Why are urban forests not replacing themselves?  For example, recent surveys demonstrate that adult trees are dying faster than new saplings are recruiting in Seattle (Washington), implying that the health of local coniferous forests is in decline.  Extensive resources are spent planting trees to combat this problem, but the underlying causes of this "lost generation" of trees remains unknown.  We investigate factors limiting seedling establishment of native conifers (Thuja plicata, Tsuga heterophylla) in Seattle, and use results to suggest strategies resource managers can use to address the "lost generation" problem.  We examine the effect of removing non-native English Ivy (Hedera helix) and adding mulch to understand what is limiting conifer regeneration, and to explore possible management techniques for improving growth and survival of transplanted seedlings.  We measured survival, general health (visually assessed), and height as response variables to the two treatments.  As of yet, no studies have empirically tested both of these factors.  We observed the highest transplant survival rates in areas where mulch was added, for both focal species.  Surviving transplants in the mulch treatment were also healthier than the transplants with no mulch added.  Ivy presence did not affect health or survival of either species, but it did affect growth of Tsuga heterophylla.  Treatments affected growth of the two species differently.  Growth of Thuja plicata was not affected by either treatment.  For Tsuga heterophylla, however, growth was highest in the ivy-removed treatment, but was not significantly affected by the presence of mulch.  Our results suggest that seedling survival is limited by a lack of mulch and that competition with invasive understory plants may limit growth for some species.  Our recommendations for park management are therefore that restoration transplants should be paired with mulch addition, as well as Hedera helix removal for Tsuga heterophylla, to maximize survival and growth.

For an animal whose brain only contains about 100,000 neurons, the fruit fly is capable of striking behavioral complexity. The goal of our work is to identify neuronal circuits that process sensory cues and transform them into changes in motor activities. Our work relies on the Gal4-UAS (Upstream Activation Sequence) system, a commonly used genetic tool that allows us to label and manipulate small subsets of neurons. The system takes advantage of different enhancer elements to drive expression of the Gal4 transcription factor in various tissues. When Gal4 flies are bred with UAS flies that contain GFP (green fluorescent protein) or RFP (red fluorescent protein), the Gal4 will bind to the UAS, and enable the production of GFP or RFP. This allows the desired cells to fluoresce, which can be tracked using a microscope. With this, we can identify Gal4 lines with interesting neuronal expression patterns and test the function of these neurons in Drosophila. Expressing the ion channel UAS-TrpA1, for example, activates neurons when the temperature is shifted from 22 °C to 27 °C. I crossed different Gal4 drivers to UAS-TrpA1. After performing a temperature shift on the progeny, I observed that various Gal4 flies became twitchy while others collapsed. These behaviors were reversible when the temperature shift ended. Fluorescing the Gal4 lines that allow for this behavior would allow insight as to the activated neurons in the brain and thoracic ganglia. The confocal images would allow us to see the neuronal expression. Through this model organism we can hope to comprehend and apply the knowledge in understanding complex systems, for example, what neurons coordinate behaviors such as navigation and locomotion.

Escherichia coli can contaminate food, such as beef and spinach, causing illnesses ranging from recurring diarrhea to death in susceptible individuals. They may produce a toxin typically associated with another harmful bacterium known as Shigella spp. This toxin is referred to as “Shiga-toxin”, and E. coli that produce this toxin are known as STECs. While E. coli O157 remains the most common STEC in the United States there are over 100 STECs that can be just as harmful. My project, conducted in collaboration with individuals at CSUEB and the USFDA, involves identifying chemical indicators that would help detect different strains of non-O157 E. coli in contaminated foods, and determining if these STECS are vulnerable to a coliphage and a Bdellovibrio sp. invasion. Specifically, nine different STECs were chosen to be screened for their ability to produce catecholate siderophores (phenolates), such as enterobactin, chelating compounds important for some pathogenic bacteria in the acquisition of iron. The same STECs were screened for the presence of acyl homoserine lactones, chemicals involved in bacterial communication, also known as quorum sensing, and expression of virulence factors. Assays were conducted to determine if a coliphage and a Bdellovibrio sp. can infect these STECs. Detection and identification of non-O157 STECS is not easy, which hinders determination of their origin and, subsequently, how to eliminate them. If new tools, such as the use of predators or chemical indicators were developed to facilitate detection of these bacteria, then determination of the source of contamination (e.g., packing plants and fields) may occur, and quicken measures that prevent or, at least, decrease further infection, disease and/or death.

Plastic pollution in marine environments has received considerable attention from scientists, policy makers and the media. Only recently, however, has much consideration been given to the unique problems microplastics (0.3 mm - 5.0 mm) present to aquatic systems. The small size of these plastic particulates makes them difficult to study and potentially more hazardous to marine and freshwater environments. While most efforts to quantify microplastic pollution have focused on marine environments, this study investigates microplastic load in a large freshwater body (Lake Washington) and the possible link between rain events and microplastic concentration. Water samples were obtained with flow meter-equipped plankton nets modified for accurate surface collection, and then processed using an oxidation reaction and visual inspection method developed by UW Tacoma. Results suggest freshwater microplastic pollution reflects average mass found in marine environments, and that there may be a correlation between rain events and higher microplastic concentrations.

Plants sense and respond to changes in their environment. For example, elongation of the embryonic stem ensures seedlings reach light after they germinate. This critical stage in a plant’s life is highly regulated by hormones, including gibberellic acid (GA) and brassinosteroids (BRs). Using the model system Arabidopsis thaliana, we discovered GA and BRs interact to control stem elongation during early development. We found that treating seedlings with both hormones resulted in a synergistic growth response during some phases of development but not others. We are exploring the genetic basis of this response using seedlings mutant in different aspects of their GA and BR pathways. For example, DWF4ox seedlings over-express a gene that encodes a BR biosynthetic enzyme. These seedlings make more BRs and have an enhanced growth response. DELLAs (RGA and GAI) repress growth and are degraded by GA. Seedlings that have reduced DELLA (rga gai) function also have enhanced growth. We are testing the growth responses of the DWF4ox rga gai triple mutant looking at hypocotyl elongation rates. By using these and other mutants that have compromised GA and BR pathways we hope to better understand the molecular mechanism of the interaction between these two hormones.

Augmenting course material to be relevant and accessible to students is a ubiquitous challenge throughout academia. We aimed to design, develop, and evaluate the effectiveness of activities meant to promote the learning of difficult concepts in Anatomy and Physiology. More specifically, we focused on the neurophysiology chapter of an open access Anatomy and Physiology course sponsored by the Carnegie Mellon Open Learning Initiative (OLI). This course is being created for pre-nursing students attending community colleges. We used the six-level schema of Bloom’s Taxonomy, a module addressing the knowledge and development of intellectual skills, to design learning activities that encouraged students to analyze, synthesize, and evaluate concepts rather than simply recall, explain, and define them. By compelling students to work at higher cognitive levels during the learning process, we hypothesized that there would be a corresponding elevation in critical thinking skills and a deeper understanding of the material. Our curriculum design strategy was informed by cognitive science research that shows increases in learning efficacy when students are motivated to work at higher cognitive levels. Assessments were designed to align with these higher cognitive level learning activities. Our learning activities were tested by Anatomy and Physiology students using Carnegie Mellon’s OLI course. The Carnegie Mellon course uses an innovative digital dashboard platform which allowed us to track individual student’s understanding of concepts before, during, and after using our materials so that we were able to analyze their success and effectiveness. We revised our learning activities based on these results.

The Middle-Late Miocene (~11 million years ago) was a period of cooling following the Middle Miocene Climatic Optimum, the warmest period in the last 35 million years. However, because of the scarcity of fossil floras of that age, our understanding of vegetation response to this climate change is poor. The Middle-Late Miocene fossil flora from Vasa Park therefore adds invaluable information that allow us to better reconstruct how the vegetation and climate has changed over time in the Pacific Northwest. Previous work on the Middle-Late Miocene in this region has focused on macrofossils and pollen/spores; this study will add information about phytolith assemblages. Phytoliths form when silica from the ground is deposited within and around various types of plant cells. When the plant dies, the phytoliths from the plant gets preserved in sediment, retaining the shapes they had in the living plant. Our understanding of phytolith production in modern day plants and their climate preferences allows us to infer, from fossil phytoliths not just what kinds of plants they represent, but also something about the climate at that site. Specifically, Vasa Park will help us understand the flora of the Middle-Late Miocene in the Pacific Northwest and how it transitioned out of a very warm time into cooler current day temperatures. Based on our knowledge of the general climate during the Middle-Late Miocene, the Vasa Park assemblage should contain evidence of a more warm-adapted flora than what we see currently in the region. Indeed, preliminary evidence from phytoliths, macrofossils (leaves and flowers), and fossil palynomorphs (plant spores and pollen) indicate that the area was a relatively warm subtropical forest. The inclusion of the Vasa Park phytolith assemblage greatly adds to our understanding of Earth’s most recent warming period and helps us infer how vegetation will change with current warming trends.

Plants respond with a sink stimulation of photosynthesis when colonized by bacterial and fungal mutualists, which compensates for costs of carbohydrate allocation to the microbes. Problems may arise when light is limited and plants cannot increase photosynthesis. We hypothesize that under such conditions the costs for maintaining the mutualism outweigh the benefits, which ultimately turns the beneficial microbes into parasites exploiting resources and reducing host fitness. We study these plant-microbe interactions under different light availabilities using lima bean plants, rhizobia (nitrogen-fixing bacteria), and mycorrhizal fungi. In our study, we apply two levels of light (full light and light intensity reduced by 50%) and four levels of microbial inoculation (sterile soil, rhizobia, mycorrhiza, and rhizobia x mycorrhiza). Fitness-relevant plant parameters are measured including plant vegetative growth as well as flower and seed production. Our study will provide first insights into potential shifts in functional interactions between plants and plant-associated microbes under light limitation.

