Castilleja levisecta (CALE; golden paintbrush) is endemic to the Pacific Northwest prairies and is an endangered plant. Recovering this species requires an understanding of its seed production and dormancy. As a hemiparasite, CALE can acquire resources from host plants by forming connections with them. I am trying to find out whether host identity can affect the number of seeds per capsule by, for example, altering pollinator visitation. My research focused on the effects of two known hosts of CALE, Festuca roemeri and Eriophyllum lanatum. CALE were grown without a host or with one of these two hosts. Two pods were harvested from each of twelve CALE per host treatment, for a total of 72 capsules. Each pod was evaluated by counting the seeds inside and weighing the components of seed and chaff separately. With these data I determined fecundity and density of the seeds. To date my research indicates CALE may be benefited by Eriophyllum lanatum as these plants have greater fecundity than the no host treatment. Festuca roemeri had no major effect on CALE when compared to individuals grown without a host. This research indicates that hosts can affect reproduction by CALE, reinforcing the importance of considering host identity when recovering this species. Variation in seed dormancy among sites and/or years can affect the success of recovery efforts. I conducted germination testing on 8 accessions of CALE by putting seeds onto petri plates on moist filter paper, exposing them to cold moist stratification, and monitoring germination. I am interested in whether different populations of CALE may differ in their need for stratification. To date my research indicates population is important for how dormant CALE is and when CALE will germinate. The factors affecting dormancy and seed production in CALE are important for reintroduction and management of this endangered species.

The development of simple and affordable diagnostics is critical to the diagnosis, monitoring, and management of disease. Nucleic acid amplification has become the gold standard for many diagnostic applications due to its high sensitivity, specificity, and low limit of detection. Fluorescent dyes typically detect the success of amplification, but require expensive optical equipment including lasers and filters. A cheap and visually distinctive method for detecting nucleic acid amplification will simplify and lower the cost of molecular diagnostics. This research project aims to develop a simple colorimetric reaction for detection of loop-mediated isothermal amplification (LAMP) reactions. LAMP produces large amounts of pyrophosphate by-product during amplification. Pyrophosphate complexes many metals and can displace dyes bound to metals in solution to create color changes. Our goal is to identify a set of chemicals consisting of colored dye and metal ions that will present an unambiguous color change denoting success (or lack thereof) of amplification. Using knowledge of metal-ligand formation constants, we have determined a group of metal-dye complexes that may be viable candidates for detection and are currently testing them in the laboratory. One example is indigo carmine dye. In the presence of pyrophosphate, the dye stays blue (a positive test), while the lack of pyrophosphate will turn the dye clear (a negative test). We are in the process of fully understanding the chemistry of this reaction and further optimizing the process to improve its viability. Exploration of other metal-dye complexes, as well as continuing investigation of the literature, will be conducted to improve the present results. This colorimetric reaction has the potential to simplify and lower the cost of DNA detection.

Von Willebrand factor (VWF) is a multimeric blood glycoprotein involved in the process blood clotting. Its primary function is to be an adhesive proteins that platelets can bind to a wound site. In the Sniadecki lab, VWF is used as a matrix protein for capturing platelets under shear flow in a microfluidic device for blood coagulation studies. Currently, VWF is purchased from a company and coated on the surface of the microchannels. High molecular weight VWF has a higher ability to form a clot, while low molecular weight VWF has a lower ability. Since the VWF comes from a commercial source, the purity of its molecular weight weight is unknown. The goal of my project is to improve the VWF coating by purifying VWF from Enzyme Research. We hypothesized that low molecular weight VWF does not support clot formation and that high molecular weight VWF will show more clot formation than the control vWF in the microfluidic device. My research efforts have been to purify VWF using the fractionation gel filtration technique developed by Professor Dominic Chung’s lab at the Puget Sound Blood Center. The gel filtration technique can isolate the vWF according to size. Gel filtration was done using a 10x10 Sephacryl 500 Column. After the filtration process, vWF of different molecular weights are then applied primarily to the surface of PDMS so that platelets can bind to them under fast flow, in order to measure forces of platelet clotting. We found that there is a link between the molecular weight of the vWF and the rate of clot formation. Low molecular weight vWF does not support clot formation and that high molecular weight vWF will show more clot formation than the control vWF.

