Fluctuating temperature patterns due to climate change could negatively impact the survival of species whose sex is determined by environmental cues rather than genetic factors. Species whose sex is determined by ecological cues or social interactions fall under the Environmental Sex Determination (ESD) category, while the Genetic Sex Determination (GSD) category includes species whose sex is determined by genotype at conception. There are numerous published examples of teleost fish species that rely on ecological factors for sex determination, but no studies have explored the extent to which the annual killifish Austrofundulus limnaeus, a species without sex chromosomes, relies on ESD. A. limnaeus inhabit ephemeral ponds in Venezuela, South America that experience extreme daily fluctuating temperatures. Climate change could affect this delicate lifecycle by disrupting seasonal temperature patterns that alter sex ratios or exposure to temperatures beyond their tolerance range. Therefore, it is essential to determine what impact temperature and social interaction may have on sex determination in developing A. limnaeus. To explore ESD, A. limnaeus larvae were exposed to constant temperatures of 20 and 30°C, and a daily fluctuating temperature regime from 20-30°C from hatching until the display of secondary sexual characteristics. Some fish were grown in isolation while others were grown in small groups. Fish grown in isolation at 30°C exhibited female-biased sex ratios with 64% developing as females whereas 69% in 20°C and 61% in fluctuating temperatures exhibited male-biased sex ratios. Ongoing experiments are being conducted to explore the possible interaction between temperature and social cues for determining the sex in this species. A. limnaeus has a relatively higher tolerance to fluctuating temperatures due to their ephemeral pond environment, and thus represents an opportunity to explore how even the most tolerant of species may respond to global climate change, especially in the context of ESD.

ovine Leukemia Virus (BLV) is a disease that affects US beef herds and large-scale dairy herds. A recent studied found correlations to the presence of BLV particles and human breast cancers. Although no actual retroviral insertion (zoonosis) was found in tissue samples, this raises concerns about BLV consumption in from infected dairy samples. To investigate this phenomenon, the prevalence of BLV from raw dairy milk samples will be analyzed. Western blotting will be used to look for the major reactionary viral glycoproteins, gp51 and gp24. Controls were designed for blotting to subclone the coding region of gp51 or gp24 into bacterial expression vectors from a plasmid containing the entire bovine viral genome. Subcloning and quality assays were confirmed first by restriction digestions, PCR then also will be sent for sequencing. The expressed glycoproteins along with infected cell culture samples will then be used in blotting procedures as positive controls while analyzing raw milk samples collected from farms.

The global network of nature reserves are intended to be refuges for wildlife species around the world. However, nature reserves are experiencing increasing amounts of human visitation each year, and it is critical to investigate how this influx in disturbance is influencing the space use, abundance, and richness of wildlife species in those reserves. We used camera trap data collected at La Selva Biological Station in Costa Rica, to understand how the presence of humans and other environmental factors are influencing the presence and distribution of various wildlife species. We deployed 9 cameras, in three blocks, on 9 different trails and accumulated 208 active camera nights in 2018. A total of 9,692 photos were obtained of 17 different animal species. We hypothesize that the occupancy rate of species will be primarily influenced by human presence on trails within the reserve. The most commonly detected species were Collared Peccaries (Pecari tajacu) with 0.837 average detections per day, Central American Agoutis (Dasyprocta punctata) with 0.409 average detections per day, and Great Curassows (Crax rubra) with 0.173 average detections per day across all camera sites. Collared Peccaries had a 72% diel activity overlap with humans, Central American Agoutis had a 66% overlap, and Great Curassows had a 78% overlap. I will use the package “unmarked” in the program R to determine the influence of humans on species occupancy and richness. These results will help inform managers at La Selva Biological Station on how to best limit the impacts of human visitation on wildlife species.