The Middle-Miocene Climatic Optimum (MMCO) was a period of rapid warming that occurred 15 million years ago. The climate change likely affected plant community composition as grasses spread across North America, but because of the scarcity of plant macrofossils from the MMCO in North America, the response of vegetation in the area to this climate change is poorly understood. Many modern crops are C3 and C4 grasses (distinguished by their photosynthetic pathway), and thus, gained understanding of the response of forest vs. C3 and C4 grasslands in deep time may help us predict the possible response of modern plants to our changing climate. Plant silica microfossils (phytoliths) are abundant in fossil soil layers however, and can be used to reconstruct the biotic response to the warming event. Polled Herefords is a site in southwest Montana that has easily accessed fossil soil layers from the MMCO that are phytolith rich. Phytolith assemblages from previously collected samples are currently being analyzed. Preliminary results show on average 2/3 – 3/4 of the phytolith assemblage in a given sample is grass silica short cells, diagnostic of grasses. The remainder of the sample is made up phytoliths indicating palms, monocotyledons, dicotyledons, conifers, and other non-grass types of plant. This suggests a fairly open habitat, and a high abundance of C3 grasses within the grass indicators indicate a climate that was warm and wet. More samples will be collected to increase the spatial and temporal resolution of the study, and to help determine if changes in vegetation reflect spatial or temporal variability, or both.

Ascidians are invertebrate chordates that live in the ocean and have an interesting life history. Normally, a tailed chordate larva hatches from the embryo, swims, becomes competent, and then responds to settlement cues, which initiate metamorphosis. Metamorphosis triggers apoptosis and tail loss. The notochord is lost as the tail reabsorbs and the juvenile settles to a substrate. In the ascidian family Molgulidae, species have evolved tailless larval development at least four times independently from a tailed ancestor. Using next generation sequencing techniques, we sequenced transcriptomes of a tailed and tailless species and their hybrid through development. We examined genes that have been shown to be expressed during metamorphosis in other ascidians and saw earlier expression of genes critical for metamorphosis in the tailless species. The number of transcripts were quantitated and we have cloned many of the genes. We show that there is early expression of metamorphosis genes in Molgulidae, including genes involved in the Mannose Binding Lectin (MBL) Complement pathway, such as MBL and MASP. We hypothesize that the maturation of the immune system early in tailless larvae could be a progression to competency and trigger to metamorphosis. We further hypothesize that a heterochrony in metamorphosis is responsible for the early activation of programmed cell death in the tailless ascidian larvae. In situ hybridizations were used to examine the localization of expression of these genes and quantitative PCR was used to independently validate RNA counts from our transcriptome data. Our ultimate goal is to elucidate the ascidian molecular trigger for metamorphosis and uncover whether heterochrony of metamorphosis leads to tailless ascidians.

Phytoliths, microscopic plant silica bodies, inform us about the types of plants that remain in the fossil record, allowing for reconstruction of past flora, climate, and ecosystems. The existing method for reconstructing vegetative structure and habitat type (open vs. closed) using phytoliths depends on the presence of presumed open-habitat grass types. This method may not be able to reliably reconstruct vegetation type when open-habitat grass phytoliths are absent, and depends on the assumption that open-habitat grasses have always been restricted to open environments. Therefore, a taxon-free, morphological approach may be more informative for inferring habitat openness using fossil phytolith assemblages. This study looks at the effects of light variation on epidermal cell shapes in modern grasses as a first step in creating a proxy for determining habitat type from the fossil record. Based on patterns observed in dicotyledonous plants, we hypothesize that epidermal grass cells will be more undulated when grown in reduced lighting condition. To test this, five species of grasses were grown in varying light treatments (20%, 60%, 100% (control), and 120% relative to the control). We took morphometric measurements of long and short cell types from epidermal peels and calculated their degree of undulation (UI). Results show three species where long cells had higher UI in deep shade (20% light) than in enhanced light (120% light), though the relationship is non-linear. With short cells only one species displayed a continuous increase in UI as light levels decreased. Thus, data from long cells show the greatest promise for use in the fossil record. To further test this method for paleoecological work, the UI of epidermal cells in soil phytolith assemblages from known modern habitat types will also need to be examined to see whether this UI and light correlation persists under natural growth conditions.

Organic matter (OM) production and decomposition is important to wetland health as it serves to cycle nutrients throughout the ecosystem. Current wetland monitoring programs in the Laurentian Great Lakes focus on structural indicators, such as fish, bird, and vegetation communities but lack the use of functional metrics such as organic matter processing. In this study we aim to create a new suite of functional indicators that focus on OM production and decomposition. Decomposition rates were measured using mesh litter bags containing dried bulrush tissue (Schoenoplectus acutus) as well as by the use of cotton strips incubated for 8 weeks at 20 coastal wetlands of Lakes Michigan and Huron. Nutrient limitation of decomposition was also measured using nutrient diffusing bags containing nitrogen, phosphorus, and a combination of nitrogen and phosphorus fertilizers. OM production rates were measured by periphyton biomass accrual on acrylic rods over an 8-week period. In addition, nutrient diffusing substrates were deployed to test the effects of nutrient limitation on algae growth. We predicted that decomposition rates would be positively associated with higher nutrient concentrations. We also predicted that algae growth rates would be greatest on substrates containing high nutrient concentrations. When developed, functional indicators could be used alongside current structural indicators to provide a better overall assessment of wetland health.

Vernalization is a mechanism by which plants induce changes in gene expression during cold winter conditions to allow flowering competency in the spring. While expression of the Flowering Locus C (FLC) gene represses genes required for flowering, vernalization inhibits FLC expression. Epigenetic modifications have been implicated in the vernalization process. Arabidopsis thaliana mutants for Histone Acetyltransferase of the CBP Family 1 (HAC1), a gene which encodes an epigenetic modifier, maintain FLC expression in vernalization conditions. We hypothesize that HAC1’s role in vernalization is to upregulate the expression of Swinger (SWN), a component of the Polycomb Repressive Complex 2 (PRC2). PRC2 is a complex of proteins known to contribute to the inhibition of FLC. Wild-type and hac1 plants were genotyped through 3-primer PCR to ensure homozygosity of the hac1 plants. To test our hypothesis, we have exposed wild-type and hac1 mutant Arabidopsis plants to cold (~5°C) as well as greenhouse (~22°C) temperature conditions. RNA was isolated from leaves of wild-type as well as hac1 plants subjected to these different conditions. Through RT-PCR, we will examine the relative expression levels of HAC1 and SWN as well as the control gene Eukaryotic Initiation Factor 4A-1 (eIF4a1)-a ubiquitous initiation factor unaffected by cold treatment. We anticipate that if our hypothesis is supported, SWN will not be expressed in the hac1 plants but will be expressed in wild type plants, supporting HAC1’s role in epigenetically modifying the SWN locus during vernalization. Future experimentation would require examination on whether SWN gene has undergone direct epigenetic modification by HAC1 or whether there is a mediating factor between the two genes.

Genetic and molecular analysis is important to understand the evolutionary trends and relationships between chordates and hemichordates. Hemichordates share significant characteristics with chordates, including pharyngeal gill slits, a worm-like body plan and a post-anal tail. We have been studying the development of the central nervous system (CNS) in hemichordates to compare development to chordates. During early development in chordates, Shh and BMP play major roles in neural tube formation and dorsal ventral CNS specification. Elav has been used as a neuronal marker in chordates and is expressed within neural plate during early neurulation. We looked for expression of Shh, BMP and Elav in hemichordates to compare the development of the CNS in each group. We cloned and sequenced Shh, BMP and ELAV in S. bromophenolosus and P. flava, using primers designed from S. kowalevskii. Our purpose is to examine the expression of Shh, BMP, and Elav in the developing neural tube in S. bromophenolosus and P. flava by in-situ hybridizations. We see expression of Elav in the CNS of S. bromophenolosus, and expect to see Shh expression in the hemichordate endoderm. BMP expression has already been reported in the dorsal ectoderm and neural tube of S. kowalevskii, but those results were interpreted as a dorsal-version in chordates. We suggest instead that this hemichordate expression pattern is identical to that of chordates during neurulation. These results suggest that hemichordate and chordate central nervous system develop by similar genetic pathways.

The process of a sperm entering an egg during fertilization is not well-understood at the molecular level for any organism. Drosophila melanogaster, the fruit fly, serves as an ideal model organism to study the normal process of fertilization and what goes awry in cases of sterility. A novel membrane protein termed GCS1 is suggested to play a crucial role in fertilization in angiosperm plants and protists. Disruptions of the gene in these organisms are sterile due to defects in gamete interactions. Comparative genomics suggest that GCS1-like proteins are conserved across a wide variety of eukaryotic species, including Drosophila, suggesting that the GCS1 gene is of ancient origin. We have cloned a genomic fragment containing a putative Drosophila GCS1-like homologue (dGCS1) and showed that it rescues mutations in a gene called pskl, which when mutated results in male sterility. This result confirms that dGCS1, like GCS1 in other organisms, plays a role in fertilization. However, our mutants also show that dGCS1 has roles in both sperm formation and sperm function during fertilization. Currently, we are using Minos, a DNA transposon inserted near a protein-coding exon of the dGCS1, to disrupt the gene. We have obtained over 100 Minos excision alleles and are comparing these derivatives genetically and molecularly to determine if we can define specific portions of the dGCS1 protein that contribute to male fertility. These ongoing studies should be informative for identifying regions of the Drosophila gene and protein that are important for function, and should shed light on how GCS1s in general function during fertilization.

