Over 50% of all Internet traffic is generated by file-sharing programs like Vuze. Suppose you are sharing files with millions of people, and you search for a particular file and the system takes 5 minutes to find it. My project is about lowering this latency so that computers in distributed systems like Vuze, other P2P systems and the Internet, can find each other faster. To decrease latency I have designed, implemented, and evaluated two new routing protocols to run on top of Harmony, a distributed system which implements a consistent lookup layer. In Harmony's initial routing layer, to find a node in the system, all other nodes had to be searched through. The new routing protocols allow less nodes to be searched. The two routing schemes adapt prior work in routing to use possibly inconsistent cached information about the system. This allows for faster routing but also allows the system to fall back to the consistent routing scheme to ensure reachability of data at all times. The first protocol is implemented purely as an additional layer on the client accessing Harmony. It creates a memory of previously accessed nodes on the client which enables the client to repeatedly access values without repeating work. The second design stores hints in the Harmony system, allowing clients to collaboratively build knowledge of the system. My routing methods increase lookup speed in Harmony.

Temperature regulation is essential to the survival of the newborn. Premature infants are at risk for poor thermoregulation due to greater skin surface area in relation to body mass ratio and decreased subcutaneous tissue. Incubators are used to create a mircro-environment suited to the infant’s thermal capabilities. Previous research in the University of Washington Medical Center NICU included thermal mapping of the unit environment, in which ambient air, evaporative, radiant temperatures and humidity were recorded in each patient cubicle across a year’s time. Findings indicated seasonal effect on room temperature was detectable. It was determined it is difficult to maintain adequate thermal support for neonates while promoting staff thermal comfort. The next step is to examine closely the effect of room temperature on incubator operation. To assess heat loss between the inside and outside of the incubator wall and determine the influence of room air, evaporative, and radiant temperature on incubator operation. Randomly generated lists of cubicles in the 32-bed NICU, across five rooms, are used to obtain periodic measurements. Incubator interior and exterior wall temperatures are being recorded. A wet bulb thermometer is used to record the room air, humidity, and wet bulb temperatures. Additional recordings such as infant’s weight gain in the previous 24 hours, supplemental heat and respiratory support, are being collected in order to draw a holistic picture of the neonate’s environment. Weekly data collection currently in process for an additional three weeks, then data will be analyzed using SPSS to determine results. Results should demonstrate the impact of the thermal environment on incubator operation. Wide variation in room temperatures potentially increases or decreases incubator heat loss, resulting in a less stable environment for the neonate. Analysis of data will inform future studies and determine our recommendations to the unit.

The purpose of this study is to use the Parent Stress Scale: Neonatal Intensive Care Unit (PSS: NICU) to describe how parents rate stressful events in the neonatal intensive care unit (NICU) based on the sights and sounds of the unit, the appearance and behavior of their infant, and the alteration in parental role. In addition, Patient Reported Outcomes Measurement Information System (PROMIS) instruments were used to understand the effects of subsequent stress on sleep, fatigue, anxiety, and depression experienced by these parents. The NICU is recognized as a stressful environment for parents. The long-term effects of this stressful experience can be detrimental to the parents and the infant. This is a descriptive study of parents, conducted in the Neonatal Intensive Care Unit at the University of Washington Medical Center (UWMC). To participate in the study, parents must be at least 18 years of age, literate in either English or Spanish, and coping well enough that this study will not augment their level of stress. Participants completed a demographic survey, PSS: NICU questionnaire, and the PROMIS surveys. This study was started in December 2010 and will end in April 2011. Thirty two completed surveys have been collected to date. Results should yield the dominant triggers of stress experienced by parents in the NICU at UWMC and describe how this stress impacts other aspects of their lives, such as sleep, mood, and energy level. This will generate knowledge available to the staff regarding the most stressful aspects of the parents' experience, and facilitate a discussion for ways to reduce stressful events for parents. It will help prepare staff to provide better support for families who have infants receiving intensive care at UWMC.