Gastric dilatation volvulus (GDV) is a life-threatening condition that occurs in large breed dogs. Previous research investigating three candidate genes of major histocompatibility complex (MHC), DLA88, DRB1 and the innate immunity gene, TLR5 found diagnostic markers that could be used to identify Great Danes at risk for GDV. The Endoplasmic Reticulum Aminopeptidase 1 gene (ERAP1), is now known to be an important up-stream component of the antigen-presentation system of the MHC, and human polymorphisms in this gene have been linked to the same inflammatory disorders. I hypothesize that Great Danes with certain ERAP1 single nucleotide polymorphism (SNP) variants on exons 3,7,12,13,16 associate with the risk for GDV in Great Danes. We conduct the experiment on two groups of Great Danes; eighty dogs are recruited for this study. Half comprise a GDV group consisting of dogs that have undergone emergency surgery for GDV, and a second age-matched gender-matched control group is dogs with no history of gastric problems. We perform polymerase chain reaction (PCR) and sequencing on subjects from both groups in the variable regions of the ERAP1, evaluating alleles from the gene for association with GDV while controlling for dog family, ages, and diet intake. The goal of this work is to establish a list of specific “risk”or “protective" alleles of ERAP1 that are associated with GDV in Great Danes. If the dominant genotype that predisposes dogs to GDV can be determined, this canine GDV model marker will provide a means to detect at-risk animals, especially in Great Danes. Early genetic testing, probiotic, drug-based approaches and gastropexy (a surgery to prevent GDV) could reduce the incidence of GDV.

Tooth sensitivity is a common affliction that leads to discomfort or even pain when teeth come in contact with a variety of stimuli, such as cold drinks or brushing. The pain is a result of the tooth's natural protective enamel layers wearing down over time, exposing microscopic channels in the teeth known as dentin tubules. These dentin tubules transmit sensation from the tooth surface to the nerves, which is why exposing them leads to greater sensitivity in the tooth. The goal of this study was to evaluate the effectiveness with which a mineral dentin desensitizer could cover and seal the dentin tubules, thereby decreasing tooth sensitivity. Samples of human teeth were cut into disks to expose the dentin tubules and applied with the desensitizer. One treatment group was immediately processed for imaging using a scanning electron microscope, while the other was stored in simulated body fluid for four weeks before imaging. Cross sectional and surface images were taken of the dentin samples, at several magnifications. Both treatment groups showed effective coverage and mineralization not only over the open tubules, but also penetrating into the tubules. These results suggest such treatments may be an effective way to treat patients with tooth sensitivity caused by exposed dentin.

Maintenance of neutral buoyancy is a challenge to all fishes. In shallow water, this is typically achieved through the use of a swim bladder, though a more favorable mechanism for deep-sea species is through a reduction in skeletal structure. Extreme environmental conditions—low temperature, high pressure, lack of light, limited food availability, and varying oxygen concentrations—exert evolutionary pressures on organisms that inhabit the deep sea. The family Liparidae (snailfishes) span the largest depth range of any marine fishes. Hadal snailfishes, the deepest-living fishes, reside as deep as ~8,200 meters. With the elimination of swim bladders, a reduction in skeletal structure has become the mechanism by which this family achieves the buoyancy needed to hunt in the water column. We used micro-computed tomography (micro-CT) scanning to study bone density across the full bathymetric range of the Liparidae with representatives across the family tree. Of these specimens, five bones were measured for density: the lower jaw, for purposes of feeding mechanics; the third vertebrae, as a control; the first left pelvic pterygiophore for studying the suction disk; the hypural plate, to study swimming and movement trends, and the sagittal otoliths. Phylogenetic analyses revealed a decrease in bone density with increasing depth. The degree of change in density with depth differed among the structures measured, implying evolutionary effects on the function and performance of bone structures in the deep sea.