Equality Mainstreaming and Intersectionality are concepts that are subject to different kinds of interpretations and are often adopted to a certain extend in public policy all over the world. Equality mainstreaming refers to the widespread incorporation of ant-discrimination into public policy. Intersectionality refers to the concept that personhood is not a monolithic experience of gender, race or another factor, but rather a combination of all these factors that affect an individual’s experience. My research focuses on the incorporation of these concepts into Brazilian public policy. Brazil is a country that is still struggling with various kinds of discrimination, namely racism and sexism, but the creation of Ministries and government agencies dedicated to fighting discrimination have ushered in innovating legislation. Through a discourse analysis of government materials, primarily public policy briefs, task forces, interviews of the Ministers involved in these public polices and informative pamphlets about public policies, I was able to distinguish between explicit and implicit discourses within Brazilian equality oriented public policy. Explicitly the Brazilian government articulates an all-encompassing equality movement. However I have encountered implicit discourses that show that gender and race concerns are at the forefront of equality oriented policies and the concept of intersectionality almost exclusively represents Afro-Brazilian women. Although this detracts attention from other vulnerable populations, Brazil’s unique history of discrimination against Afro-Brazilians and women calls for this increased consideration.

Traumatic injuries can often lead to massive blood loss. When a patient’s mean blood pressure (MAP) drops due to bleeding, the standard protocol is to administer intravenous fluids with the goal of restoring normal blood pressure. However, increasing blood pressure may also increase the rate of bleeding, causing even more overall blood loss. There is no current evidence to show that MAP correlates directly to the velocity of blood exiting a wound. We hypothesize that a direct relationship exists between blood pressure and velocity of bleeding. We test this hypothesis using direct ultrasound imaging of a bleeding artery in a swine model of trauma. N=8 anesthetized pigs underwent bleeding from a femoral artery punch wound and were simultaneously given intravenous fluids to increase blood pressure and cardiac output. Ultrasound measurements of blood flow velocity from a femoral artery punch wound were taken at regular intervals. We found that MAP did not correlate with the bleeding velocity at the wound (R=0.27. p=0.068). However, cardiac output was significantly correlated with bleeding velocity (R=0.37, p=0.012). MAP and cardiac output were then stratified into normal or low categories based on their median values for the entire group. Average bleeding velocity was not different when MAP was low compared to when it was normal (normal=255.7 cm/s vs. 221.8 cm/s, p=0.18). However, bleeding velocity was significantly decreased when cardiac output was low vs. normal (normal=253.6 cm/s vs. 196.1 cm/s, p=0.01).This data does not support our hypothesis of a direct relationship between MAP and wound bleeding velocity. Alternatively, cardiac output as a measurement of blood flow, rather than pressure, may better reflect bleeding after injury and may be a more-useful guide for fluid resuscitation during active bleeding.

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.

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

Ischemic preconditioning (IPC) is the neuroprotective phenomenon in which a brief ischemic episode induces genetic reprogramming of affected brain cells, causing physiological changes that minimize cell death from subsequent, sustained ischemic insult. We hope to characterize the cellular and molecular mechanisms of IPC. Our lab previously has shown that IPC is dependent on Toll-Like Receptor 4 (TLR4) and involves the production of interferon stimulated genes (ISGs) in microglia. Following ischemia, damaged neurons release endogenous TLR4 agonists called damage-associated molecular patterns (DAMPS), which induce interferon (IFN) release by microglia. The IFNs initiate a biochemical signaling cascade that activates transcription factors which bind to the interferon-stimulated response element (ISRE), promoting ISG transcription which alters the immunological function and phenotype of microglia, mediating neuroprotection. My project is to confirm the cell signaling mechanism by which hypoxic/hypoglycemic (H/H) conditions induce ISG expression in vitro. To accomplish this, I am using a reporter assay to detect ISRE activation with luciferase protein expression, looking specifically at the effects of H/H experimental stimulations on ISRE activity in microglia from the N9 cell line. I am first optimizing a transfection protocol using green fluorescent protein (GFP) as a reporter. I will use the optimized protocol for experiments, transfecting microglia with DNA encoding the luceriferase enzyme linked to the ISRE promoter, then incubating the cells under H/H conditions, and finally using a plate reader to quantify fluorescence. We anticipate that H/H stimulation will result in increased luminescence in wild-type microglia, and we would interpret these results as indicating that ISG expression in H/H-stimulated microglia is mechanistically due to transcriptional activation at ISRE sites. Identifying the genetic actors in IPC will reveal intracellular targets for pharmaceuticals that can mimic this neuroprotection. We can then administer these medications pre-emptively to patients at risk for stroke or emergently to acute stroke patients.

