Benefits of nature on children’s health and development are becoming increasingly recognized across the globe. Norway is revered for putting this research into practice, centering nature in early childhood education and setting precedents for ways in which preschools and kindergartens can get their children moving beyond the traditional classroom and up into the trees, down into the mud, and everything in between. Norway has taken a progressive stance on multicultural learning as well. The Norwegian Framework Plan for the Content and Tasks of Kindergartens defines education as an inclusive cultural arena to promote respect for the diversities of all children. I explored this intersection as a landscape architecture student. How might nature itself be important to children’s development and expression of cultural values? In what ways was learning facilitated differently outdoors vs indoors, and what implications might that have for the design of outdoor learning environments? Over the course of three weeks in Trondheim, Norway, I visited three barnehage (preschools) and conducted interviews on the connections between the landscape and the Framework Plan’s goal of inclusion. I found that outdoor environments could be less culturally coded than indoor classrooms, creating an unfamiliarity conducive to curiosity. This curiosity, coupled with undefined materials found in nature or man made objects placed outside of typical contexts, encouraged children to use play to design, communicate, and participate in imaginary worlds together, rather than having to rely on language or common frames of reference. Consequently, some Norwegian educators saw nature as a critical component of promoting children’s inclusion, tolerance, respect, and understanding of the diversities among one another, a revelation frequently overlooked in the U.S. More broadly, my findings point to that missed opportunity, where educational goals for children are similar but neglect serious consideration of the landscape as part of the approach.

Viral pathogens, especially those that undergo rapid mutagenesis, pose a significant threat to public health. Viruses that exemplify this issue include influenza, HIV, and Ebola. Given the low efficacy of seasonal vaccines for influenza, our project focuses on improving existing influenza vaccinations. Instead of using conventional methods, such as injecting inactivated pathogens or viral subunits, mRNA sequences encoding the viral hemagglutinin (HA) fused to our recently-developed self-assembling I53-dn5 nanoparticle platform will be administered in vivo. The organized array of the protein platform can lead to stronger B-cell crosslinking and a robust immune response. However, characterization of I53-dn5 in vitro is critical before use in vaccination studies. My work focused on optimizing the expression, secretion, and assembly of the I53-dn5 protein platform. To mimic in vivo conditions, I transfected DNA encoding HA-fused I53-dn5 into HEK293F cells. Past experiments have shown that when dn5A and dn5B are transfected separately, they express at disproportional concentrations. To resolve this, we encoded both components onto one DNA plasmid for transfection. However, with this new approach, we also needed to cleave the two components after expression. To do so, we incorporated different cleaving peptides, such as T2A and Furin cleavage sites. Through western blots, SDS page electrophoresis, SCC protein purification, and electron microscopy, I analyzed how these cleaving peptides impacted assembly and secretion of the protein platform. Once we are able to consolidate an effective model, we will be able to start in vivo studies. Furthermore, if effective, our model can be used to create vaccinations against other viral illnesses, including HIV and coronavirus.

Prescribed opioids are the most common analgesic used for alleviating acute and chronic pain. Despite this positive attribute, opioids are also highly abused drugs that can lead to tolerance and dependence. This abuse has caused a dramatic increase in opioid overdose-related deaths over the past couple of decades, deeming it a crisis. However, cessation of opioid use in tolerant individuals who wish to detoxify can precipitate severe withdrawal symptoms, often leading to relapse in order to avoid experiencing these negative symptoms. In recent studies, modulation of neuropathic and neuroinflammatory responses have been linked to withdrawal symptoms. As a result, we hypothesized that microglia, the resident immune cell of the central nervous system, serve as a potential target for withdrawal treatment. In order to test this, we reduced microglial activity by inhibiting the purinergic signaling pathway. This was achieved by first exposing mice to escalating doses of fentanyl over the course of a few days to create tolerance. Then, we administered clopidogrel, a selective antagonist of the P2Y12 receptors which are expressed in microglia, before inducing withdrawal using naloxone. Subsequently, in order to quantify whether inhibition of microglial P2Y12 receptors mitigated naloxone-precipitated withdrawal in fentanyl-tolerant mice, we measured avoidance of the withdrawal context with the conditioned place aversion (CPA) test, and evaluated somatic signs of withdrawal with EthoVision video analysis. Avoidance of the negative emotional and physical symptoms of withdrawal is a key driver of relapse, therefore the results from this experiment can provide prospective molecular pathways to target for future studies in treating opioid withdrawal symptoms. Reducing the severity of withdrawal would thus allow ease in discontinuing opioid use and diminish relapse.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide which has been associated previously with stress, fear, and post-traumatic stress disorder (PTSD). One region that strongly expresses Adcyap1, the gene encoding PACAP, is the lateral habenula (LHb), a node of stress in the brain. Studying PACAP and its role within the LHb provide insight into the aid and treatment of a variety of stress disorders by understanding the mechanisms of these conditions. In order to investigate the function of PACAP within the LHb we used a 2x2 experimental design. We injected a virus expressing either a Cre-inducible excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hM3Dq and RiboTag or Cre-inducible RiboTag alone into the LHb of Adcyap1-2a-Cre mice to activate and quantify gene expression in these neurons specifically. Mice were injected with either the DREADD-specific drug clozapine-N-oxide (CNO) or vehicle just prior to contextual fear conditioning, a behavioral procedure in which mice are placed in a novel chamber and given repeated foot shocks in order to elicit a fear memory. The following day, mice were placed in the same chamber without CNO and their time spent freezing indicated the strength of their fear memory. We hypothesized that mice which have their LHb PACAP neurons activated will have increased time spent freezing within the contextual fear chamber, indicating they have a stronger fear memory than the control groups. This study could shed light on the mechanisms of PTSD and other stress disorders.

