Protection of old buildings is one of the tools for reinforcing well-being of our communities and ensuring their sustainable development. Today, older built stock is challenged by demands for rapid urban growth and modernization, and so are challenged cultural identities of our neighborhoods. Preservation of historic urban fabric is necessary as transformations and growth occur. The question this project seeks to answer is how can we secure balance between protecting authentic character and ensuring economic vitality in historic urban cores? The primary methods are assessment of historic properties in the Old Town Neighborhood of Redmond and research of effective placemaking solutions among downtown revitalization case studies. The final report features a set of design suggestions regarding the public realm within Old Town's Historic Core and is meant to help preserve, promote, and celebrate the neighborhood. This project is a part of a planning process intended to develop architectural design standards for Redmond’s Historic Core.

The vertebrate body forms through a process called embryogenesis. In my research, I focused on the establishment of body length during embryogenesis of the model organism zebrafish (Danio rerio). Under normal conditions, the cells of the developing body intercalate towards the back, or dorsal side, of the embryo, and extend longitudinally to grow the body axis. One observed defect in this process blocks cell intercalation, thus leading to underdeveloped bodies, which is known as a convergent extension phenotype. In my research, I studied the role of Microtubule-Associated Serine/Threonine kinase-Like (MASTL) RNA in body axis formation. Because of a known role for MASTL in regulating chromosome segregation, I hypothesized that overexpression of MASTL would interfere with normal cell function and lead to a convergent extension phenotype with a shortened body axis in the developing zebrafish embryo. To test this hypothesis, I injected embryos with different concentrations of RNA and allowed them to develop. Overexpression of MASTL shortened body lengths at all but the lowest concentration of RNA. I then chose a low but effective concentration of MASTL RNA, injected embryos and quantified embryo length. Finally, I performed RNA in situ hybridizations, a process by which labeled probes are used to localize specific RNA transcripts in embryos. Using a variety of labeled probes, I found in addition to shortened body length, the distribution of the prospective spinal cord cells and muscle cells were abnormal in MASTL-injected embryos. The results of these experiments clearly show that overexpression of MASTL leads to a convergent extension phenotype. To continue this research, I will look at whether or not MASTL affects body length formation through Protein Phosphatase 2a, a known target of MASTL. The results of my research will lead to a better understanding of the role MASTL in cell intercalation and vertebrate body elongation.

Sexually transmitted infections are a major health concern, both in the developing and developed world. Despite the fact that vaccines have been developed and are under development for many STIs, there is currently no efficient method of topical delivery for these vaccines. The barrier posed by the vaginal epithelium and the difficultly of achieving intracellular delivery hinder their ability to elicit immunity. The optimal topical drug delivery system would ideally transport vaccines through the epithelium and into immune system cells. There is a naturally-occurring pathway in the vaginal epithelium in which the FcRn receptor, which is present on epithelial cell surfaces, binds and bidirectionally translocates IgG antibodies across the epithelium. We hypothesize that a nanoparticle decorated with IgG could be transported by the same mechanism. Loading the nanoparticle with the desired vaccine would then enable delivery to cells populating the vaginal submucosal tissue, providing the topical delivery that is desired. Several methods of conjugating fluorescent polystyrene nanoparticles to IgG have been investigated. I have selected two methods to pursue. The first of these is a passive adsorption method, in which the antibody was allowed to stick to the nanoparticle surface through hydrophobic interactions. The second of these employed commonly used (thiol-maleimide) conjugation chemistry. Now that these methods have been identified as potentially viable, I will assay them to determine if the IgG protein can still bind FcRn while conjugated to the nanoparticle. If this yields positive results, I will move on to animal models to determine the biodistribution of the particles. This will allow me to see if the IgG modification has increased the particles’ ability to reach immune system tissues. If this project’s aim is successful, it may become as useful system for the delivery of vaccines against a variety of diseases.