Microfluidic devices containing microposts, which are used to measure platelet contractile force, are fabricated using a flexible polymer called polydimethylsiloxane (PDMS). PDMS possesses many advantageous characteristics including being non-toxic, optically clear, and typically desirable mechanical properties. However, PDMS is limited as a microfluidic device given its hydrophobicity, or strong ability to repel water. This characteristic is especially problematic when PDMS is used in microfluidic devices intended to analyze blood samples. Blood is protein rich, so the hydrophobicity in PDMS leads to significant biofouling, or biological accumulation, on the surface. Moreover, the range of mechanical properties available with PDMS is limited. This limits the utility of microposts as a platelet force sensing tool since the microposts may be either too stiff or too soft to adequately measure contractile force. The work presented herein sought to circumvent these challenges by replacing PDMS with polyurethane (PU). PU was chosen because its mechanical properties can be modified to adapt to a range of platelet contractile forces from, for example, hyper- and hyporeactive platelets. First, a range of PU types was explored. From these, a commercial grade PU entitled ClearFlex was found to be the best. As a result, it was used in subsequent characterizations, and an optimized protocol for casting microfluidic devices from ClearFlex was developed. The next steps are to test the mechanical, optical, and biological properties of ClearFlex compared to PDMS. The final PU microfluidic device will be developed into a diagnostic tool specifically for clotting disorders.

A major application of micropost technology is the measurement cellular traction forces to quickly diagnose potential medical issues. Currently, polydimethylsiloxane (PDMS) is the material used to fabricate micropost arrays using an intensive three-day double casting procedure. The process begins with casting a negative PDMS mold from a very delicate silicon master array. Negative molds cast from this master require surface treatment overnight via silanization in a vacuum, and then final casting requires an additional overnight cure. The silanization step cannot be forgone in the current process; doing so would result in a micropost array that is inseparable from its negative mold. Additionally, the double casting process must be repeated for every new array in each experiment since PDMS negative molds are not reusable due to degradation by repeated surface treatments. In order to improve upon the micropost fabrication process and widen the scope of applications beyond academia, my colleagues and I are developing a technique to cast PDMS micropost arrays directly from a reusable polyurethane negative mold. Our primary goal is to determine the reusability of polyurethane negative molds in casting PDMS micropost arrays without repeated silanization. To characterize reusability, I will use scanning electron microscopy to measure and quantify the dimensions of the microposts in the arrays in comparison to the silicon master. This step will be repeated for each array reproduced from the polyurethane negative mold as a quantitative measure of its reusability. The creation of a reusable polyurethane negative mold would completely eliminate the intermediate PDMS negative mold and subsequent silanization requirements. Therefore, this replacement would reduce the time required to create a PDMS micropost array from three days to eight hours. Moreover, a polyurethane negative mold could also allow for the simultaneous fabrication of multiple arrays, thus further reducing the time requirement for fabrication significantly.

Polydimethylsiloxane (PDMS) micro and nanoposts have been widely used to measure traction forces in cells, however, the mechanical properties of PDMS micro and nanoposts remain unclear. The current measurements of our lab’s PDMS posts are calculated based on the bulk or global properties of PDMS versus the local modulus, which matters at the cellular scale. It is important to denote this difference because cells actively sense their local physical microenvironments, not the global environment or bulk modulus. In this project, I fabricated specified polydimethylsiloxane micro and nanoposts, which are used in lab to measure cellular forces, and examined the mechanical properties (spring constant and Young’s modulus) of these micro and nanoposts, taking into account the fact that cells only sense their local microenvironments which means experimentally finding the properties of PDMS at the micro and nanoscale. To fully characterize this hydrogel, I utilized atomic force microscopy (AFM) to provide a local constant force (0-60nN for calibration), scanning electron microscopy (SEM) to calculate the dimensions of specified posts of the micro and nanoposts at 110⁰ C at different curing times. Future research utilizing PDMS micro and nanoposts to measure cell forces will take into account these correction values for exact force measurements. These findings provide an update and more thorough way to calculate the mechanical properties of the PDMS micro and nanoposts used in lab.