The introduction of non-indigenous marine species has been an increasing problem around the world. Many introduced species are negatively impacting the ecosystems in which they invade. Ascidians (commonly known as tunicates) are a group of sessile marine invertebrates that have received much recent attention due to their capacity to impact non-native habitats and their ability to proliferate quickly. Ciona savignyi is a species of solitary tunicate that is rapidly invading estuarine habitats around the world. The present study addresses the C. savignyi invasion of Puget Sound, WA. It will act as a follow up to attempts documented by The Washington State Department of Fish and Wildlife (WDFD) to eradicate another invasive tunicate Didemnum vexillum by exposing it to low levels of dissolved oxygen (DO). To assess the lethal tolerance of DO to C. savignyi, individuals will be subjected to a suite of DO concentrations under controlled laboratory conditions. The method developed by WDFD of generating hypoxic conditions under sealed tarp wraps in a marina will also be examined. Fouled dock surfaces at Elliot Bay Marina in Seattle, WA containing C. savignyi will be sealed off with 1m tarp wraps and the rate of decrease of DO under each wrap will be monitored until it reaches the lethal range determined in the laboratory. This study may have implications for developing large scale eradication programs for non indigenous tunicates in the Puget Sound region and elsewhere.

2D reflection seismic data acquired across Ventura fault in Ventura Basin, California reveal a potentially active thrust system that extends from approximately 4 km at depth to the near surface. The strata north of the basin dip approximately 50° south and the strata south of the basin dip approximately 57° north. Using kinematic and geometric models, the profile was restored to its pre-deformed state to obtain a balanced cross-section. The same profile was also compared to a forward-modeled section using a few plausible kinematic models. The results predict that the section can be constrained by fault-propagation-fold model; as the fault ramp propagated upsection, it formed an anticline-syncline pair in the foreland. This interpretation is consistent with the south-verging anticline present on the surface. The development of the fault ramp formed a blind thrust which could possibly lead to an increased seismic activity in the area. Further processing and interpretation work is underway in order to better constrain the geometry of the thrust system in the area.

 To better understand the process of language learning in human infants, animal model studies, particularly in avian species, have investigated vocal mimicry in which infants modify their own vocalizations to imitate those presented by a “tutor.” Due to their advanced social structure and wide vocal range, the big brown bat, Eptesicus fuscus, provides a mammalian model species in which to study vocal imitation and learning through auditory input. Our project will examine how communication and echolocation calls are developed and processed by infant bats. Subjects will either develop listening to calls of mothers and conspecifics, or will not be able to hear these natural vocalizations. They will instead be provided with digitally altered recordings of calls, in which the frequency range is shifted to differ from calls that are emitted by conspecifics yet still within the audible range. In both groups, the infant bats will be allowed to remain with their biological mothers in order to prevent stress due to isolation. Experimental subjects will have earplugs inserted binaurally, which will only be removed at times when the bat is exposed to the altered playback recordings. The vocalizations of the experimental group will be compared to the group of normally raised individuals. If the vocalization patterns of the experimental group resemble the digitally altered recordings, it will provide evidence that auditory input is necessary for development of echolocation and/or communication calls. Additionally the effects of atypical auditory stimuli will be tested through comparative electrophysiological recordings of neural responses in the inferior colliculus (IC), a midbrain structure responsible for integrating auditory stimuli. We hypothesize that the tonotopic organization of the IC in bats exposed to atypical vocalizations will reflect the spectral properties of the altered playback.

Interfacial tension (IFT) is a fundamental parameter in any situation where two fluids form a boundary. Traditional tensiometry methods (e.g., Wilhelmy Plate, Du Noüy Ring) require a bulk sample for testing, which limits which fluids can be measured. This project sought to implement a new method to determine the IFT in nanoliter sample sizes, which facilitates testing a wider variety of fluids. Building on work by Cabral and Hudson, we set out to design and demonstrate a microfluidic device and method to measure the IFT of two oils with two common surfactants in a multiphase flow. The device generated monodisperse aqueous droplets and deformed them as they passed through channel constrictions. These deformations were recorded by a high-speed camera through a light microscope and were analyzed by custom software using Taylor theory to give the IFT. We present the change in IFT with change in surfactant concentration in the oils tested. These results will become part of the literature database of values that facilitate the analysis and design of multiphase systems.