The Blue River of south-central Oklahoma is a spring-fed stream that drains much of the eastern Arbuckle-Simpson Aquifer, and is one of only two free-flowing rivers in Oklahoma with little to no anthropogenic influences on the flow of this river. Not much is known about the structure and composition of fish communities in the upper reaches of the Blue River. In collaboration with The Nature Conservancy, assessments of fish in riffle habitats of the Blue River were conducted adjacent to the Oka’ Yanahli Nature Conservancy Preserve in the summer of 2018. Fish samples collected were processed, individual fish were identified to species based on morphology, and biological metrics were calculated. A total of eighteen species of fish were collected from the riffle stream habitats in the upper reaches of the Blue River. The relative abundance of each species was calculated, and the Central Stoneroller (Campostoma anomaum) was highest in abundance, the Orangebelly Darter ranked second in abundance, and the Orangethroat Darter ranked third. The Orangethroat and Orangebelly darter were more likely to be found in areas in the riffles where river bed particles were in the small to large cobble size range (60 to100millimeters). The two darter species did not show any difference in preference for current velocity in the riffle habitats. The two darter species co-occurred in riffle microhabitats as indicated by the positive correlation between the numbers of each darter species collected across all seine hauls. Looking at biota in this river could give insight into how different habitats function in a free-flowing river, and more specifically, what is could potentially happen in riffle habitats of the Blue River because these areas will be the first stream habitats affected if flows are reduced from anthropogenic withdrawals of water from the Arbuckle-Simpson Aquifer.

Wildfires play a major role in the structure and composition of landscapes and the general ecology of the Pacific Northwest. The Klamath-Siskiyou eco-region in northern California and southern Oregon has been experiencing an increase in the frequency, scale, and intensity of wildfires in recent years. Understanding the effects of wildfires on small mammal communities is an important, yet understudied, aspect of the response of wildlife to wildfires. In 2014, two wildfires burned areas on and adjacent to a long-term study area of wildlife. We will investigate the effects of these wildfires on the occupancy of small mammals such as Neotoma fuscipes, Glaucomys sabrinus, and Tamiasciuris douglasii, using occupancy analyses of data collected during fall field seasons using track plate stations. The longitudinal data set that we have includes years of data before the fires occurred, allowing us to disentangle any effects of the wildfires from any naturally occurring variation. This research is important in determining what lasting effects these increases in wildfire frequency are going to have on predator-prey dynamics.

Stem cells are known for their ability to self-renew and differentiate into specialized cells. A key factor that drives self-renewal and differentiation is the specific localization of cell-fate determinants inside the cell cytoplasm. In this work, we focused on a protein called Numb, commonly known to inhibit the Notch signaling pathway responsible for the regulation cell-fate. We sought to understand the correlation between cell spreading, morphology and Numb localization. We studied human adipose-derived stem cells (HADSCs) in two conditions: 1) Cells fully spread after 24 hours and 2) Cells that were actively spreading or retracting. In each condition we analyzed the distribution of Numb fluorescence expression inside the cell cytoplasm. We found that in cells that were allowed to fully spread, Numb was highly localized only on cells with pseudopodia (“arm-like” cytoplasmic protrusions). Numb was evenly distributed when cells were fully spread, had a large area and circular morphology. In the second condition, cells that were actively spreading or retracting were small and circular in morphology. Interestingly, these cells had a high fluorescence expression of Numb specifically localized at the cell edges. These results seem to indicate that Numb localization was found in areas of membrane activity, such as pseudopodia and spreading/retracting cells. This could be due to the fact that Numb has been found to participate in integrin turn-over (cell adhesion). Based on these results our next step will be to modify membrane activity and cell adhesion and observe the effects on Numb localization and stem cell differentiation. Regulating stem cell fate via protein localization will allow us to enhance the production of differentiated cells for regenerative medicine applications.

Human T-Cell Leukemia Virus type 1 (HTLV-I), a retrovirus that currently 10 to 20 million people are infected with, has been shown to be associated with the Adult T-cell Leukemia (ATLL) and HTLV-associated myelopathy-tropical spastic paraparesis (HAM/TSP). 1 After an extended period of time, 3-5% of those infected with HTLV-1 will develop ATLL or HAM/TSP. The viral Tax protein is thought to have involvement in the development of these diseases. The Tax protein inhibits telomerase and topoisomerase-I which in turn inhibits the process of DNA repair. Tax prevents apoptosis and does not allow cells to enter the G0 phase of mitosis. The oncogenic properties of Tax are correlated to the interaction of Tax with host cellular proteins which are strongly influenced by the large amount of Tax protein made from the integrated provirus. We are using viral promoter magnetic pull-downs to investigate HTLV viral-host interactions involving minimal -306bp viral promoter containing vCRE enhancer sites and Tax-recruited host proteins. Experiments have used electrophoresis to determine whether or not the magnetic beads bind the promoter DNA. Absolute quantitation is necessary for consistency and reproducibility in analysis of proteins suspected of recruitment to the promoter.