Purkinje cells are large neurons with intricately elaborate dendritic spines inside the cerebellum of the brain, where they play an important role in controlling motor movement. The migration of Purkinje cells during morphogenesis of the cerebellum requires multiple signaling pathways. Prior research has shown that the extracellular matrix protein Reelin promotes Purkinje cell migration by triggering tyrosine phosphorylation of the cytoplasmic adaptor protein Dab1. Reelin signaling is terminated by the proteasomal degradation of phosphorylated Dab1. Cullin5/RING ligase (CRL5) is the E3 ubiquitin ligase that targets active Dab1 for degradation and its absence causes Dab1 accumulation and promotes Purkinje cell mispositioning. SOCS (suppressors of cytokine signaling) proteins are substrate adaptors for CRL5 that bring phosphorylated substrates to the ligase. SOCS7 has been previously described to be the substrate adaptor that recruits phosphorylated Dab1 to CRL5 during neocortical layering. However SOCS7 is not required for ubiquitylation and turnover of Dab1 in the cerebellum. Thus, the identity of the SOCS protein that regulates Dab1 in Purkinje cells during migration remains to be explored. A novel in utero electroporation method in the cerebellum enables us to manipulate expression level of different genes without the need to generate mutant animals. We will inject specific shRNAs to silence SOCS expression in Purkinje cells to address which SOCS recruits phospho-Dab1 to CRL5 in the cerebellum. Our expectation for this experiment is that knockdown of one or more SOCS proteins will mimic the same misposition phenotype of the Purkinje cells as described for CRL5 mutant animals. Thus we will identify the SOCS-CRL5 complex that regulates the migration of Purkinje cells during cerebellum development. Purkinje cell mispositioning may contribute to diseases like spinocerebellar ataxias and autism, pointing to the exigency of conducting experiments on the mechanism of Purkinje cell migration.

Stroke is the fourth leading cause of death and the leading cause of long-term disability in the United States. Microglia are the resident immune cells in the central nervous system and play critical roles in stroke pathophysiology. During ischemic stroke, cytokines known as interferons (IFNs) are released from CNS cells and bind to microglial cell surface receptors. This binding initiates a signaling cascade that leads to increased expression of Interferon-Stimulated Genes (ISGs). We hypothesize that increased expression of ISGs contributes to post-stroke neuroprotective pathways. There are three types of IFNs, and Type 1 includes IFNs α and β, which are well-known immunomodulatory factors. Type 2 IFNs include IFNγ, a prototypical pro-inflammatory activator. Type 3 IFNs (IFNλs) are the most recently discovered and least well characterized. IFNλs are effective in treating several viral illnesses and they are better tolerated (fewer side effects) than Type 1 IFNs. We have recently shown that Type 1 IFNs can induce increases in ISG expression in microglia. My project goal was to characterize the ability of IFNλs to induce ISG expression in microglia and compare their pharmacological potency to that of Type 1 IFNs. To do this, I stimulated primary microglia with varying dosages of IFNλ3 and for varying amounts of time. I extracted RNA and prepared protein lysates from the stimulated cells. ISG expression was quantified by qRT-PCR and immune (Western) blotting. I also collected conditioned medium and assayed for the release of specific ISG chemokines, such as CCL5 and CXCL10 using ELISA. I obtained dose response curves and time course data. Our results showed robust up-regulation of multiple ISG mRNA transcripts and increases in release of specific chemokines following stimulation of microglia with IFNλ3. These studies will help us continue to characterize important cellular and molecular targets for pharmacological intervention in stroke.

The discipline of Pathology has an enormous scope and students are constantly challenged to determine which topics are essential for clinical patient care and for the National Medical Board exams. There is a significant gap between the material presented in pathology textbooks and the material outlined in medical board review books. In addition, much of the practical knowledge that is most useful for medical students and physicians falls in between this gap. To address this need, we created PocketPath, a web-based modular teaching system, to help students and physicians consume Pathology material more efficiently. PocketPath is designed in an easy to read format that delivers an optimal amount of high yield pathology content, which includes USMLE Step 1 Board Review-style questions and important research papers. Using student surveys, interviews and web usage data, we determined how students consumed PocketPath’s content and improved their pathology test scores. Initial data suggests that PocketPath may replace lecture time, promote active learning, and potentially change the way students prepare for Board exams.