Opioid abuse leads to over 40,000 annual deaths in the United States; even more individuals are impacted by anticipatory withdrawal anxiety and subsequent treatment avoidance. Despite the magnitude of this issue, there is a lack of effective treatments that address opioid dependence and withdrawal. Molecular responses to opioids have traditionally been linked to neuronal activity, but recent literature suggests that microglia also play a role in opioid addiction. Recent experiments we conducted reveal that opioid dependence and withdrawal have inverse effects on the microglia translatome, with morphine treatment correlating to decreased gene expression and withdrawal correlating to increased gene expression. We found a dramatic change in genes relating to cyclic AMP signaling during withdrawal, which has been shown to modulate microglia motility and potentially their interactions with nearby neurons. From this, we sought to further investigate the molecular basis of microglia morphology during opioid tolerance and withdrawal. To do so, we constructed four experimental groups consisting of mice who received saline followed by saline, saline followed by naloxone, morphine followed by saline, and morphine followed by naloxone. After obtaining the mouse brains through perfusion, we took sections of the striatum. In order to visualize and quantify microglia morphology, we performed 1ba1 immunohistochemistry to stain the slices, then imaged mounted slices on a confocal microscope to acquire confocal stacks of the striatum. This was followed by 3D reconstruction of individual microglia for analysis using 3DMorph software. The results of this experiment are a step towards clarification of molecular mechanisms behind opioid dependence and withdrawal for future work on mitigating the effects of opioid addiction. Alleviating withdrawal symptoms through translational research would allow users to more easily cease opioid use and therefore reduce opioid abuse mortality.

Nicotine is the addictive substance found in various tobacco products. CYP2A13 is an enzyme localized in the lungs, metabolizes tobacco-specific nitrosamine carcinogens that contribute to lung cancer. Therefore, pinpointing CYP2A13 inhibitors is an approach to lower tobacco-based lung cancer risk. Cinnamaldehyde is a common flavoring agent in the fluids of electronic nicotine vaping devices. Cinnamaldehyde was found to be a potent inhibitor of CYP2A6, another enzyme that metabolizes nicotine. Because CYP2A13 and CYP2A6 exhibit overlap in substrate/inhibitor selectivity, the goal here was to evaluate the inhibition of CYP2A13 by cinnamaldehyde. A time-dependent inhibition coumarin assay was performed to determine the kinetic parameters for cinnamaldehyde in recombinant CYP2A13. Primary incubations contained cinnamaldehyde, CYP2A13 Supersomes, and potassium phosphate buffer. Incubations were initiated with NADPH. Secondary incubations contained coumarin, NADPH, and potassium phosphate buffer. At selected time points, an aliquot of the primary incubation mixture was transferred to the secondary incubation tubes, which were terminated with trichloroacetic acid after heating at 37°C for 5.5 minutes. A linearity study was conducted to determine the appropriate termination time. CYP2A13 activity was measured by the detection of hydroxycoumarin using high-performance liquid chromatography (HPLC) and a fluorescence detector. Hydroxycoumarin formation decreased with time and inhibitor concentrations. Maximal inhibition following an 18-minute incubation was 38.3 ± 1.6 and 4.0 ± 0.6%. The maximal rate of inhibition was 0.109 per minute. The results provide evidence that cinnamaldehyde is a time-dependent inhibitor of CYP2A13. Furthermore, cinnamaldehyde appears to be a more potent inhibitor of CYP2A13 than CYP2A6, based on the maximal rate of inhibition. The results imply that cinnamaldehyde could interfere with the bioactivation of nitrosamine lung carcinogens. Additional kinetic studies are needed to confirm the results of this study and to evaluate the safety and toxicity profiles of cinnamaldehyde in complex physiological models.