Methanol is a common pollutant in freshwater ecosystems due to its widespread use in industrial operations, fuel, antifreeze, paints, and insecticides, and being a result of the burning of garbage. As a bioindicator species, Daphnia magna health indicates changes in aquatic ecosystem health. They are an important part of the food web, eating algae, and as a food source to insects and fish. No previous studies have examined the effects of the pollutant methanol on Daphnia heart function and mortality. This experiment examined the effects of varying methanol concentrations on heart function and mortality of Daphnia magna. It was hypothesized that methanol significantly affects the heart and mortality rate of Daphnia. Fifteen experimental biospheres were used in each trial, with three jars for each of the five concentrations. The biospheres were filled with deionized water and methanol in concentrations of 0 ppm (control), 822.6 ppm, 1644.5 ppm, 3289.0 ppm, 7119 ppm. In a second trial, the concentrations were 0 ppm, 822.6 ppm, 1644.5 ppm, 205.7 ppm, and 411.3 ppm. Heart and mortality rates were observed and recorded for each trial over 4-8 days. For each jar, videos were recorded for 15 seconds each and heart rate was counted, and mortality counted every other day. Based on the survival and heart rate differences, it was concluded that as methanol concentrations increase, the heart rate of Daphnia varied but didn’t respond linearly to concentration levels. Additionally, higher concentrations of methanol negatively affect survivability, although the initial control groups had somewhat high death rates. This study highlights the negative impact of methanol pollution on Daphnia because a negative impact on Daphnia affects the whole freshwater ecosystem. Indeed, in Lake Washington, Daphnia represent a major source of nutrients for juvenile Sockeye Salmon, making Daphnia indispensable for their growth.

Growing reliance on antibiotics in livestock production, commercial agriculture, and human healthcare has created evolutionary pressures on bacteria. These pressures have given rise to a new threat to public health, drug-resistant microbes. In addition, antibiotics eliminate all microbes, including the beneficial ones, and are not advisable for long-term use. Recent advances in the understanding of bacterial virulence via quorum-sensing (QS) has presented the scientific community with a promising new approach for alleviating the ongoing overuse of broad-spectrum antibiotics. Quorum-sensing is a form of coordinated gene expression mediated through detection of specific population density. Bacteria use QS as a way to regulate behaviors including biofilm formation, virulence, and motility. Quorum-sensing inhibition (QSI), is the ability to prevent QS and is used by organisms across kingdoms. This prevalence suggests that disrupting prokaryotic communication is either a common defense tactic against infection or acts as a competitive advantage in resource acquisition. Fungi, a kingdom in direct competition with prokaryotes are prime candidates for broadening our understanding of the mechanisms behind quorum-sensing. With its abundance of endemic fungi, the Puget Sound region provides a unique opportunity to screen species for QSI compounds. In this study, fungi representing diverse ecological niches were collected from local forests around the Puget Sound and isolated on potato dextrose agar plates. To screen fungal isolates we used C. violaceum, a bacterium that forms purple colonies when able to quorum-sense and changes to white when a QSI is present. Our results suggest fungi are an underexploited and unexplored source of novel bioactive molecules that could provide a method to inhibit virulent effects of bacteria without damaging an organism’s microbiome.

I present a baseline assessment of the plant family Melastomataceae along an elevational gradient of the Llanganates-Sangay ecological corridor in central Ecuador. Conservation efforts within this corridor aim to preserve the high levels of diversity and endemism present in the region. Melastomataceae, as the third most diverse plant family in Ecuador, contributes valuable biodiversity, biomass, and ecological services to the ecosystems of this corridor. On Cerro Mayordomo (max elevation: 3,383 m), 300 m transects were established at four different elevations, and six 5x5 m plots were assessed along each transect (600 m² assessed total). All melastome species encountered in plots were grouped and counted based on morphological characteristics. My findings reveal a positive correlation of melastome diversity and abundance with elevation up to 2,500 meters. At 3,000 meters, species diversity decreases sharply, but total abundance remains high. Jaccard, Sorenson, and Morisita-Horn similarity indices indicate dramatic changes in melastome community composition over even slight differences in elevation. Eighteen species of flowering melastomes belonging to four genera were found, revealing a subset of the family's astounding diversity in this region. The results of my study will be used to inform future conservation efforts and ecological studies in this incredibly unique and important Andean corridor.