Infection depends on the successful colonization of a host by a pathogen. For a virus to infect its host, it must come into contact with the host, gain entry into target cells, and replicate. There are a variety of factors that could influence the likelihood of a host becoming infected. For example, a higher concentration of virions in the environment will increase the probability that a host will come into contact with one. The relationship between viral concentration and the number of hosts infected could exhibit various forms, such as linear, logarithmic, or exponential. Viral genetic differences could also influence infectivity if certain genotypes are more effective at initiating infections. In this study we examined the relationship between dose size and the proportion of hosts infected by the fish rhabdovirus Infectious hematopoietic necrosis virus (IHNV), and whether this relationship differed between virus genotypes. We compared the minimum infectious dose of two genotypes, one with higher virulence (HV) and one with lower virulence (LV), to determine how infectivity is associated with virulence. Previous studies indicate that at 100% infection levels, HV reaches higher viral loads than LV, in addition to having a host entry advantage, but to our knowledge the minimum infectious dose of either genotype has not been investigated. We hypothesized that the more virulent genotype would have a lower minimum infectious dose. We infected groups of fish with 10¹, 10², 10³, 10⁴, and 2x10⁵ pfu/mL of one of the two genotypes. After allowing the virus three days to initiate an infection, the fish were euthanized and their viral RNA was extracted and counted with quantitative real-time PCR to determine whether each fish was infected. The information gained about infectious dose can be used to produce a more refined understanding of viral fitness, and thus, viral evolution.

Fungal endophytes live cooperatively within the tissue of nearly all land-plants. Symbiotic fungi can provide great benefits to their plant host, often observed as increased growth or stress tolerance. I hypothesize that fungal endophytes increase the invasive ability of agressively invasive plant species. Phragmites australis is a common wetland reed with a global distribution. Roughly 200 years ago an agressive European genotype was introduced to North America. This non-native genotype out competes native genotypes and forms dense monocultures along the edge of both salt and fresh water. These monocultures not only reduce plant diversity but manipulate the ecosystem by altering soil chemistry and changing the physical structure of the land-water interface. P. australis is a considerable problem along the Great Lakes in Michigan and in Grey's Harbor, WA. I have isolated and identified the most abudnant filamentous fungal endophytes from both WA and MI populations of invasive P. australis. The endophyte communities at each location show almost no similarity, suggesting that P. australis aqcuires endophytes from the environment rather than carrying endophytes with it. By innoculating rice and turf grass with P. australis fungal endophytes and growing them under various conditions, I aim to discover the symbiotic funciton of these fungi. Additional experiments investigate the effectiveness of various field tested fungicides on individual P. australis fungal endophytes grown in culture. The long term goal of this project is to develope a novel method for erradicating invasive plants by targeting vital mutualistic fungal endophytes. P. australis fungal endophytes in culture are sensitive to field tested fungicides and the next phase of this projet will investigate the effect of fungicides on symbiotic plant.

Populations of P. menziesii on islands should exhibit reduced genetic diversity and canopy coverage, lower population age, increased reproductive succession and reduced understory species richness with respect to Mainland populations. Populations sampling within the same region and elavation should exhibit similar patterns. Seedling density as well as distribution of trees in multiple size classes and their diameter to height ratios were presented for each site. Species richness represented plant community structure and constituted of understory plants and percent coverage for each species within the plots. Genetic analysis consisted of laboratory analysis for selected individuals within the sites. Despite the vulnerability of small trees in establishment due to predation and greater sensitivity to environmental factors, high energetic costs to maintain large support structures of older trees create an additional unexpected source of vulnerability within the population. As genetic variation is component of successful reproductive succession and establishment, reduced variation in populations could result in greater sensitivity to more extreme environmental conditions as might be expected with climate change.

The immune response of individuals with disrupted sleep such as night-shift workers has been shown to decrease relative to individuals with regular sleep patterns. Cytokines are small molecules secreted as part of the immune response to infection. Certain cytokines have been shown to regulate sleep, and this regulation likely constitutes a critical component of the immune response. A common consequence of exposure to unusual light-dark (LD) cycles, such as those under jetlag or shift-work schedules, is the internal desynchronization of circadian rhythms. This desynchronization is associated with disruptions in the sleep-wake cycle and we hypothesize that this disruption may be linked to abnormal secretion of cytokines. Using an animal model of internal desynchronization, we exposed rats to a 22-hour LD (11L: 11D) cycle which leads to desynchronization of the sleep stages known as Rapid Eye Movement Sleep (REMS) and nonREM sleep (NREMS). In these animals, the temporal sequence of REMS and NREMS changes stably between days of alignment, in which the sequence is normal, and days of misalignment, in which this sequence is disrupted. We will analyze levels of three cytokines, interleukins 1, 6, and tumor necrosis factor (TNF) over a 24-hour period in animals either bled during days of alignment or during days of misalignment. Our hypothesis predicts lower production levels of these cytokines in misaligned rats.The implications of this study may indicate that the alignment of circadian rhythms, particularly of the sleep-wake cycle, are critical to maintaining normal cytokine levels and to sustaining normal immune response.

Neurons contain two highly specialized compartments: the dendrite, which receives synaptic or sensory inputs, and the axon, which relays these signals to other cells. Different types of neurons often have unique, type-specific dendrite morphologies and proper dendrite patterning is important for normal neuronal function. Underscoring the importance of dendrite morphology to neuronal function, defects in dendrite patterning likely contribute to numerous neurological disease states and the cognitive decline that accompanies normal aging. However, the many genes that contribute to the complex dendrite patterns remain largely uncharacterized. To identify genes involved in this process, we conducted a genetic screen for mutants that alter the patterning of sensory neuron dendrites that innervate the body wall of fruit fly larvae. Briefly, we mutagenized fruit flies with EMS, a chemical mutagen, and screened for late-larval stage lethal mutations that perturb dendrite patterning using a fluorescent protein that allowed us to visualize a single functional class of sensory neurons in living larvae. Here, we report our analysis of a novel mutant, messi. Using live cell imaging and quantitative analysis, we found that messi leads to exuberant dendrite branching and intermingling of dendrites in sensory neurons. This latter defect is of particular interest since “self-avoidance” may represent a general mechanism for organizing sensory dendrites and little is known about the genetic basis for this phenomenon. Using time-lapse microscopy, we found that messi causes progressive dendrite defects: the dendrite patterning of messi mutants is indistinguishable from wild type early in development, but becomes abnormal later in development. Similarly, dendrite defects in many neurological disorders are progressive, therefore further analysis of messi may contribute to our understanding of how dendrite maintenance is deregulated in diseased states. Currently, we are working towards identifying the gene affected in messi mutants and determining whether messi affects dendrite patterning in other functional classes of sensory neurons.

Spontaneous activity (SA) in the developing brain is responsible for neural network formation processes such as proliferation, migration, axon extension and pathfinding, and synapse formation and maintenance. In embryonic mouse hindbrain, SA role in forming networks tends to be the most critical at specific developmental stage E11.5, when the activity is most robust and synchronous. The SA is thought to initiate in a group of serotonergic raphe nuclei located in the midline former rhombomere 2 (r2) region of the hindbrain. Voltage-dependent calcium waves propagate rostrocaudally in cells along the midline and laterally into the surrounding tissue (Hunt et al. 2005). Over time, SA decreases in frequency, later disappearing by stage E15. This project aims to investigate the mechanisms for this disappearance. Our experiments are done using electrophysiology and calcium imaging. Electrophysiology has shown that SA disappearance correlates in time with an increase in conductance as well as a hyperpolarization of the membrane potential. Calcium imaging has shown that application of BaCl2, a potassium channel blocker, can induce activity at stage E15. Activity at this stage can also be induced by raising extracellular potassium concentration, which effectively depolarizes membrane potential. This evidence suggests the up regulation of potassium channels in the mechanism of SA disappearance.

Understanding stem cell maintenance is crucial for delineating conditions such as tissue loss, aging, and cancer. Using the Drosophila female germ-line stem cell (GSC) model to observe stem cells in vivo, several extrinsic and intrinsic factors have been demonstrated to regulate maintenance of Drosophila GSCs; they have also been found to be conserved in mammalian systems. The search is on for novel regulatory factors of stem cell maintenance with the hope of extending our understanding to stem cells in mammalian systems. My project involves the characterization of the molecular function of Pineapple eye gene (Pie), a novel regulator of GSC maintenance. Through a loss-of-function screen in GSCs, it has been demonstrated that mutations of pie result in GSC maintenance failure. The amino acid sequence of pie revealed that it contains ring-domains suggesting possible E3 ubiquitin ligase function. Such proteins are key components of the ubiquitination pathway, which are involved in protein tagging and degradation. To test pie’s function as an E3 ligase, I am using a novel assay that employs the fact that bacteria do not have endogenous ubiquitination pathway and therefore utilizes Escheria coli as an in vivo system to express the components of the ubiquitination pathway and detect the formation of ubiquitin chains, a key indicator of E3 Ligase activity. I have demonstrated that the expression of pie’s ring-domains without a substrate is not sufficient for the detection of ubiquitin chains. I will now test whether the full length pie is necessary for proper ubiquitination. Further, I will also test whether pie’s functional domain needs a substrate in order to function effectively. Successful characterization of pie can reveal new pathways to target for conditions involving stem cell maintenance.

Embryonic stem cells are isolated from the early developing embryo and are capable of forming the different cell types found in the body. Understanding how these cells develop and maintain their specialized state is critical to understanding how they function. We hypothesize that embryonic stem cells acquire a unique metabolic state that aids them in maintaining their specialized state. Using two distinct mouse pluripotent states, embryonic stem cells (ESC’s) and epiblast stem cells (EpiSC’s) representing cells isolated from the inner cell mass of the blastocyst and the epiblast, a later stage in embryonic development, we found that EpiSC’s are much more glycolytic in terms of their energy usage compared to ESC’s as measured by oxygen consumption and extracellular acidification rates. Furthermore, using quantitative polymerase chain reaction experiments, we found that genes key to regulating and maintaining high rates of glycolysis are significantly upregulated in EpiSC’s compared to ESC’s. We showed that the overexpression of HIF1α, a known key regulator of these glycolytic genes, in ESC’s is able to shift the ESC’s towards a more glycolytic metabolic state and cellular morphology reminiscent of EpiSC’s. In addition, transition of ESC’s to EpiSC’s using the known chemical inducers Activin and Fibroblast Growth Factor 2 revealed that changes in the expression of metabolic genes occur prior to changes in expression of cell fate markers, which suggest that a metabolic transition in the ESC’s may be key in its transition to an EpiSC-like state.