HIV/AIDS is a pandemic that affects over 33 million people globally, and it is estimated that the HIV virus has claimed over 22 million lives. It has been shown that HIV therapeutics are most efficacious when delivered in combinations that target multiple points of the HIV life cycle. Small molecule therapeutics must remain within a narrow range of concentrations in the blood and tissues known as the therapeutic window to be effective. With traditional drugs the patient will spend a large amount of time out of the therapeutic window as concentrations of drug rise, peak, and then fall as the drug is metabolized, leading to inefficient treatment. Therefore to maximize efficacy it is necessary to develop technologies that are able to stabilize the serum concentration of multiple small molecule drugs simultaneously. Controlled combination delivery of antiretroviral agents promises to vastly improve the quality of care for HIV positive patients. However modern biomaterial platforms have fallen short of this goal. I propose the use of an adapted lipid-based, nanoscale biomaterial platform (ICMV) for the controlled and simultaneous delivery of multiple small molecule antiretrovirals as well as an antiviral protein. I further propose future work for the evaluation and optimization of the ICMV platform for sustained drug release, in hopes of developing a long acting HIV treatment. The ICMV system will significantly improve antiviral drug regimes, as well as the scope of deliverable medications. Controlled release platforms also play an essential role in maintaining systemic drug concentrations within the therapeutic window. These advances in therapeutic technology will lead to significant improvements in the treatment and prevention of HIV, which will directly impact millions of human lives.

 Over 90% of cancer-related death is caused by metastasis of invasive cells from one organ to another. Discovering more about this proliferative capability will be influential in creating new cancer therapies that will not only diagnose cancer, but may soon eventually fight it. Cancer metastasis involves migration of cells along bundles of extracellular matrix (ECM) fibers. In this context, understanding topotaxis through various kinase pathways which regulate cell migration is essential. An example of a kinase pathway is the PI3K pathway, one of the most mutated and well-studied pathways. When this pathway is over-expressed, proliferation of cancer cells is increased, while apoptosis of the cells is decreased. Additionally, cancer cells exhibit a more rigid nature suggesting that there are durotaxis involved for metastasis. Understanding these two mechanotaxes and how a particular oncogene influences the cellular response to these factors are vital in our quest to understand the process of cancer invasion. In our experiment, we studied two specific oncogenes in this pathway: PIK3CA and PTEN. Since PIK3A and PTEN are up-regulators and down-regulators of the PI3K pathway respectively, they will be an excellent model to investigate how PI3K is regulated by oncogenes. We hypothesize that cells not containing the down-regulated PTEN oncogene (knockout) and cells containing up-regulated PIK3CA oncogene (knock-in) will result in directional cell migration and increased migration speed and persistence time. To test this hypothesis, we utilized nano-scaled platforms of increased stiffness to simulate the ECM collagen fibers found on cancer micro-environments. Coupled with polymers such as polyurethane acrylate, polydimethyl siloxane, and poly(lactic-co-glycolic) acid, these platforms can vary the stiffness of cancer microenvironments, ranging from kiloPascals of normal tissue to gigaPascals of cancerous tissue. Thus, these platforms give us the opportunity to design a cancer tissue model to study effects of oncogenes on cancer invasion.

Stem cell-based therapies provide new avenues to create cardiac tissues for in vitro screening and regenerative medicine applications. In order for stem cell-derived cardiomyocytes to effectively recapitulate the function of native tissue, these approaches must account for the mechanical properties of the cardiac tissue extracellular matrix (ECM). Nano-fabricated scaffolds that mimic the structural and mechanical properties of the myocardium may be an effective method of deriving clinically relevant stem cell-derived cardiac tissues. It is necessary to quantitatively understand how these nano-fabricated scaffolds affect the structure and function of the cells. Focal adhesions are specialized protein complexes that enable cells to sense mechanical forces and transduce signals from the ECM. We hypothesize that stem cell-derived cardiomyocytes will form larger focal adhesions on nano-fabricated scaffolds compared to traditional flat substrates, resulting in a more mature cardiac phenotype. We designed a computational method of quantifying these focal adhesions, which bears information about the stability and adhesion of the cardiomyocyte on its substrate. The method simultaneously measures shape and size of the cardiomyocyte, which indicates its state of cell maturity and ability to integrate with host tissue. We used confocal microscopy to obtain images of fluorescently labeled focal adhesions and cell membrane. Using ImageJ and MATLAB, fluorescent intensity was used to reconstruct 3D cell shape and measure expression of focal adhesions. Using this method of quantitative analysis, we can quantify both adhesion and maturation of cardiomyocytes, which will better allow us to characterize nano-fabricated scaffolds. The ability to quantify focal adhesions and cellular morphology will be useful in analyzing how cell-ECM interactions affect maturation, and ultimately aid the development of more effective stem cell-based methods for cardiac tissue engineering. This method can also be used to better understand the mechanisms involved in focal adhesion formation and the interplay between mechanical signaling and cell function.