In the inferior colliculus (IC), the principal midbrain auditory nucleus, many neurons experience stimulus-specific adaptation (SSA), a reduction in response to a sound that is repeated frequently (Perez-Gonzalez et al., 2005; Malmierca et al, 2009). There is reason to hypothesize that SSA is at least partially shaped by GABAergic inhibition (Kutscher A, Covey E, 2009). This study will use neuropharmacological methods to evaluate the role of inhibitory synaptic input in IC neurons that show complete or partial SSA. The IC of the echolocating big brown bat, E. fuscus, is large, readily accessible, and highly developed. Our lab has developed methods for electrophysiological recording in awake animals, making the bat an ideal model organism in which to study auditory SSA (Covey et al., 2009; Simonton et al., 2009; Koch, Covey 2010). Using extracellular recordings and piggyback microelectrodes, cells displaying SSA in the bat IC are identified and SSA is quantified using an “oddball” stimulus paradigm in which a “standard” stimulus presented with high probability and a novel, low-probability, “oddball”, stimulus is inserted at random times during the sequence (Malmierca et al., 2009; Simonton et al., 2009, Koch, Covey 2010). After a cell’s SSA value has been determined, drugs that block GABA receptors are locally applied, and the SSA measurements are repeated. The neuron is then allowed to recover and SSA measurements are again repeated. These experiments should lead to a greater understanding of the role of GABAergic inhibition in producing SSA in the IC, and hence to a greater understanding of how adaptation and attention in auditory processing are achieved.

The ecological phenomenon of life-history tradeoffs has been studied across a diverse range of topics and organisms, from pathogen resistance tradeoffs in fruit flies, to photosynthetic tradeoffs in trees. Female sockeye salmon (Oncorhynchus nerka) face tradeoffs related to reproduction: What is the optimal age and size at which to mature and reproduce? Larger bodied, older females produce more eggs and so can achieve higher reproductive success, but they are also at a greater risk of being eaten by bears or stranding in shallow water. On the other hand, smaller, younger females have a higher chance of surviving to reproduce but produce fewer eggs. We studied these tradeoffs in female sockeye salmon from Hansen Creek, Alaska. From 1999 through 2010 female adult sockeye salmon (N = 58,760) were categorized by cause of death and measured for length. These data established the relationship between body size and likelihood that females would die of senescence, having completed spawning, or die prematurely from stranding or attacks by glaucous-winged gulls or brown bears. We then used data on individually-tagged fish (N = 881) to determine the probability of successful spawning by fish that died prematurely. These data indicated that 65.0% of the bear-killed females died prior to spawning, as did 45.8% of the gull-killed fish and 70.6% of those that stranded in shallow water. We then established the relationship between length and age at maturity and the production of eggs. Finally, for any given length, we will calculate the reproductive potential as a function of average egg production and likelihood of pre-spawning death. This will allow us to compare the calculated optimal length with measured average length, and therefore assess the tradeoff between fecundity and predation.

Recently, research on human sex differences has been commonplace with typical topics being differences in sexual arousal, use and opinions of erotica, and use of modern technology. Despite this rich literature, there is a gap regarding the combination of the three. In this quantitative study, surveys addressing how often a piece of technology is used to access a certain type of erotica were distributed to 72 college age men and women. Based on past research that states men are more likely to use technology and erotica than women, I predict the analysis will support my hypothesis of there being human sex differences in the frequency of accessing sexually arousing stimuli when using modern technology. Due to the fact that technology is ever-changing and effects most aspects of life, it is important to conduct this research to see how modern technology is effecting human sexuality.