MicroRNAs (miRNAs) are small noncoding RNA molecules that post-transcriptionally regulate gene expression. MiRNA biogenesis involves two key ribonucleases, Drosha and Dicer. I am investigating the Drosha enzyme. Previous research from the Ruohola-Baker lab reveals that reducing levels of Drosha in stem cells significantly reduces levels of some miRNAs, while only slightly reducing levels of another group of miRNAs. This surprising result led us to conduct a bioinformatics analysis to identify characteristics that differentiate these two groups of miRNAs. We discovered structural differences, specifically that bulges in the central region of miRNAs were more frequent in heavily reduced miRNAs than in miRNAs that were not affected. My mentor, Henrik Sperber and I propose a model explaining why miRNAs with mismatches (bulges) in the central region of the sequence are more sensitive to reduced levels of Drosha than rigid miRNAs with no mismatches in that region. To test whether a bulge causes differential miRNA processing when Drosha is the limiting factor, I am adding and removing bulges to primary miRNA transcripts, overexpressing them in cells with low Drosha levels, and comparing their mature levels using qPCR. In addition, I am testing whether in nature cells vary Drosha levels as a mode of regulation. A previous study shows that T cells at specific stages of differentiation reduce Drosha expression by as much as 10 fold. My own bioinformatics analysis reveals that certain tissues in mice have an 8-fold reduction of Drosha. To see whether reduction of Drosha levels correlates with differential microRNA processing, I am using microarray data to find subsets of microRNAs that have dramatically reduced levels when Drosha expression is significantly low. With my research, I hope to discover a potentially novel mechanism of regulation in which cells alter Drosha levels to selectively process a subgroup of miRNAs.

Paleontological evidence has shown that during large scale extinction events, hydrogen sulfide (H2S) rises to abnormal levels in Earth’s atmosphere. To survive these extinction events, an organism would be required to counter-act the negative effects of H2S by evolving unique adaptations. It can be hypothesized that many of these unique adaptations still exist in present-day lineages. Modern plants, for example, may have developed a method of utilizing H2S in order to survive, and possibly thrive, in what would be considered a highly toxic atmosphere. Through experiments involving species Phaseolus vulgaris and Pisum sativum, it was discovered that applications of H2S to seedlings increased growth at rates of up to 200% within 1 week. In addition to the increases in growth, increases in plant mass and photosynthetic activity were also observed. In order to better understand the effects of H2S on P. vulgaris and P. sativum, seeds of the species were also exposed to H2S, under the same controlled conditions. The effects of the seed experiment were similar to the initial seedling tests. Time to germination decreased with exposure, and the overall rates of germination increased. Large scale implications of these findings present benefits such as greater crop yields and perhaps a more efficient method of bio-fuel production. Continuing research of H2S and its effects on plant life could someday create a powerful means to deal with our ever-expanding human population…and the ever-expanding human footprint that goes along with it.

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

The geographic ranges of species are strongly influenced by climate. As temperatures increase, species are expected to shift poleward in latitude and upward in elevation, a prediction supported by observations of range shifts in recent decades. Species interactions also play a crucial role in setting geographic ranges of global biomes, and could be critical in determining the response of species to climate change. Due to limited data on interspecific competition in the boreal biome, Earth's largest land biome, it is largely unclear how climate change will affect the range limits of the individual tree species that occupy it. In recent decades, spruce trees (Picea engelmannii) have increased in abundance at high elevation locations formerly dominated by larch trees (Larix lyallii). This change is thought to result from improved climatic conditions for spruce establishment but has not been tested. I hypothesize that spruce have higher establishment rates than larch where the two co-occur when temperatures are high. To test this hypothesis, I will harvest 120 stems of each species at locations in the North Cascade Mountains in the summer of 2012. I will then count the number of annual growth rings for each sapling to determine establishment year. Next, I will model the relationship between establishment rates and climate in different decades of the last ~100 years for each species. Finally, I will compare the establishment-climate relationships of the two species to infer how the relative abundances of the species might shift with warming. These results have large-scale implications for changes we should expect to see in global biomes as warming continues. In particular, this hypothesis suggests that spruce will replace larch as climate change progresses.

Plants use hormone-signaling pathways to regulate growth and development. Brassinosteroids (BRs) are critical plant hormones that regulate growth such as the elongation of the embryonic stem. We study the specific mechanism of this process by testing the hormone-induced growth responses of both wild type and mutant Arabidopsis seedlings. We are testing whether growth repressor SPATULA is involved in BR growth response. We hypothesized that the growth repressor SPATULA is downstream of the BR pathway. To test this hypothesis, we acquired mutants that lack SPATULA function. After confirming the genotype of the mutants by PCR, we used a high-resolution camera to observe growth throughout early development in response to hormone treatments. Data is recorded by the camera over the course of 132 hours. The images are then analyzed and growth rates are calculated. Using these data, we determined if SPATULA is involved in the BR growth response.

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

As carriers of globally prevalent diseases such as Malaria and Dengue Fever, mosquitoes are an important model for research in vector biology. Previous studies have shown that many mosquito species are capable of changing their host-seeking behavior to match environmental shifts in host availability. However, the sensory mechanisms that mediate the shifts in hosts remain unknown. Because many other insect species are capable of learning novel associations throughout their lives, we hypothesized that this behavioral shift in mosquitoes was an indication of a similar capacity for learning. This study examines the degree, speed, and accuracy with which female Anopheles stephensi mosquitoes respond to Pavlovian conditioning. We paired seven different odors with a blood reward to gauge the change in the insects’ reactions to these scents over time. We found that mosquitoes were capable of Pavlovian learning, but not when stimulated with odors known to be naturally repellent to mosquitoes. Moreover, compared to other insects, mosquitoes quickly learned the association between the reward and the novel odor. They also demonstrated selectivity towards the odors they were trained on, and showed little response to odors that were similar to the trained odor, suggesting that their learning process is highly odor-specific. These findings suggest that learning behaviors may be an important factor to mosquito survival in natural conditions. Pavlovian learning in mosquitoes has never before been shown, and further research into the neural mechanisms mediating the shift in host preferences could very well lead to advancements in the prevention of mosquito-borne pathogenic infections.

The fossil record in western Montana and central Oregon of the late Oligocene-early Miocene period, about 20-30 million years ago, presents a rich fauna of gophers (Geomyidae) of the subfamily Entoptychinae. These burrowing creatures may represent up to 25% of the mammalian fauna of certain fossil assemblages. Understanding the ecology of this large population is thus important for interpreting community-wide ecological changes. Prior to investigating their ecology, it is necessary to understand the taxonomic and morphological diversity of these fossil gophers. Previous attempts have been made to differentiate these gophers by using cranial and dental material. However, skulls and dentaries of such small rodents are rarely preserved. Isolated teeth are sometimes the only material available for identification. As a consequence of their abrasive diet, geomyids wear down their teeth over time, erasing potentially diagnostic features of the occlusal surface of the tooth. Therefore, several authors have suggested that differences in the shape of the teeth could be used as a means of species differentiation. This hypothesis has yet to be quantitatively tested. Geometric morphometrics allows such analysis of shape independently of size. This method involves the statistical comparison of geometric coordinates of landmarks placed on an object of interest. I will apply this method to the enamel band of the last premolar and last molar of 140 fossil geomyids. The goal of this study is to test the hypothesis that the shape of Entoptychine teeth can be used as a tool for species differentiation. Preliminary results of the analysis of M3s reveal statistically significant differences between genera. More specifically, the angles of the tooth (tilted anteriorly or posteriorly), its shape (rectangular versus square), and the proximity of its two lophs, appear taxonomically diagnostic. This analysis will allow me to distinguish between gopher taxa, and allow the investigation of their ecology and potential roles in the community.

Differentiated human embryonic stem cells (hESCs) demonstrate the potential for regenerative medicine and drug discovery utilizing rationally assigned cell fate. But hESC are limited by the technical difficulty of recapitulating an ever growing set of cellular phenotypes using a finite set of pluripotent cell lines. The resulting molecular deficiencies limit the ability of differentiated cells to integrate into host tissue, and diminish the translational potential of findings based on differentiated hESC. Induced pluripotent stem cells (iPSCs) can be derived from patients with or without genetic disorders, and have much greater use for drug discovery and basic research, and patient derived iPSCs are less prone to rejection by host tissue. However, iPSC generated by insertion of genes into the host genome, increasing risk of rejection and limiting maturation of iPSC-derived cells. RNA-induced pluripotent stem cells (RiPSC) offer a solution to problems of genomic modification by inducing pluripotency exclusively through the transfection of mRNA. MicroRNA (miRNA) exhibit widespread regulation of mRNA translation and degradation, and do not directly alter the genome. A bioinformatics study curating publicly available microarray data for components of the miRNA biogenesis pathway, pluripotency factors, and miRNA expression will generate hypotheses about the functions of miRNA and relevant proteins, with a focus on differentiation and neural cell fate. Quantification of miRNA and mRNA expression in hESC and RiPSC during retinal differentiation will support or disprove hypothesized relationships between protein and miRNA expression. Transfection of miRNA will be used to determine the ability of these miRNA to enhance differentiation and maturation of retinal cells. Additionally, the resulting retinal progenitors will be implanted into mouse retina, directly testing the potential of miRNA to improve clinical outcomes of stem cell based therapies for the nervous system.