Human alpha defensins, a component of the innate immune system, are small cationic peptides that possess antiviral activity against non-enveloped viruses. The effect of defensins on human adenoviruses (HAdV) is serotype-dependent, infection by some serotypes is enhanced while for others it is neutralized. Enhanced infection correlates with increased cell binding; however, the mechanism of increased binding is unclear. One hypothesis is that defensins mediate receptor-independent binding. Inhibitor studies support this hypothesis, although formal proof is still needed. To test this hypothesis, we used CRISPR/Cas9 lentivirus to knockout the primary receptor, coxsackie adenovirus receptor(CAR), in A549 lung cells. In order to vet these cell lines, they were infected with different HAdV serotypes that use either CAR or an unrelated molecule, sialic acid, as their primary receptors. As expected, the sialic acid-utilizing but not the CAR-utilizing serotype was able to infect the CAR KO A549 cells. We have used these cell lines in combination with integrin co-receptor inhibitors to measure binding and infection of wildtype and mutant adenoviruses in the presence and absence of defensins. These experiments allowed us to determine the extent to which defensin-mediated attachment and entry is receptor-independent.
 

Non-invasive prenatal testing is a recently approved form of genetic testing for pregnant women to screen for chromosomal problems such as Trisomy 21 (i.e., Down syndrome [DS]) without the risk of miscarriage. In the event that this test indicates DS, people may consider whether to continue pregnancy, and perceptions of DS may influence this decision. The present study examines whether positively and negatively framed information about DS influences perceptions of DS and the imagined decision to continue or terminate pregnancies when testing indicates a fetus has DS. It was hypothesized that participants who receive positively framed information would be more likely to imagine continuing pregnancy and have more positive perceptions of DS. Participants were given the same set of facts about DS, either framed positively (e.g., “50% have no heart problems”) or negatively (e.g., “50% have heart problems). The participants reported their perception of the syndrome, including personality traits, adult achievements, and level of learning disability. The participants were then asked whether their feelings toward a pregnancy might change if they were aware their child would have DS. Contrary to the hypothesis, there was no significant effect of framing on perceptions of DS or imagined decisions about pregnancy. However, perceptions of positive personality traits were significantly related to greater expected adult achievement (r=.33, p<.03) and lower expected level of learning disability (r=.39, p<.01). Although this study did not show framing effects, the framing manipulation studied was subtle. Given the possible implications for genetic counseling, more research—with a stronger manipulation—could be done to examine these effects.

Antibiotics are an environmental contaminant increasingly found in aquatic ecosystems, adversely affecting wildlife and contributing to antibiotic drug resistance. Sources include untreated agricultural runoff entering rivers and estuaries, outdated or leaking septic systems in rural areas, and large urban populations excreting unmetabolized medications into sewage systems. Current wastewater treatment methods are unable to effectively mitigate the release of these environmental toxins, thus new approaches are needed. White-rot fungi produce lignin-modifying enzymes which can degrade persistent organic pollutants, including antibiotics. Previous studies have demonstrated that the mycelia of turkey tail (Trametes versicolor) and shiitake (Lentinula edodes) mushrooms can reduce concentrations of the common antibiotics erythromycin and cefuroxime, respectively. In this study, three species of fungus (turkey tail, shiitake, and oyster mushrooms/Pleurotus ostreatus) were combined to create a more dynamic and effective approach to removing antibiotics from wastewater, using commonly available equipment and low-maintenance growth conditions. The fungi were cultivated at room temperature in modular bins, connected in series with removable tubing. The mycelia of the fungi were exposed to antibiotic solutions (erythromycin and cefuroxime dissolved in water) and tested for rates of removal in two phases. The first phase established a baseline rate of removal for each single fungus/antibiotic pair; the second phase optimized the sequence of fungus species and method of exposure (continuous flow vs. batch mode) to improve filtration of a synthetic wastewater solution containing both antibiotics. Results suggest that combining fungal species may be a more efficient method of filtration compared to methods using a single species. This is a promising step towards advancing the practical technologies available for complex wastewater treatment in various settings.