Autistic adolescents are often perceived as socially isolated (Lasgaard, Nielsen, Eriksen, & Goossens, 2010; Tantam, 1988). The perception of social isolation by those outside of the Autistic community can also have far-reaching effects within the scientific community. This misunderstood perception leads to non-scientific based conversation and thinking and to stereotyping the adolescents (Draaisma, 2009; Taylor, Fiske, Etcoff, & Ruderman, 1978). The implication of stereotyping can have a negative impact on self-efficacy in adolescents diagnosed with Autism Spectrum Disorder (A.S.D.), especially in those adolescents who have been diagnosed with High Functioning Autism and Asperger’s Syndrome when they are themselves aware of the social awkwardness that is exhibited through their perceived behavior (Carter, Sweden, & Kurkowski, 2008). Although it is recognized that deficiencies in areas such as social cue recognition and empathic recognition are part of the diagnostic criteria (American Psychiatric Association., 2000), it is important to make a distinction between socially and culturally accepted “societal niceties” versus having the capacity to create and maintain friendships and to be an accepted member of a social structure. This study explores the idea that A.S.D. adolescents do have social networks of friends, and that technology and social networking can support the formation of close relationships (Lenhart, Madden, Macgill, & Smith, 2008; McPherson, Smith-Lovin, & Cook, 2001; Summerskill, 2009). This pilot study will examine social networks of A.S.D. youth in the Portland, Oregon metropolitan area. Using self-report survey methods, the social structure of the autistic adolescent youth can be mapped via Social Network Analysis methods. The study will include 20 autistic youths between twelve and eighteen years of age. The data collection procedure will include reports from autistic youths, as well as from parents and from the reported friends, using a snowball (convenience) sampling strategy. Analyses will rely on the UCINET program and similar methods.

Mechanical forces play a critical role in biology from cellular differentiation to the formation of blood clots. However, there are currently limited quantitative methods of measuring these intrinsic molecular forces. The development of fluorogen-activating proteins (FAPs) that are capable of allowing normally dark fluorogen dye molecules to fluoresce offers a unique possibility for the design of a novel force sensor with piconewton sensitivity. These proteins consist of two single-chain antibodies that must dimerize in order to bind and activate a fluorogen dye molecule for excitation. The force sensor itself consists of a single FAP with a flexible linker connecting the two chains such that they are held in close proximity to each other. In the presence of tensile mechanical force applied across the complex, the two antibody chains will be pulled away from each other, disrupting the binding of the fluorogen dye and subsequently decreasing the fluorescence intensity. The purpose of this project is to develop an in vitro testing system to demonstrate that the protein is responsive to applied forces. In order to apply tensile forces in a controllable manner, two high-affinity attachment tags (biotin and SNAP-tag) were added on the N and C termini of the complex. These ligands will be utilized to attach the force sensor to paramagnetic beads and a streptavidin-coated surface. Introducing a magnetic field will result in force simultaneously being applied across a large number of proteins and a detectable decrease in fluorescence intensity as the fluorogen dyes unbind. Positive confirmation of the relationship between fluorescence and force will validate the protein as a force sensor and allow for future development in implementing the probe in a cellular system.

Pulsed, low intensity ultrasound (PLIUS) has long been researched as a possible method to accelerate cell growth. More recently, PLIUS has been used to boost the growth rates of several species of bacteria. Boosting bacterial growth rates could have a significant impact on increasing the rates of vaccine and pharmaceutical production. In order to study the effects of PLIUS on bacteria growth more completely, we have proposed the design of an ultrasonic bath that can fit into a standard bacteria incubator. The bath is powered by a 9 volt battery, and creates pulses of ultrasound at a 1/2 duty cycle. Users can vary the pulse frequency and intensity of the ultrasound supplied. The primary goal of this project is to enable further research of PLIUS, using a low-cost, low-power device that does not require specialized bacteria culture techniques. We hope to use this bath to confirm that PLIUS can increase the growth rate of E. coli. Then, we hope to determine which implementable parameters (application time, pulse frequency, intensity etc.) will optimize E. coli growth.

