2) contrast growth with estimates of survival across elevation.

Fire damage is a stressor that plants have evolved around (to varying degrees) for thousands of years, and is known to induce a signaling/repair pathway that is not fully understood. Illuminating the transcriptional regulatory mechanisms of this pathway could not only give us insight into a basic biological system that is applicable to all forms of life that experience aging, cancer, and disease, it could also provide us with information about plant-specific fire resistance and healing that could be utilized to fortify our crops in a warming world. This study explores possible interactions between histone acetyltransferase (HAT) proteins produced by HAC1 and RNA synthesis enzyme RNA Polymerase II (RNAPII) in Arabidopsis thaliana plants damaged by fire. HAT proteins upregulate gene expression by chemically loosening histones (tightly-wound "spools" of DNA) and exposing coding regions to increased transcription, and RNAPII regulates overall transcription by recruiting and manipulating numerous factors of RNA transcription and synthesis, but current understanding of complementarity or antagonism between HAC1 and RNAPII remains tenuous at best, especially in plants. In this experiment, I either exposed both Arabidopsis thaliana mutants (deficient in functional HAC1) and their non-mutated counterparts (containing normal HAC1) to flame wounds, or left them alone, and then collected non-burned leaf samples twenty minutes later. Because gene expression can be measured by quantifying proteins, I used Western Blotting to detect levels of RNAPII in an attempt to elucidate any interaction between these two transcriptional regulators. My preliminary results suggest that less RNAPII is produced when plants are burned, but that the HAC1 gene is not necessary for the regulation of RNAPII after burning, though replications are necessary to confirm this.