We are focusing our study on mushrooms in the genus Cortinarius that have a symbiotic association with the roots of trees. Our specimens are from an Oregon White Oak forest located in the Columbia River Gorge, Washington. The Cortinarius species associated with Oregon White Oak form a unique combination of species in comparison to those in conifer forests from the Pacific Northwest. Cortinarius species from Oregon White Oak strands do not reproduce every year, consequently the data we have from Oregon White Oak strands collected over a period of three years provides a special opportunity to study these unique species. We have multiple collections of a large number of species, and it is likely that many of them will be new to science. DNA sequences of the ITS-28S gene regions are being used to determine species diversity and the relationships among species. Molecular analyses provide an excellent tool for studying relationships among mushroom species and will allow us to catalogue the diversity of the oak associated species in the Pacific Northwest. This project is part of a larger study of Cortinarius in the Northern Hemisphere.

Ongoing neurogenesis in the adult brain is a fundamental process of neural plasticity. The songbird is an established model for studying neuroprotection, neurogenesis, and neuronal turnover due to dramatic plastic changes in morphology and function in the nuclei that control avian song production. HVC, a region of the brain that times the production of song, doubles in size at the beginning of the breeding season, largely as a result of an increase in new neuron incorporation. The new neurons also send projections to and stimulate growth in their target nucleus, RA. In the non-breeding season the song control circuit rapidly regresses in size. Full growth and regression of HVC occur within 7 days of a rapid transition between breeding and non-breeding conditions, and neuron number changes by around 25% (> 68,000 neurons) due to neuronal recruitment or apoptosis, respectively. We examined the time course of neuronal turnover in relation to changing rates of apoptosis and neuronal recruitment during periods of rapid growth and regression of HVC. To test the hypothesis that turnover of new neurons is elevated during non-breeding season, we labeled two cohorts of new HVC neurons with thymidine analogs at one, two and four month intervals in birds maintained in breeding and on-breeding condition. We determined turnover rate by measuring the number of new neurons in each cohort of neurons at the different intervals. Preliminary data suggest that turnover rate is higher in non-breeding conditions. We also find that the balance between neuronal incorporation and apoptosis in HVC changes rapidly during transitions between breeding and non-breeding conditions. These studies show that neuron number in HVC results from a shifting balance between neuronal birth and death.

Essentially all growth and developmental processes in plants require action of the hormone auxin. Auxin regulates gene expression by mediating the degradation of repressor proteins called Aux/IAAs. At low concentrations of auxin, these IAA proteins repress auxin-responsive genes by binding to auxin response factor (ARF) activating transcription factors. A subset of ARF and IAA proteins are involved in the initiation of new lateral roots, which are important for overall root architecture and function. Mutant phenotypes in these ARFs and IAAs fall into two separate classes. Based on this evidence, we hypothesize that the structural properties of specific ARF and IAA proteins cause them to have specific binding preferences. For example, we predict that ARF5 has stronger binding preference for IAA12 than IAA14, while ARF7 has stronger preference for IAA14. To test interactions between these proteins, we are expressing them in the yeast, S. cerevisiae. This allows us to look at individual protein interactions at single cell resolution, which is not possible using a plant based system due to the ubiquity of auxin responses and the presence of multiple IAAs and ARFs. We are using a bimolecular fluorescence complementation approach, in which two complementary halves of fluorescent proteins are fused to two interacting proteins of interest. Specifically, we have fused halves of different fluorescent proteins to IAAs and ARFs, such that each specific IAA-ARF complex exhibits a different color of fluorescence. Using this system, we will compete two different IAA proteins for binding of a single ARF. This approach will allow us to analyze the binding preferences of proteins through quantitative comparison of fluorescence of protein complexes. The results of my research may explain some of the specificities between ARF and IAA mutant phenotypes found in Arabidopsis and how distinct protein properties lead to differences in gene expression and development.

The diatoms are a prolific and productive group of phytoplankton belonging to the Phylum Heterokontophyta. They are responsible for ~23.5% of global primary production and their high photosynthetic capabilities depend on their abilities to synthesize chlorophyll. A key step in the biosynthesis of chlorophyll is catalyzed by the enzyme 'light-dependent protochlorophyllide oxidoreductase’ (POR). Early in the evolution of diatoms, the gene encoding this enzyme (por) was duplicated, resulting in two homologous but later divergent genes: por1 and por2. We aim to create a comprehensive heterokont por gene phylogeny to determine whether the gene duplication event occurred before diatoms diverged from sister taxa in the Heterokontophyta, early in diatom evolution, or in only one diatom lineage. Additionally, this phylogeny will allow us to determine the effects of natural selection in shaping the evolution of these genes. Preliminary studies suggest that positive selection acted in the past on both por1 and por2, though these protein sequences are now conserved within diatoms. To create this por gene phylogeny, we will sequence the por genes of various organisms representative of the major orders within the diatom class, Bacillariophyceae. Additionally, we have successfully sequenced numerous representatives of other Heterokont classes that will serve as the background to the Bacillariophyceaen gene phylogeny. These studies require the cultivation of various strains of algae, DNA extraction, PCR with por specific primers, and gene sequencing.

Our goal is to introduce a transgene into the plastid genome of maize instead of the nuclear genome because a plastid-localized transgene is not transfered via pollen. Plastid transformation will be performed using vectors based on plastid DNA (ptDNA) replication, terminal sequences, and and maintenance within plant tissues, work previously done in our lab. The first step is to extract ptDNA from maize seedlings. PCR is used with the ptDNA to get a product containing the plastid genome targeting left and right border sequences (LBS and RBS, respectively). To be more specific, the region amplified corresponds to nucleotides 128500 to 131000 of the maize plastid genome and designated 5'End2-to-3'End1. The second step is to insert 5'End2-to-3'End1 into the cloning plasmid pUC18, creating the plasmid, pZMCP101. It also must be mentioned that three other vectors will be tested, using slightly different LBS/RBS (3'End2-to-5'End1, 5'End2-to-5'End1, and 3'End2-to-3'End1). The next series of steps adds expression control regions and reporter genes between the LBS/RBS. We use the16s rRNA promoter and psbA 5'-and 3'-UTRs regions from maize ptDNA. For reporters the gfp gene, a visual marker, and pmi gene, metabolic selection, are used. PMI will be used because a plant without the pmi gene will not grow on mannose. So, after we combine gfp, pmi, and the control regions with pZMCP101, we now have pZMCP201-the final plasmid vector. Then we will shoot pZMCP201 into maize plastids using particle bombardment. We will use both the circular and linearized vectors and expect greater efficiency with the latter. We will be able to determine if the vector was incorporated into the chloroplast genome by visualization of GFP expression and growth of transgenic tissue on manose selection medium. 

Many plants, as part of their growth process, accumulate groundwater-derived silica that is deposited into inter- and intracellular spaces, forming what are known as phytoliths. Due to a combination of preservational and diagnostic characteristics, phytoliths found in the fossil record serve as a powerful paleontological tool for the reconstruction of past environments and climates. For this study, rock samples containing phytoliths were collected from a site called Everson Creek in southwest Montana, once part of the Great Plains region of the United States, which spans in age from 25.3 to 24.5 million years and corresponds to an interval of warming worldwide. The samples have been processed for phytoliths using heavy liquid flotation, whereby silica bodies are separated from other material by means of their unique density. Currently, the phytoliths are in the process of being counted and divided into various diagnostic groups according to morphology. The results of these counts, when viewed in temporal order throughout the site, will shed light on the floral and climatic history of the region based on the prominent plant groups and their growth preferences. This research, which covers a time interval previously unstudied for phytoliths aims to clarify the timing of the rise to dominance of open-habitat grasses in the region, a subject that is currently disputed in the literature. The spread of grasslands is an important event in plant history because it represents the development of open habitats and is intimately linked to the expansion and evolution of various other biota. Since grasses are large producers of highly diagnostic phytoliths, any change in their ecological role will easily be seen in this study.

The evolution of a complex structure in organisms that share only a distant evolutionary history provides an opportunity to study the phenomenon of convergent evolution. One such convergent structure is a particular kind of laterally compressed blade-like shearing tooth in mammals known as a plagiaulacoid molar or premolar. Plagiaulacoid dentition has evolved independently in at least four clades of mammals: the rodent-like Mesozoic multituberculates, early primates known as carpolestids, modern and extinct South American shrew opossums, and several families of Australian diprotodont marsupials, including modern and extinct kangaroos, possums, and others. To understand the selective forces that led to the independent evolution of this specialized trait in multiple lineages, I correlated measures of tooth complexity with the known diets of modern plagiaulacoid marsupials, using this data to then infer diet in extinct taxa and compare trends in complexity among these distantly related groups of mammals. To quantify dental complexity I created 3-D digital models of the lower cheek teeth of representative taxa and used custom GIS software to generate orientation maps of the tooth surfaces. Contiguous pixels with the same orientation were then grouped into patches that approximate the number of different shearing surfaces available to mechanically process food. Orientation Patch Count (OPC) is a measure of complexity that allows me to place mammals into broad dietary categories based on the amount of plant material in the diet. This framework can be used to infer the feeding ecology of extinct mammals from digital models of fossil teeth. If the plagiaulacoid phenotype is an adaptation to similar selective pressures, I expect to find a similar range of OPC values and a similar amount of variation in complexity between plagiaulacoid clades, indicating comparable dietary range even though these groups vary widely in geographic location, temporal distribution, and evolutionary history.