Mercury (Hg) is widely known to be a neurotoxin. Mercury in our environment is found in many different forms, and the key difference between them is evident in the way they are absorbed by organisms. Hydrophobic methylmercury (Me-Hg) readily bioaccumulates in the tissues of all organisms, leading to Hg exposures involving higher doses. Recent research shows that sulfate-reducing bacteria (SRBs) play a role in methylating mercury only when they possess the hgcAB gene cluster. Gaining a more comprehensive understanding of the aqueous conditions required for SRBs to thrive and consequently methylate inorganic mercury is essential for addressing the ongoing problems associated with Hg toxicity. In the initial phase of this study, ion chromatography was used to quantify sulfate (SO42-) concentrations along an urban river in an industrial region of Seattle (the Duwamish/Green River Watershed). The measurements revealed that SO42- concentrations as high as 5300 ppm were present at several sites along the lower portion of the Duwamish River. A number of previous studies also showed significant amounts of mercury in this region’s sediments and fish tissues. The secondary phase of this study involved an investigation determining whether the measured high sulfate concentrations were related to the production of Me-Hg and additionally examined if SRBs likewise played a role. River sediment was analyzed for the presence of the hgcAB gene cluster. Ongoing studies are focused on quantifying Me-Hg at sites where SRBs were found and on the identification of key indicators for mercury methylation conditions along a watershed. This study provides further insight into the relationship between mercury, sulfates, and SRBs when found in combination in an aqueous environment.

Probiotic bacteria grow on the intestinal lining and are essential for optimal digestive health. Understanding the optimal growth conditions for these probiotics is particularly important, since healthy probiotic populations in the gut have been shown to positively impact health in a number of ways. In order to determine the effect of pH and nutrient availability, the following methods were performed; first, probiotic growth was achieved by inoculating Sigma-Aldrich (MRS) and Luria-Bertani media (LBm) at different pH’s ranging from 2 to 7.5. For both MRS and LBm, live cultures of probiotics have been proven to grow successfully in both media while incubated at 37 degrees Celsius at the pH of +/- 6.2. The Bradford protein assay was used to determine protein concentration in samples of both media, and the tryptic digestion of the bacterial cultures were analyzed by liquid chromatography mass spectrometer. These findings will be reported, highlighting important differences in the proteomic data sets in relation to different environmental variables.

Armadillidium vulgare are terrestrial isopods characterized by their ability to conglobate (roll into a ball) as a mechanism of defense from predation. It is theorized that conglobation functions as both a defense mechanism against predation and a means to avoid desiccation in arid environments. This project tests conglobation as a mechanism of water conservation. Water-loss in A. vulgare, was investigated in the free and conglobated states. In experiment one, 40 specimens of A. vulgare were suspended in their conglobated state using dental rubber bands and kept at 30.5°C and 30% relative humidity for 22 minutes along with a control group of additional 40 specimens of A. vulgare in their free state. Water-loss rate decreased by 16.5% when specimens were in their conglobated form. In experiment two, we first varied the humidity and then temperature to test whether different conditions triggered voluntary conglobation. Four groups of 15 specimens of A. vulgare were kept at 25°C in environments of varying humidity: 18%, 30%, 46%, and 99% respectively for one hour. No voluntary conglobation was observed in response to humidity change. Finally, we observed that high temperature triggered conglobation beginning with 6% conglobating at 31.5°C and 100% at 50°C. Our findings suggest that while conglobation behavior may help to conserve water, A. vulgare conglobate primarily as a defense mechanism. Further research is needed to clarify the function and purpose of the conglobation response in A. vulgare.