Myocardial infarction remains the leading cause of death and disability in developed nations. While current therapeutics are able to slow the progression of heart disease, there are no viable treatment options to repair damaged myocardium. As such, cardiac stem cell therapy has received much attention for its promise to repair damaged heart tissue. However, there currently is no ideal stem cell source for cardiac stem cell therapy. Cardiovascular progenitors (CVPs) are stem cells that can differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells, but CVP cell fate determination is not well understood. It is well known that stem cells respond to the physical, geometric, and chemical cues provided by the microenvironment. Herein, we report the development of a platform that allows specific design and control over the in vitro stem cell microenvironment. Polyurethane acrylate (PUA) is a synthetic polymer with tunable rigidity and can be used to form nanopatterned substrates through the use of capillary-force lithography (CFL). Bifunctional PUA-binding peptides that contain the RGD peptide sequence are used for presentation of cell adhesion sites. We report precise control over the physical rigidity, nanotopographical dimensions, and RGD peptide presentation. We anticipate that our platform will be able determine the optimal conditions required for directing CVP cell fate. Thus, by determining the conditions responsible for CVP cell fate determination, CVPs could be a viable cell source for cardiac stem cell therapies.

Diagnosing infectious disease in the developing world continues to be a challenge due to the lack of infrastructure and resources needed to conduct state-of-the-art methods of nucleic acid detection. Surface enhanced Raman spectroscopy (SERS) is a potential alternative to current point-of-care (POC) nucleic acid tests because it could offer similar sensitivity. However, current SERS substrates that possess the reproducibility and stability needed for translation to POC applications are too expensive. Electrospinning gold nanorods (AuNR) into polymeric nanofibers was employed as a method to reduce the cost, improve reproducibility and lengthen shelf life of SERS substrates for POC applications. Electrospinning also offers a facile method of aligning anisotropic metallic nanoparticles, which can improve SERS sensitivity over non-aligned particles. AuNR size and dimension was varied alongside fiber diameter to optimize SERS enhancement. For the detection of target nucleic acid (or oligonucleotide), a sandwich-like design was used: Oligonucleotides complementary to the target strand were conjugated to SERS active fibers in a 96 well plate format, allowing for multiplex detection with different probes. After the sample was administered, probe oligonucleotides conjugated to a Raman reporter molecule were washed over the SERS substrate to complex with the bound target strand before Raman testing. Electrospinning produced homogenous fiber meshes with uniformly deposited AuNRs to create reproducible enhancement across the substrate. In addition, the SERS fibers showed prolonged shelf-life suggesting that the polymeric fibers offer stability for AuNRs. Successful conjugation of probe oligonucleotides to the polymer mesh is shown. Initial experiments showed presence of signature Raman spectra associated with the probe molecule, indicating specific binding of the target strand. However, additional experiments are necessary to improve sensitivity. Creation of this technology provides the initial framework that electrospun fibers can offer a mode of translation of SERS to sensitive detection of biologics for POC diagnostic applications.