Understanding modern climate changes hinges upon our ability to assess past climates. While relatively few analogs to present climatic shifts are represented in recent geological history, the Paleozoic Era (542-251 Ma) may offer more. Investigating such transitions in the more distant past though will require expansion of existing techniques. Several methods use fossil leaf morphology to infer past climatic conditions. One such approach, DiLP (Digital Leaf Physiognomy Approach) uses leaf margin serration (toothiness) and leaf shape to infer past mean annual temperature. This technique has proven successful in using modern plant communities to estimate regional climates but can only assess past temperatures as far back as 120 Ma since it currently only utilizes leaves of recently-evolved flowering plants. However, other, more ancient groups of vascular plants have also evolved leaves with toothed margins. With toothed leaves and a fossil record exceeding 350Ma, lycopsids (clubmosses and allies) and ferns have potential to extend paleotemperature assessments back to the Late Paleozoic. To determine whether climate (in particular, temperature) influences leaf shape in living representatives of these lineages, three species of lycopsid (Lycopodium clavatum, Lycopodiella alopecuroides, and L. appressa) and two species of fern (Polystichum munitum and Blechnum brasiliense) were cultivated in a growth chamber under two temperature regimes: 15°C and 25°C, each trial lasting five months. Several leaves were selected from each specimen, laminated on overhead transparencies, and scanned for digital measures using a modified DiLP measuring protocol. While quantitative leaf measurements remain ongoing, preliminary qualitative assessment has shown that lycopsids radically change their growth habit when grown under different temperature regimes. If leaf margin toothiness or shape changes significantly in the leaves of these plants in response to temperature, fossil representatives of these ancient lineages could potentially be implemented to better assess climatic changes long-preceding the age of flowering plants.

Rhododendron is an aesthetically valuable plant genus primarily distributed in Northern hemisphere. Previous work by collectors and taxonomists has revealed that Rhododendron taxa have a scattered pattern of distribution around the globe. Yet, the evolutionary history that led to this modern distribution pattern is not well understood. Fossil records of once extant Rhododendron populations can be a key component for inferring the diversification and migration of Rhododendron. Macrofossils such as leaves and stems have more restricted requirements in their formation, and hence are rarely discovered. On the contrary, microfossils of pollen are more widely preserved and found. In this study, I explored the potential of using pollen microfossils of Rhododendron to understand how the modern distribution pattern of Rhododendrons came about. To that end, I compared and contrasted modern Rhododendron pollen from three subgenera and twenty four subsections in order to discover the degree of consistency in pollen morphology within and unique combination of traits of each taxon. All observations were performed under light microscopy. Experimental data include both structural differences and quantitative measurements. Preliminary results demonstrate promising level of variation in measurements such as the structure of apperture edges, pollen wall sculpture, overall size, and wall thickness. These palynological features are then fitted into a Rhododendron phylogeny based on molecular data to reveal any potential taxonomic significance of variation in pollen morphology. 

Within the previous year, over half of a million people died from malaria and ninety one percent of these deaths occurred in Africa. My research studies the living conditions of the Maasai tribe in correlation to the prevalence of malaria, intestinal parasites, and head fungus. During field research in Tanzania these were the three most widespread diseases among the three hundred patients treated. The life cycles and causes of each disease were studied individually to distinguish how specific environmental and household factors contributed to the transmission of the diseases. Relevant factors of the living conditions were identified. I hypothesized that the incidence of disease would be reflected in the prevalence of the elements relating to the transmission of disease. I collected data on the living conditions in Tanzania by visiting Maasai households and evaluating the following criteria: the presence of a fire inside the house, the presence of feces and/or garbage in the household, the number and size of the windows in each household, the number of people living there, the number of people who were sick living there, the presence of uncontained animals in the compound, the presence of mosquito nets, the type of material used to make the house, the presence of a toilet, and the presence of a cover on the toilet; if applicable. Analysis of the results suggests that for each disease, the relevant factors of living conditions matched expectations in all three diseases. These results suggest that by making certain improvements to the living conditions of the people in the Maasai village, it would be possible to reduce the prevalence of the tropical diseases of malaria, intestinal parasites, and head fungus.

Cooperation is a fascinating paradox of evolution. Why would an organism incur a cost to itself in order to benefit another individual? The Achilles’ heel of cooperation is that it can be exploited by cheaters. Cheaters reap the benefits of cooperation, but do not incur the same costs as the cooperators. Cheating is therefore considered a major problem in the evolution of cooperation. One possible way of reducing the vulnerability of cooperating organisms to cheating is through communication. Communication allows individuals to cooperate only when the conditions are favorable. My research involves a form of bacterial communication called quorum sensing, which allows bacteria to accomplish cooperative tasks that require a collective action. Pseudomonas aeruginosa, an opportunistic pathogen that causes severe infections in immuno-compromised individuals and patients with cystic fibrosis, uses quorum sensing to regulate the production of toxins and enzymes. We used a wild-type cooperating strain of P. aeruginosa and several non-cooperating cheater strains to investigate the relationship between cooperators and cheaters. We predicted that the cheater strains would benefit when grown together with cooperators in a well-mixed environment, but suffer in a structured environment. We tested this prediction through competition assays in both a shaken liquid environment (unstructured) and a soft-agar environment (structured). Combined with further research, our results may suggest that communication helps a cooperating population avoid collapse when faced with cheaters.

Cooperative interactions are observed at all levels of biological organization. Cooperation facilitates major transitions in evolution such as from unicellularity to multicellularity. How a cooperative system evolves from its incipient state is unknown. I have used a synthetic cooperative system termed CoSMO (Cooperation that is Synthetic and Mutually Obligatory) as a model for incipient cooperation. CoSMO is an engineered yeast system consisting of two non-mating strains, differentially labeled with fluorescence. It relies on direct exchange of distinct metabolites between the two cooperators. Evolved CoSMO cocultures exhibit an improvement in viability, defined as an increased ability to survive reductions in population density. I have determined that this viability improvement is heritable, by measuring the viability of reconstituted evolved CoSMO pregrown in rich media which allow cells to grow in the absence of cooperation. Furthermore, by mixing an evolved strain with its ancestral partner, I determined that a single evolved cooperator was responsible for community-level viability improvement. I am characterizing phenotypic changes in evolved cooperators that contribute to increased viability. The phenotypic changes can potentially be categorized as either “self-helping”, such as through increased starvation tolerance and higher affinity for metabolites, or “partner-helping”, such as through increased metabolite release. In all cases, I will identify the molecular basis for these adaptations through genome-wide re-sequencing. Furthermore, genomic analysis on entire evolved CoSMO cocultures will be performed to reveal the diversity and frequency of relevant mutations in the population over time. Lastly, I will perform similar analyses on replicate populations. If similar adaptive changes that result in improvement in system viability are observed, a deterministic process to improve viability is implied. Otherwise, diverse mechanisms can achieve improved fitness. In both cases, we will gain insights into how cooperative systems evolve from inception.

Retrotransposons exist as genetic elements within chromosomal DNA that can replicate themselves and move to new locations within a genome, using a process involving an RNA intermediate. They are a widespread class of transposable elements, abundant in plants, fungi, mammals and yeast. One type of retrotransposon is a Long Interspersed Nuclear Element (LINE). Unlike most retrotransposons, LINEs have no long terminal repeats (LTR) and they possess a poly A tail which defines the 3’ terminus of the element. They are several kilobases long and contain two open reading frames (ORFs) encoding a gag protein (ORF1) and endonuclease and reverse transcriptase domains (ORF 2) conferring the element’s ability for retrotransposition. Due to incomplete insertion attempts, many 5' truncated copies of LINE elements are present in the Rhododendron genome. A family of LINE elements called RLINE and several related 5’ truncation fragments have been discovered in Rhododendron williamsianum. I used PCR primers to amplify the regions of the genome in R. williamsianum where truncated RLINE insertions were found. The amplified DNA was then sequenced by Sanger sequencing. I am analyzing ten 5’ truncation fragments ranging from 692- 2918 base pairs. By phylogenetic analysis of aligned sequences common to the various full-length and deleted RLINES, I will endeavor to determine the evolutionary relationships of the truncated fragments to the full length RLINE and to one another. So far, I have sequenced and compared 8 different RLINE fragments of Rhododendron williamsianum, and will continue characterizing RLINE insertions.

Flight control in dynamic environments is a complicated task that many animals accomplish with ease. It requires rapid integration of sensory input as well as balancing long-term planning with reflexive behaviors. The giant hawk moth Manduca sexta is a useful model organism for studying flight control because its physiology is well understood and it is easy to rear in captivity and work with experimentally. In order to make flight control adjustments, Manduca constantly integrates information from slow-sensing visual sensors and fast-sensing mechanosensory organs. Both visual motion and mechanical rotation elicit strong abdominal reflexes in Manduca during tethered flight, however the exact role of the abdomen in flight is unclear. Previous work has suggested that abdominal reflexes help maintain flight stability, but it has not been shown that the abdomen is actively used for control. The goal of my research is to determine if Manduca can actively use their abdomen to stabilize the visual field. To answer this question, I have created a virtual flight arena composed of a projection system, magnetic tether, and IR sensor. The visual stimulus is projected onto an acrylic dome within which the moth is suspended by the tether. The experiment consists of a system where the moth controls the velocity of a horizontal bar displayed on the dome with its abdominal position. The bar has an initial velocity that the moth counteracts by moving its abdomen in order to stabilize the visual field. The precision of the moth’s abdominal control capabilities can then be tested by differing the initial velocities of the bar and varying the way that abdominal position controls the bar’s velocity. In addition to answering my research question, this project provides a framework to study the general multi-input multi-output problem in neural control.