A series of ultraviolet exposure tests were conducted on a high-oil content, paraffin-based wax binder used in an operational U.S. Thoroughbred horse racetrack to simulate multi-year outside exposure. This type of hydrocarbon binder is commonly used in synthetic granular composites used in North American Thoroughbred horse racetracks and other equine sports surfaces. The function of the binder is to hold together the sand, polymer fiber, and rubber particles that constitute the surface. Previous research on this binder extracted yearly from the same racetrack over a six-year pear period, during which the track was not altered compositionally, showed that environmental oxidation increases over time. This degradation can potentially affect the mechanical performance of the overall surface which may impact the safety of both horse and jockey/rider. The goal of this work is to understand how the binder changes over time and replicate the atmospheric aging of racing surfaces in a much-reduced timeframe (from years to days). Tests conducted include Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), gas chromatography (GC-FID), and rheometry.

This presentation discusses, through a series of historical case studies, how the issue of nuclear waste on indigenous lands is a reproductive justice issue. Drawing on bioethical theory, secondary historical and sociological analysis, and primary source accounts, the presentation demonstrates that the impacts of nuclear waste on indigenous lands and communities are the result of systemic racism against indigenous communities, and that those impacts, including high rates of miscarriage and reproductive cancers, remove bodily autonomy and reproductive choice. Negative health outcomes make communities unsafe places to raise children, and the potential for increased exposure to toxins through traditional cultural practices impacts a community’s ability to raise children with those cultural practices. This history and attention to nuclear waste as an issue of reproductive justice must be part of the conversation as energy and waste storage policies are developed to address climate change.

In order for cells to generate copies of themselves they must undergo a highly complex process called mitosis. During mitosis, many enzymes called protein kinases work together to ensure both daughter cells inherit the correct number of chromosomes when the cell divides. Polo-like kinase 1 (Plk1) is a protein kinase that regulates several events during mitosis including centrosome maturation, spindle assembly, sister chromatid cohesion, and cytokinesis. Recently, the A-kinase anchoring protein Gravin (AKAP12) has been implicated in regulating Plk1 function at mitotic centrosomes. Specifically, loss of Gravin has been linked to defective protein signaling at centrosomes, chromosome misalignment, and increased incidence of micronuclei (small nuclei, an aberration often seen in cancer). However, while previous studies used shRNA-mediated knockdown to reduce Gravin levels in cells, it remains unclear how complete loss of this scaffold in human cells influences mitotic signaling events. To test this, our lab generated Gravin knockout U2OS (osteosarcoma) cells using CRISPR/Cas9 genome editing. First, I employed a combination of immunohistochemical staining and quantitative imaging tools to assess how Gravin loss affected chromosome alignment, micronuclei formation, and gamma tubulin accumulation at centrosomes. I found that loss of Gravin in U2OS and HeLa cells caused misaligned chromosomes and micronuclei. Additional experiments I conducted revealed that Gravin-depleted U2OS, HeLa, and MEF cells presented aberrant gamma tubulin accumulation at mitotic spindle poles. Next, a local drug-targeting approach was used to specifically inhibit Plk1 activity at mitotic spindle poles in U2OS cells. I determined that localized inhibition of Plk1 produced similar mitotic defects as observed in cells lacking Gravin. Collectively, these findings suggest that Gravin is required for coordinating proper Plk1 signaling at centrosomes during mitosis while the loss of this scaffold protein leads to mitotic defects. Future work will uncover downstream substrates of Gravin-anchored Plk1 that becomes dysregulated in cells lacking Gravin.

Leigh syndrome (LS) is a pediatric form of mitochondrial disease which affects the central nervous system (CNS). LS is partly characterized by symmetric necrotizing lesions in the brainstem and cerebellum. Our laboratory utilizes the Ndufs4 (KO) mouse model of LS, as it closely resembles human LS, including the characteristic CNS lesions and the age of onset of disease. Ndufs4, which is also a causal LS gene in humans, is deleted in these mice. In addition to progressive CNS lesions, the Ndufs4 (KO) mice show ataxia and weight loss, and death occurs at a median of 55 postnatal days of age. Information regarding the temporal specificity and mechanisms underlying the pathogenesis of CNS lesions are unknown. Previously, our laboratory has discovered that the loss of Ndufs4 in the VGlut2 expressing glutamatergic neurons drives the CNS lesions and the aforementioned phenotypes of the KO mice. In order to characterize the early events in lesion formation in the CNS, we assess necrosis in the major cell types including VGlut2, GFAP, and Gad2 using GFP reporter lines. This is done by staining and imaging by confocal microscopy of the brain tissue of both the Ndufs4 (KO) and control mice from the cell-type specific GFP reporter lines at postnatal days 30 and 55, corresponding to pre- and post- disease respectively. Additionally, we will be collecting data from mice in more age groups, including at 25, 35, and 45 postnatal days of age. We expect that this will allow us to determine how defects in mitochondrial function lead to diseases such as Leigh Syndrome with tissue, region, and temporal specificity, and in turn, may allow a proposal for Leigh Syndrome treatment.