In 2012 there were an estimated 35.3 million people living with HIV globally and 2.3 million new infections. In Sub-Saharan Africa, the region of the world with the highest incidence of HIV, women are disproportionally infected with HIV. We hypothesize that the use of an electrospun polymer nanofiber vaginal drug delivery system encapsulating cyanovirin-N and etravirine can provide a method to combine two anti-HIV agents with different physiochemical properties and mechanisms of action into a signal microbicide product to improve prevention efficacy through improved potency and limiting the impact of drug resistance. Poly(vinyl) alcohol nanofibers encapsulating cyanovirin-N and poly(vinyl) pyrrolidone nanofibers encapsulating etravirine were electrospun. The cytoxicity and bioactivity of the nanofibers was determined in vitro in TZM-bl cells using the CellTitar blue assay and HIV-1 BaL assay, respectively. We found that our poly(vinyl) alcohol nanofibers encapsulating cyanovirin-N, poly(vinyl) pyrrolidone nanofibers encapsulating etravirine, and the composite nanofiber formulations showed limited cell cytotoxicity in vitro. Comparable inhibition rates of HIV infections in vitro to the free drug formulations were found, indicating that electrospinning does not impact the antiviral activity of cyanovirin-N and etravirine. Additionally, we found that the composite nanofiber formulations displayed enhanced potency compared to the single agent nanofibers, indicating a complimentary effect of combining cyanovirin-N and etravirine into a single microbicide. These results demonstrate the potential for the use of polymer nanofibers encapsulating cyanovirin-N and etravirine as a vaginal microbicide to prevent HIV infections.

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

Treatment for osteoarthritis is currently limited to pain management and surgical intervention, including arthroplasty. Long-term, these treatments fail as viable solutions as many metal implants erode bone and pain management does not address continued joint deterioration. To progress the treatment of arthritis, our group has begun to design an implantable scaffold to resurface arthritic joints. We have applied a nano architectural approach to designing a scaffold, which recapitulates the native extracellular matrix of cartilage tissue. To fabricate our scaffold we have developed a method to orient nano fibers in three-dimensional space within a hydrogel, thus allowing us to mimic the orientation of native collagen. Currently, this scaffold is undergoing mechanical and biochemical analysis to evaluate it’s potential for cartilage tissue formation. I have focused on mechanically characterizing our scaffold by determining the compression modulus across several architectural variables, and in comparison to native cartilage tissue. I have demonstrated reproducibility in our fabrication method by obtaining statistically independent groups based on varying nano fiber material, spacing and hydrogel concentration (p<.05). In comparison to native cartilage, I have identified a combination of materials and geometry (polylactic-co-glycolic acid nano fibers, Norland Optical Adhesive 76 polymer, and 200 micrometer nano fiber row spacing), which are equivalent to native cartilage tissue compression values (2-4 megapascals). I am currently following up mechanical characterization with live dead staining and confocal microscopy to evaluate the biocompatibility of the scaffold. In the near future, we hope to evaluate stem cell differentiation using mesenchymal stem cells (MSCs) by evaluating glycosaminoglycan and collagen type II concentrations. Thus far, our mechanical characterization and methodology for synthetically engineering a mechanical construct has demonstrated potential for therapeutic use in the treatment of arthritis and joint degradation.

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

 Despite decades of research, our best attempts at formulating a vaccine for the human immunodeficiency virus (HIV) have failed. Recent work in mucosal immunology and virology has highlighted the importance of the mucosal immune system in the successful infection of a host after mucosal exposure to HIV. A vaccine formulation that effectively induces humoral and cellular immune responses in the vaginal mucosa could clear local HIV populations before the virus is able to gain a foothold in the body. The proposed project will develop a novel vaginal mucosal HIV vaccine using therapeutic cell encapsulation. Briefly, a transgenic cell line producing HIV virus-like particles (VLPs) will be entrapped within a nanoporous polycaprolactone (PCL) capsule optimized to release VLPs. Release of the VLPs and viability of entrapped cells will be studied over long periods of time and controlled by an electrospun PCL membrane. Finally, the ability of the cell-based vaccine to interact with dendritic cells will be studied through the bone marrow dendritic cell uptake assay and the mixed lymphocyte reaction assay. This work would represent the first use of cell encapsulation technology to create a vaccine. If successful, this project could generate essential data that could be used to develop novel mucosal HIV vaccine for human testing.