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

Deuterostome adult body plans greatly differ between echinoderms, chordates and hemichordates. Genetic analysis has helped to understand the evolution of deuterostomes and the relationships between species. In this project, we have sequenced the Hox genes in Ptychodera flava, an indirect developer of the hemichordates, which has larvae that are morphologically similar to echinoderms. This information, along with gene expression data, gives insight to the similarities in developmental patterns between phyla as well as insight to their evolution. Once the genomic organization of Ptychodera flava Hox genes and Hox gene expression is complete, we can begin to deduce the developmental pattern of the common ancestor of hemichordates and echinoderms. Since the Hemichordate phyla contains both indirect and direct developers that are equally related to echinoderms, which are indirect developing, a deep comparison between gene expression and genomic organization will help us understand their common ancestor. We approached this by sequencing Bacterial Artificial Chromosomes (BACs) that contain the P. flava Hox complex and by examining gene expression via in situ hybridization. We are specifically interested in whether the Hox complex is rearranged, as in echinoderms, or co-linear, as in chordates and where the genes are expressed during different stages of development. Results show that P. flava Hox 5 through Hox 11/13c are collinear on the chromosome, but the arrangement of Hox 1 through Hox 4 is still unknown.

Presently, the United States uses 19.6 million barrels of oil per day. This amount represents 25% of the world’s total consumption, making the development of an alternative biofuel one of the world’s top energy and environmental priorities. Rose Ann Cattolico’s laboratory is working to create that alternative fuel through the use of algae lipids. A number of different tests are being conducted on physiology of selected algal strains with the goal of increasing fatty acid production. A most promising algal strain has been identified. This organism (referred to as P3) was recovered from a lake in Colorado. P3 was put through reiterative flow cytometry selection, creating what is now called P4. The P4 cultures were then subjected to antibiotic selection, creating P5.5 and P5.6. My study is to: (a) determine the taxonomic identity of this organism using molecular techniques. I hypothesize that this alga is a new species in the taxon Chrysochromulina and (b) document whether P3 has mutated while in culture. To achieve these goals I will do the following experiments. (a) The closest taxonomic relatives to Chrysochromulina sp (eg. C. parva) will be compared by sequencing the 18S ribosomal RNA, psbA , and nad5 genes. The results of these experiments will help determine the relationship of Chrysochromulina sp. and other members of this taxon. (b) The divergence of “derived strains” from P3 will be determined, which all iterations of P3 – P5.6 will be examined, using the same gene set presented above. Gene sequencing will also be used to check for other iterations of P3 that have been maintained in different media. My studies will reveal whether P3 has mutated as a result of directed evolution in the laboratory.

During early development, spontaneous waves of electrical activity propagate across many different structures in the central nervous system. These waves are a critical feature of neuronal development, regulating neuronal migration, physiological maturation, and synaptic connectivity. In slices of mouse cerebral cortex taken at early stages (embryonic day 18 – postnatal day 1), we observed circular clusters of cells (domains) that were synchronously active during times when the slice was not generating spontaneous waves of activity. The domains consisted of approximately 10 to 50 cells and activity appeared to initiate from a center cell before the synchronous activation of the surrounding cluster of cells. We hypothesize that domains may form the functional basis of activity that propagates across the entire cortex. We have applied a range of drugs to elucidate the mechanism that triggers these small coactive domains. The domains do not appear to be dependent on electrical activity, they are not mediated by the neurotransmitters gamma-aminobutyric acid (GABA) or glutamate, and they are not dependent on calcium channels or the release of internal stores of calcium. Application of the gap junction blocker octanol inhibited the appearance of domains, but questions remain as to the specificity of octanol. Future work will identify the type of cell that triggers the domain and further study the relationship between the presence of domains and development of the neocortex.

Evolutionary biology studies how populations change over time. For evolution to occur, there must be genetic variation, ultimately from mutation. Any mutation in a given environment may have a positive, negative, or neutral effect on fitness. Evolving populations tend to increase in fitness. A metaphoric “fitness landscape” is useful for visualizing this, with genotypes linked by mutations and elevation being their fitness. Fitness “peaks” denote genotypes from which all mutations are deleterious (downhill). If the landscape is smooth (possessing only one peak) then populations will always converge there by natural selection. However, if the landscape is rugged (multi-peaked), populations might get stuck on a low-fitness peak. This population must cross the “valley” between peaks to reach the higher peak. We are studying how migration patterns between subpopulations of Escherichia coli influence adaptation. Structured populations only allowed migration between nearby subpopulations of bacteria, while unstructured migrations occurred between any of the 95 other subpopulations of bacteria. We found that structured populations reached a higher fitness than unstructured ones. My project is investigating the genetic mechanisms behind these results. Using Illumina technology, I have completed whole genome sequencing and targeted sequencing on these populations. I am using these results to determine the type and frequency of mutations acquired in structured vs. unstructured populations. We have two hypotheses explaining structure’s effect on adaptation: the Mutational Distance Hypothesis and the Diversity Hypothesis. The Mutational Distance Hypothesis predicts that there will be more mutations accumulated in the structured populations than the unstructured, whereas the Diversity Hypothesis predicts that structured populations will have greater genetic diversity than unstructured populations. This work will lead to a greater understanding of adaptation and may have medical implications concerning the understanding bacterial evolution.

Cell migration and cell adhesion are crucial processes for embryonic development, homeostasis and cancer progression. Regulated changes in adhesion are coordinated with cytoskeletal change, which result in morphogenesis. Conversely, the loss of cell to cell adhesion can promote tumor cell metastasis and change in cell motility. Thus, examining the specific regulator of the adhesion and cytoskeleton is important in terms of furthering our understanding of the development and disease progression. We specifically study an important protein regulator called p120 catenin and its involvement in cell migration in the zebrafish embryo. An extensive literature implies that p120 catenin both positively and negatively regulates cell adhesiveness through phosphorylation. Cell cultures show that phosphorylation of serine residues strengthens the adhesion and that phosphorylation of tyrosine residues increases the cell mobility. After the morpholino knockdown of normal p120 catenin, I will co-inject the mutant Y228F p120 catenin. Then I will observe whether the mutant without the tryrosine phosphorylation rescued normal development. For this I will use in situ staining of the somites with myoD at the 3 to 15 somite stage. To observe migration and protein localization, we will use a confocal microscope with the embryos' cells labeled with cherry fluorescent p120 catenin, green fluorescent Rho GTPase, or a green fluorescent plasma membrane. If mutant p120 catenin, which cannot be phosphorylated, can rescue successfully, we conclude the phosphorylation site does not play an important role in cell-cell adhesion or cell migration. However, if it does not rescue the morpholino knockdown, tyrosine phosphorylation at the 228 site is important for p120 catenin’s modulation of cell–cell adhesion and cell migration.

The Dickinson lab studies the neurobiology of fruit flies. Particularly, we tested the effects of the neurotransmitter octopamine on a fly's ability to respond to visual stimuli while flying and walking. Evidence shows that a set of visually sensitive cells that are thought to be involved in stabilizing reflexes during flight have different responsive properties with and without octopamine. To further investigate the role of octopamine in this system, we looked for optomotor response deficits in flies that do not produce octopamine. We used mutant Drosophila melanogaster flies that no longer have the gene for enzyme tyramine-beta-hydroxylase, which converts tyramine to octopamine. As a result, these flies build up an excess of tyramine and are unable to produce octopamine. We tethered a fly to a metal pin, and placed it an LED light arena, where we presented it with different moving stripe patterns. We placed an infrared light above the fly, and an infrared sensor below it. Infrared light was used because it is outside the visual range of the fly and would not interfere with our intended visual stimuli. The fly cast a shadow on the sensor, from which we computed the wingbeat frequency and amplitude. We used the left and right wingbeat amplitudes to determine the fly's torque, which indicates its turning response. We analyzed this turning response to measure the fly's ability to respond to the visual stimuli. Lastly, we compared the octopamine-null fly's response to wild type flies. These experiments will further our understanding of the role of octopamine in the optomotor response system of fruit flies.

Interspecific hybridization is common in flowering plants.  Hybrids tend to exhibit characteristics intermediate between both parents, and to grow more vigorously.  A novel specimen of the genus Heuchera (alumroot) found in a garden setting in Seattle is thought to be a hybrid between the commonly cultivated Heuchera sanguinea and the PNW native H. cylindrica.  In nature, H. cylindrica and H. sanguinea have non-overlapping geographic distributions.  Heuchera cylindrica is native to the western United States and Canada, whereas H. sanguinea is native to New Mexico and Arizona and it is not known if interbreeding between the two species is possible.  The unknown specimen in question shows intermediate characteristics of both species, which include flower color, inflorescence architecture and more vigorous growth.  To determine the relationship of the putative hybrid specimen with the two suspected parents, fixed differences in the Internal Transcribed Spacer (ITS) regions of nuclear ribosomal DNA of H. cylindrica and H. sanguinea were compared .  ITS regions were amplified using PCR and were then sequenced.  The results were edited, assembled and aligned to look for differences in the parents' ITS regions.  The differences in the parents' ITS regions are present in combination in the unknown specimen's DNA and shows that it is the hybrid of H. cylindrica and H. sanguinea.  A further test, using the maternally inherited chloroplast genome (cpDNA), was conducted in a similar fashion, with the expectation that the putative hybrid should contain the cpDNA of the species that is the maternal parent.

Large waves of synchronized, spontaneous electrical activity (SSA) play an important role in many aspects of mammalian central nervous system development, such as regulating changes in neurons' ion channel properties and strengthening synapses through coincidence detection mechanisms. Questions still remain about the mechanisms of wave generation and propagation, and it is unclear what factors limit SSA to only a short critical period in development. Around the time of birth, much of the SSA is generated by neurons that release the neurotransmitter GABA. Our aim in this project is to clarify the roles of two ionotropic GABA receptor subtypes, GABAa and GABAa-rho, which respond differently to GABA stimulation and may have different distributions within the cell membrane. We selectively block either the GABAa or GABAa-rho receptor subtype in cultured slices of neonatal mouse brain and use calcium imaging to observe changes in the spatial and temporal properties of SSA. Current data suggest that activation of GABAa receptors is the dominant factor in SSA generation, but GABAa-rho receptors (or receptors with a mixed subunit composition) are also necessary for normal wave frequency.