Many non-immunoglobulin binding proteins mediate specific high-affinity interactions and find applications in medicine and biotechnology. These antibody-like binding proteins represent alternative scaffolds for design of surface display libraries of novel binding molecules but with advantages over antibodies. Here, we propose to develop an activatable recognition protein (actibody) library using the affinity regulated bacterial adhesive FimH scaffold. FimH binding has been engineered to respond to different regulatory stimuli without altering its specificity. Therefore, FimH represents an attractive starting point for the development of display library of activatable antibodies. The diversity library will be constructed by varying the amino acid composition of three complementarity determining regions (CDRs) that make up the ligand-binding pocket of FimH. A set of skewed degenerate oligonucleotides will be designed to introduce variations in the CDRs while maintaining a wild-type bias in conserved, structurally-important or solvent-inaccessible residues. Further optimizations will condition for adequate FimH expression, proper folding and maintenance of the scaffold conformation prior to screening. Avidity enhanced, competitive surface adhesion assay was developed to specifically screen the FimH actibody library. Kinetic and equilibrium screening parameters were derived from competitive surface elution model of bacteria FimH libraries detaching from glycosylated surface in the presence of soluble glycan. In artificial test experiments, the selection method allowed for identification, isolation and amplification of one high performing variant among one million non-binding variants. The bacterial fimbriae display of pre-conditioned FimH libraries combined with the competitive adhesion screening and additional fluorescence-activated cell sorting (FACS) presents remarkable potential for isolating variants that bind to novel targets in a regulated manner. The mechanical properties of the FimH scaffold and its ability to form catch bonds could be utilized in biological systems for a number of useful recognition purposes such as molecular force biosensors and force-regulated selective and microfluidic diagnostic tools to detect pathological agents.

Mechanical force regulates essential cellular processes in humans including blood clotting and bone restructuring. To study this mechanical regulation on the subcellular level, a protein-based force sensor is being developed to measure forces within cells. The force sensor consists of two fluorescence activating protein (FAP) subunits connected by an amino acid linker. The sensor loses its ability to induce a dye known as a fluorogen to fluoresce if the two subunits are separated from one another. In order to make quantitative measurements with the sensor, the force at which the two subunits separate must be known. To determine the force of separation, force must be applied to the construct in a controlled manner. Experimental results have shown that most of the interactions between the tags on the protein and the antibodies specific to these tags are not stable enough under tension to allow the separation measurement to be made. The exception to this finding is the interaction between streptavidin and biotin. To create a stable mechanical anchor for the protein, a SNAP-tag is being expressed with the protein. The SNAP-tag enzymatically attaches itself to benzyl guanine-biotin, which can be bound by a streptavidin. Currently, experiments are being performed to determine the functionality of the SNAP-tag. If the tag is functional, force can be applied to the resulting protein through these mechanical anchors, which may ultimately enable quantitative measurements of force with the sensor. These force measurements may add to the understanding of mechanically regulated cellular processes.

 Mechanical forces play a significant role in protein activity. Although force spectroscopy enables researchers to artificially apply forces to and observe proteins in vitro, there is no direct method for detecting and measuring intrinsic forces. The overall goal of this project is to design a genetically expressible biosensor that can measure forces via fluorescence. This force biosensor adapts a fluorescence-activating protein (FAPs), a two-domain peptide which dramatically enhances the fluorescence of a single fluorogen molecule by trapping it within an interdomain cleft. Interdomain interactions at the interface can be ruptured by applying tensile force across the peptide. The specific aim of this project is to predict mutations that will modify the force-responsiveness of this protein. Domain separation is modeled by molecular dynamics (MD) simulations and analyzed to determine force-bearing residues and associated rupture forces. MD analysis informs mutations that can alter the force-responsiveness of the FAP. These mutations will be expressed and validated using MD simulation and fluorescence microscopy. With these mutants, we can design an array of biosensors that are sensitive to different levels of forces.