In natural environments, animals seeking resources face potential perils associated with foraging behavior, namely predation. Thus, animals must balance their needs with risks by utilizing adaptive behavioral strategies, such as adapting foraging behavior in the presence of environmental threats. The periaqueductal gray (PAG) has been implicated in the generation of defensive behaviors as part of this fear system. Specifically, the dorsal PAG (dPAG) is involved in escape when a threat is imminent (proximal), while the ventral PAG (vPAG) is involved in freezing when a threat is relatively remote (distal). Previous research has shown that the stimulation of the dPAG is an effective unconditioned stimulus (US) in fear conditioning because it transmits aversive US information to the amygdala, a crucial component of the fear conditioning system. Electrolytic lesions of the dPAG have shown to enhance defensive freezing in fear conditioning paradigms, supporting the view that the dPAG is involved in a circa-strike adaptive response to escape a predatory attack. In order to investigate the role of the dPAG in a risky foraging paradigm, I trained two groups of rats, sham controls (n=2) and dPAG lesions (n=4), to venture into a foraging area in which a remotely controlled robot surges and snaps as the animal approaches the food pellets placed at varying distances from the safe nest zone. The sham control animals learned quickly that the robot could not physically harm them as it always surged a fixed distance, successfully retrieving the pellets at all distances after the first two trials. The dPAG lesioned animals had a lower success rate of retrieving the pellets due to compromised escape behavior. These results suggest that the dPAG mediates threat detection and defensive responses in semi-naturalistic environments, shedding more light on the fear system to better understand anxiety disorders.

Despite the increase of international students on university campuses in the United States and promotion that encourages international admissions, there is a lack of care and resources to ensure their academic success and overall satisfaction. Transitioning to the college environment is challenging for all students, especially for international students who encounter extra challenges such as language barriers, greater financial burden, and homesickness. In this study, I investigate how social support from campus clubs, friendship, family, and mentorship programs impact academic success. Pulling from an extensive review of the literature, the main goal of this project is to construct a social survey that will be administered to 100 undergraduates at the University of Washington to measure academic success and measures of social support. Academic success is measured as self-reported cumulative GPA. I hypothesize that students with stronger social support networks will result in higher GPAs. Students with greater life satisfaction are also likely to have higher GPAs compared to those with lower life satisfaction. Future work will focus on how these international students’ lifestyle and education impacted how they react to the transition.

Recent pharmaceutical development that targets specific cell signaling pathways has been in the rise. However, many of the drugs can also induce off target effects especially in cardiovascular system. Current in vitro cardiotoxicity screening utilizes genetically modified cell lines, which do not characterize the biochemical and electrophysical parameters of human heart cells. The discrepancy of cell characteristics produces false negatives and false positives resulting in the release of potentially lethal drugs and the loss of valuable drugs, respectively. The project aims to improve the predictive value of the cardiotoxicity screening by creating an assay with well developed cardiac tissue composed of matured human pluripotent stem cell-derived cardiomyocytes. Made possible by well-established nanopatterning techniques, cardiomyocytes are cultured on a poly(dimethylsiloxane) thin film with topographical features layered with multiple functional substrates using spin coating and soft molding. The thin film with tunable dimensions is employed as a substrate upon which force generation of cardiomyocytes can be characterized. Under the poly(dimethylsiloxane) layer, poly(N-isopropylacrylamide), a thermal responsive polymer that transforms from a hydrophobic form to a hydrophilic form, is spin coated and utilized as a sacrificial layer that will release the thin film when the temperature is lowered. After the release of the thin film, measurement of the deformation of the thin film allows for the calculation of force generation and gives a direct representation of the effects of cardiotoxicity on the overall cardiovascular system functionality. Optimization of the thin film fabrication was completed and upon scanning electron microscopy observation, high fidelity of the nanopattern was observed. This biomaterial platform better mimics the native human heart tissue and has the ability to characterize the force generation of cardiomyocytes. Furthermore, this platform could be utilized as a drug-induced cardiotoxicity assay which could potentially benefit the pharmaceutical drug development.