Genus Rhododendron consists of over 1000 species of woody plants.  While the overall phylogeny of the genus is known, the relationships between species are not.  My research focuses on a small and unusual retrotransposon, Cassandra, and its possible use for evolutionary studies.  Retrotransposons are sequences that are able to move to new DNA chromosomal locations via mechanisms simlar to retroviral DNA replication.  Transposition involves transcription of an integrated DNA copy through an RNA product that acts as a template for reverse transcription, and finally integration of the resulting DNA at new sites in the plant genome.  Like other retrotransposons, Cassandra has no mechanism for complete excision from a site it has occupied.  Thus, once the element is inserted into a genomic site, all or part of it is retained at this position as a stable marker.  Consequently, a Cassandra insertion at a given site is a shared derived trait, suitable for evolutionary tree-building.  Ultimately, knowing these sequences of transposition may shed light on the pattern of evolutionary divergence of Rhododendron and Ericaceae plants.  Based on one insertion site, my data suggests that thus far, only a solo LTR version of the Cassandra element is found in most species of the subgenus Hymenanthes (primarily in East Asia), with an empty site found in the subsection Pontica (more geographically diverse).  Interestingly, for a few species within Hymenanthes that lack the element, another conserved insertion (approx. 450 bp) is found in the DNA instead.  Moving forward, I will look at additional insertion sites, to gain further information on Rhododendron phylogeny.

Pedaliaceae (Sesame Family) are a small family of plants, about 14 genera with approximately 70 species that occur mainly in arid regions and deserts of the Old World, with their center of distribution in Southern Africa. Contained in Pedaliaceae are two widely known species: Sesamum indicum, which is cultivated and provides sesame seed and Harpagophytum procumbens, Devils Claw, an endangered species which is harvested wild and used for medicinal purposes. Evolutionary relationships (phylogeny) within the family are currently not very well understood. Previous classification systems have included the Martyniaceae, a small New World family, which occurs in similar environments and exhibits similar fruit morphology, with the Pedaliaceae but this inclusion of has since been questioned. In order to infer a phylogeny for the Pedaliaceae I am using the chloroplast loci ndhF and tnrL-tnrF which generally provide good resolution to determine the relationships between families and major lineages within a family of plants. Preliminary analyses based on these two loci indicate that the genus Sesamum is not monophyletic. To achieve resolution of the relationships of the species within Sesamum, I am sequencing two commonly used nuclear loci: ETS and ITS, the external and internal transcribed spacers of the nuclear ribosomal repeat, and one of the many loci in the pentatricopeptide repeat (PPR) which have only recently been developed for phylogenetic study in the Olmstead Lab. The goal of this project is to infer a phylogenetic tree of the Pedaliaceae on the species level, resolve their relationship to Martyniaceae and the genus Trapella, as well as the placement of these groups within the larger tree of the Lamiales.

The harvest of oil from large-scale algal cultivation has become an attractive source of alternative and sustainable biofuel to replace traditional fossil fuels. Understanding how algae store and breakdown their synthesized oils sequestered within specialized organelles known as lipid bodies is key to improving algae oil production. My study is to: a) propose a model alga, b) cytologically probe lipid body architecture, c) and document lipase activity gaining access to oils within lipid bodies. To select a model alga for my studies, I conducted a survey of my laboratory’s algae culture collection looking at organisms across a variety of taxa, lipid body morphology, spatial arrangement, and total number of lipid bodies. BODIPY 505/515, a lipophilic dye, was used to integrate the lipid bodies of these organisms. Chrysochromulina sp. was identified as the best model organism for its simple compliment of two large lipid bodies. Counterstained lipid bodies indicate a strong co-localization of an endoplasmic reticulum membrane with lipid bodies. Fluorescein diacetate was used as a fluorescent indicator for lipase activity. Over a 24-hr time course study, flow cytometric data showed fluorescein diacetate fluorescence increasing as BODIPY 505/515 fluorescence decreased. Cytological studies also show fluorescein diacetate localizing to lipid bodies during its peak fluorescence. These results indicate an endoplasmic reticulum membrane surrounds Chrysochromulina sp.’s lipid bodies and lipases are involved in gaining access to fatty acids through the endoplasmic reticulum membrane. In determining effective algal lipid harvesting protocols it is paramount to first understand cellular lipid usage. Developing the most efficient methods of converting algal lipid bodies to biofuels will accelerate the viability of the global transition to alternative fuel sources.

Gastrulation is a crucial step in embryogenesis, creating differentiation between ectoderm, mesoderm, and endoderm, as well as forming necessary organs. Transmembrane and juxtamembrane proteins located at the adherens junction, such as cadherins and p120 catenin, work together to either promote cell motility or adhesion. Using Danio rerio embryos as a model organism, we hypothesized that when p120 catenin is at the plasma membrane it binds and stabilizes the cadherins, causing cell clustering and increased adhesion. When p120 catenin is in the cytoplasm it activates Rac1 and Cdc42 (two members of the Rho GTPase family), which causes cell motility. By using a “depletion-rescue” experimental approach, p120 catenin levels are depleted by injecting a splice-morpholino, then “rescued” by coinjecting wild-type Cdc42 or Rac1. This indicates that p120 catenin in healthy embryos works to activate Cdc42 and Rac1 during gastrulation, resulting in cellular motility. When dominant-negative and constitutively-active mutants of Cdc42 or Rac1 are injected in combination with morpholino, the embryos do not “rescue” well, if at all. We postulate that wild type GTPases “rescue” well because the can receive a signal and activate migration directionally while the constitutively-active GTPases activate migration in all directions. These studies on the regulation of cell motility can help to elucidate larger signaling pathways, leading to additional effects on cell survival, growth, and invasiveness, all of which may be caused by the upstream p120 catenin.

Gastrulation is the process during which cells migrate and tissue layers differentiate in early embryo development. This process requires a balance between cell-cell adhesion mediated by cadherin-catenin interactions and stimulation of cell migration. Our research focuses on p120 catenin (p120ctn) dependent cell migration during gastrulation in zebrafish. P120ctn stabilizes cadherins at the plasma membrane and interacts with Rac1 and Cdc42 GTPases to stimulate cell migration, and with RhoA GTPase to regulate cell-cell adhesion. P120 binds and translocates RhoA to the plasma membrane where a RhoA gunanine nucleotide exchange factor stimulates release of GDP and binding of GTP. GTP-RhoA activates RhoA kinase which activates myosin light chains, inducing cross links to stabilize actin filaments, thus increasing the strength of cell-cell adhesion. In this project we aim to address what the role of RhoA GTPase is in the p120ctn signaling pathway as well as how phosphorylation of p120ctn affects the RhoA-p120ctn binding interaction. By knocking down p120ctn and attempting to rescue developmental deficiencies, we can elucidate how p120ctn interacts with RhoA to regulate cell-cell adhesion. We hypothesize that injection of GDP-bound (DN) RhoA will rescue a splice-morpholino p120ctn knockdown allowing low concentrations of p120ctn to activate Rac1 and Cdc42 GTPases that promote cell migration. Our microinjections of DN-RhoA mRNA into one-cell zebrafish embryos have shown that DN-RhoA, and not WT-RhoA or CA-(GTP-bound) RhoA, is able to rescue p120ctn knockdown. Additionally, p120ctn is regulated by phosphorylation and we are currently interested in how tyrosine phosphorylation of p120ctn affects its ability to bind and regulate RhoA. We hypothesize that tyrosine phosphorylation site mutants will be unable to rescue splice-morpholino p120ctn knockdown, and therefore be essential regulation sites. Using confocal fluorescence microscopy and a binding assay to measure GTP-RhoA, we hope to elucidate whether these sites are directly involved in the p120ctn-RhoA binding interaction.

Ocean Research College Academy (ORCA) students partake in monthly State of Possession Sound (SOPS) cruises to collect data from various sites in the Snohomish River estuary near Everett. The focus of this SOPS research is on dissolved oxygen fluctuations in Possession Sound and the Snohomish River. Large fresh water inputs into Possession Sound bring sediment, organic matter, nutrients, and pollutants from both natural and human influences, which may impact the dissolved oxygen (DO) levels of the water. In addition, the tides affect the amount of DO in the Snohomish River during flood tide and ebb tide. The hypothesis formulated for this research was that less populated areas upriver would have a higher DO content rather than more populated areas downriver. Measurements were taken at four different locations along the Snohomish River and at the delta during ebb tide to negate the tidal effects. The parameters collected with a YSI 85 were DO, temperature, and salinity in order to find the influences that determine levels of DO in the areas of interest. Future temporal studies based off of this research could directly link anthropogenic and seasonal influences on specific respiring marine organisms in the ecosystems of Possession Sound.

Cues from predation can have strong effects on prey behavior and development, but antipredator responses can reduce fitness in other ways. Costly responses to predators should be, and often are, proportional to the level of risk (i.e., threat-sensitive); however, the mechanisms by which prey assess risk are not fully understood. In mesocosm experiments, red-eyed treefrogs (Agalychnis callidryas) show threat-sensitive growth reduction in response to predation cues from giant water bugs (Belostoma) and dragonfly naiads (Anax). We reared A. callidryas tadpoles from hatching in individual 400 ml microcosms in an open-air laboratory in Panama to assess their growth responses to variation in water-borne chemical cues from predation on conspecifics. In an experiment manipulating cue concentration, cues from predation by both Anax and Belostoma elicited a similar, dose-dependent reduction in growth. We then investigated effects of the frequency of chemical cueing by holding the total cue amount constant across treatments, but delivering it in fewer larger doses or more frequent smaller doses. We found a strong negative correlation between tadpole growth and the frequency of cue events. These results suggest that the frequency of predation cue events is more important than the biomass of prey consumed, or amount of cue delivered per event, for assessment of predation risk. This is consistent with results from mesocosm experiments wherein tadpoles responded more strongly to predation on small tadpoles, via multiple predation events, than they did to predation of an equivalent biomass of larger tadpoles.