Cardiomyocytes are the optimum cell source to model and study heart diseases and to screen drugs for cardiotoxicity in vitro. Cardiomyocytes differentiated in vitro from human pluripotent stem cells, however, are structurally and functionally immature. It is necessary to accurately recapitulate the behaviors of cells in the native myocardium for pathogenesis and drug-screening assays. Therefore, before the application of human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs), we must address their maturation state. Physical cues presented to neonatal rat cardiomyocytes in a specific scale matching their native microenvironmental niche have been shown to profoundly regulate their structure and function, as characterized by elongated anisotropic morphology, well-organized contractile machinery, and faster action potential propagation. It is unknown, however, whether the same nanotopographical cues would have a similar positive influence on the maturation of hPSC-CMs. In this study, we tested the hypothesis that anisotropic nanopatterned substrates would enhance the maturation of hPSC-CMs, resulting in cardiomyocytes more suitable for in vitro cardiac tissue engineering applications. To test this hypothesis, we seeded hPSC-CMs differentiated from the matrix sandwich method on nanopatterns of various dimensions and compared their maturation with hPSC-CMs cultured on traditional flat substrates. To characterize maturation, the structural and functional phenotypes of hPSC-CMs were analyzed from their morphology/alignment, calcium handling properties, and gene expression. It was anticipated that, similar to neonatal rat ventricular cardiomyocytes, hPSC-CMs would exhibit a size-dependent change in their structure and function and that there would be an optimal substrate dimension size resulting in the most mature hPSC-CMs. The experimental result could yield a hPSC-CMs culturing protocol for deriving mature human cardiomyocytes for future drug screening assays and heart disease modeling. After optimizing substrate dimensionality, other culturing aspects such as chemical and mechanical cues and their temporal introduction to cells can be tested to further enhance the maturation of hPSC-CMs.

Cardiovascular disease remains the leading cause of death worldwide. The heart is one of the most resistant organs to self-repair from injury, e.g. myocardial infarction. Stem cell therapies, and tissue engineering have had limited success in inducing heart tissue to regenerate owing to the failing incorporating morphological, electrical, mechanical cues that are important for physiological functionality of transplanted heart tissues. Pre-engineered functional tissue on scaffold prior to myocardial transplantation enables recapitulating topological, mechanical, electrical and biological cues of healthy myocardium extracellular matrix (ECM), while providing cites for adhesion and migration of cells. In this study, we investigated one of the potential novel materials, Graphene Oxide, for pre-engineered cardiac tissue maturation. We performed various analyses to confirm maturation of cardiac tissue on graphene oxide physiologically, morphologically, and functionally. Graphene Oxide showed positive effects on maturation of cardiac tissue. Neonatal Rat Ventricular Myocyte tissue showed anisotropic cooperation with contraction when it was on the material. Interestingly, Graphene Oxide substrates were reduced during cell culture, which was confirmed by Raman spectroscopy. However, mechanisms for the reduction are still need to be explored. We would like to continue our study on finding underlying mechanisms and role of Graphene Oxide on cellular redox reaction. We are cooperating this material with nanogrooved substrates to build a platform for cardiac tissue maturation for tissue implantation.

The demand for efficient and sustainable agricultural crop growth has increased with growing world populations. Water and nitrogen are essential, but often limiting, factors for plant growth; understanding nutrient and water relations within a whole-plant system is important for larger-scale planning. Recent research has shown that bacterial endophytes, microorganisms living symbiotically within plants, may alleviate resource limitations by fixing supplemental nitrogen from the atmosphere. This dinitrogen is allocated to the plant in exchange for photosynthetically fixed carbon, altering source-sink relationships within the plant system. The change in substrate flow may also affect water movement in the plant by shifting osmotic potential. The purpose of this study is to investigate the effects of bacterial endophytes on: 1) partitioning of photosynthetically fixed carbon within the whole plant, and 2) leaf water potential. Sunflowers (Helianthus annuus) will be inoculated with WP5 (Rahnella sp.), a bacterial endophyte strain isolated from native poplar (Populus trichocarpa), and grown in a controlled environment. I will use ¹³CO₂ pulse labeling on one mature leaf of each plant (from both inoculated and control groups); plant tissues from the entire plant will then be analyzed for labeled δ¹³C content. Water potential will be measured using a pressure chamber and psychrometer. Measurements of nitrogen content, photosynthetic capacity, and biomass will also be performed. My hypothesis is that in the inoculated plants, carbon will be allocated locally to endophytes in the target leaf, subsequently decreasing osmotic potential in the apoplastic cellular spaces. Gaining a more detailed understanding of endophyte-plant relationships can have profound impacts on agricultural practices and inform resource management.

Behavior in mammals follows a 24 hour clock, wherein some activities like locomotion and feeding are prominent in certain periods and inconspicuous in others. These behaviors are said to follow a circadian rhythm and are controlled primarily by a region in the brain called the Suprachiasmatic Nucleus (SCN), known as the “Master Clock” of the body, that uses light as a zeitgeber, or entrainer, for its circadian output. Previous work has shown that non-photic entrainability of feeding is possible, suggesting that light to the SCN is not the only zeitgeber affecting circadian activity. Additionally, other studies have shown stress to alter certain circadian activities, implicating it as a possible zeitgeber. Although much research has been done on how the SCN affects bodily functions, not as much work has been done identifying other zeitgebers that may directly influence or compete with the SCN. Our experiment sought to find the effects fear has on circadian rhythms and if it could act as a zeitgeber in the absence of the SCN. We studied rats living in a closed environment with a safe bedding area and an aversive foraging area consisting of an electrical shock grate where food and water could be obtained. The room was kept on a strict 12 hour light/dark cycle, with random shocks occurring during either the light or dark cycle. We found that compared to control animals, SCN lesion rats had significant changes in their circadian feeding behavior but not locomotion. Although our results show that fear cannot act as a zeitgeber in the absence of the SCN, they do indicate fear as a modulator for circadian behaviors. Given the close link between fear and stress, our findings are especially relevant for students, showing the negative effects of stressful environments.

 Eptesicus fuscus, an insectivorous bat, vocalizes extensively in social communities and during nightly foraging. In their natural environment, bats are frequently exposed to calls from other members of their species. These calls are processed in the bats’ central auditory system, which is highly specialized and adept at processing sounds within the frequency range of its own species’ vocalizations. To investigate the effects of altering the characteristics of the calls that bats were exposed to during postnatal development, we reversibly deafened infant bats with earplugs, presented calls that were higher or lower in frequency range than normal calls, and looked at calls emitted by the bats and physiological changes in the inferior colliculus (IC), the auditory midbrain center. We hypothesized that bats exposed to calls that differed in frequency range from normal calls would learn to vocalize in a frequency range consistent with that to which they were exposed and that there might be a corresponding alteration of the frequency map in the IC. Experimental pups’ earplugs were removed only during the presentation of pre-recorded bat calls that were manipulated in frequency. Following call presentation sessions, vocalizations were recorded. After eight weeks, electrophysiology was used to study the activity of neurons in the IC. The IC is organized tonotopically, meaning that neurons that respond to a particular frequency are grouped together, forming a systematically organized frequency map. While recording neuronal activity, auditory stimuli were presented to determine the preferred frequency of neurons. Using data from recording sites throughout the IC, we found that the tonotopic organization of the experimental pups differed from that of control pups. This implies that after birth, the auditory centers of the brain remain plastic for a period of time and that communication in Eptesicus fuscus is not a purely innate behavior.