Plants, like other multicellular organisms, possess a complex set of defensive measures deployed upon stimulation of the immune system. In the case of insect herbivory, these defensive measures depend upon specific pattern recognition receptors (PRRs). These PRRs enable the plant to detect herbivore associated molecular patterns (HAMPs). The Inceptin Receptor (INR) is a known herbivore receptor found in legumes, which detects Inceptin 11 (In11), a peptide fragment unique to the oral secretion of Spodoptera exigua. Beyond the plasma membrane much of the pathway from In11 detection to immune response is unknown. This research investigates possible signaling kinases downstream from INR. Receptor-Like Cytoplasmic Kinases (RLCKs) are known to mediate both specific immune and developmental signaling cascades in Arabidopsis thaliana. This suggests that they may play a role in the signaling network of herbivore induced immune responses. In this work we systematically investigate seven RLCKs of interest from three clades within the legume RLCK protein family which are upregulated by In11 treatment in the common bean (Phaseolus vulgaris). To better understand their effects, selected RLCK’s are transformed into the plant model organism Arabidopsis thaliana via Agrobacterium tumefaciens, and homozygous transgenic lines overexpressing the RLCKs are isolated. To examine the effects of over-expressing RLCKs on immunity, classic immune assays will measure their effects on Reactive Oxygen Species (ROS) and the phytohormone Ethylene production during immune activation. This research provides insights into plant immune signaling specific to caterpillar herbivory. In the future, this research may enable the intentional amplification or modulation of plant immune response to reduce losses in crops susceptible to herbivore infestation, a growing concern for food security in the near future.

Plants' defense mechanism against herbivory and disease is integral to both natural ecological balance as well as global food supply. Induced plant responses to these threats are often triggered by specific molecules such as Herbivore Associated Molecular Patterns (HAMPS) and Pathogen Associated Molecular Patterns (PAMPS). In this project, we are refining a Luciferase Reporter Assay (LRA) and then using that assay to categorize novel HAMPS and PAMPS. This assay’s first main part is the HAMP/PAMP Receptor (HPR). By inoculating a Nicotiana benthamiana (NB) leaf with an Agrobacterium containing a Plasmid with an HPR, we are expressing specific HPRs. We are then using the second part of the LRA, a Luciferase Reporter, whose promoter is tied to an immune response related gene to measure levels of immune activity without the need for Transcriptomic Analysis. After we have infiltrated the NB leaf with the vector containing Agrobacterium and induced a response by adding a HAMP/PAMP, we are measuring Luminescence as a proxy for immune response via an imaging machine, then using R to run an analysis on the levels of immune response, helping us characterize an HPR and its signal pathway. We recently optimized time points for HPR and Luciferase imaging and have found that infiltration by Agrobacterium only takes 24 hours for sufficient plasmid integration. As such we are using this assay to run an experiment on the molecular mechanisms of an HPR in only a few days. Refining this LRA and the information we have gathered has helped shed light on the underlying mechanisms used in induced plant defense. In the future, we are planning on expressing genes of interest using this technique to seek novel activators and suppressors helping us further understand mechanisms of plant defense.

Plants utilize cell surface protein receptors to recognize insect herbivory through the detection of Herbivore Associated Molecular Patterns (HAMPs) Following the detection of HAMPs, plants initiate specific immune responses, often measured by the increased production of the hormone ethylene gas and by Reactive Oxygen Species (ROS). The Inceptin Receptor (INR), which is specific to legume plants, recognizes the HAMP Inceptin11 (In11). The binding of In11 to INR initiates a signaling cascade, leading to an immune response. However, the signaling mechanism activated by INR is unknown. The Receptor-Like Cytoplasmic Kinase (RLCK) gene Herbivore Induced Kinase 1 (HIK1) is upregulated by In11 treatment in the bean species Phaseolus vulgaris. The goal of this research is to determine if HIK1 and other RLCKs are downstream proteins required for INR signaling. Because of the genetic intractability of P. vulgaris, I transform Arabidopsis thaliana with RLCK genes using Agrobacterium tumefaciens infiltration. Isolated genetic lines are then used to analyze the effect each RLCK has on immune signaling. Transgenic plants are treated with bacterial associated molecular patterns to trigger an immune response, then tissue samples of the leaves are measured for ROS and ethylene gas production. Results are then compared with ROS and ethylene gas production of wildtype plants. If the studied RLCKs are involved in downstream INR signaling, the transgenic plants will have increased ROS and ethylene gas production. I anticipate HIK1 to have the strongest increase in ROS and ethylene gas production due to the upregulation of HIK1 after In11 treatment in P. vulgaris. Understanding the INR signaling pathway is vital for engineering of plants that are resistant to insect herbivory without the use of pesticides.

Plants recognize herbivore-associated molecular patterns (HAMPs) during herbivory that activates signaling to induce immune defenses. Caterpillar oral secretions contain Inceptin 11 (In11) which is a HAMP recognized by legumes such cowpea via Inceptin Receptor (INR). Thus, In11 and INR are a model system to study proteins involved in HAMP induced defenses, including Kunitz trypsin inhibitors (KTIs). It is known that KTIs are serine protease inhibitors with anti-herbivore activity; however, the precise role of In11 induced KTIs and the effect of cysteine content variation in cowpea KTIs remains unknown. Here, we show that selective removal of cysteines has a negative effect on KTI function in cowpea experiencing herbivory from the fall armyworm (Spodoptera frugiperda). We found that cowpea KTIs act as antiherbivore proteins against the fall armyworm when expressed in Nicotiana benthamiana, as we saw reduced weight gain on larvae feeding on leaves expressing wildtype KTI. Furthermore, we found that KTI function was negatively affected by the removal of cysteines, and larvae fed leaves expressing any of the mutant gained more weight than those feeding on wildtype. We hypothesize that these findings are due to reduced protein stability because we did not detect mutant KTIs in frass samples by westernblot. Understanding KTI protein structure and how it influences protein function is important for designing and selecting antiherbivore proteins to be used for plant defense in agriculture.

On the morning of September 10th, 1990, the body of seventeen-year-old Maria Soledad-Morales was found on the outskirts of San Fernando de Valle, the capital city of Catamarca province in Argentina. What followed in the aftermath of the murder and botched investigation was the Catamarcazo, one of the largest and most publicly salient protest movements in the decades following the Argentinian Dirty War, the military dictatorship that murdered 30,00 people between 1977 and 1983. In this project, I examine genealogies of resistance among women-led protest movements in Argentina from 1977 to 1992 and argue that linkages between movements contributed to a modern Argentinian understanding of gendered violence in the present day. Through analysis of newspapers, interview transcripts, and photographs, I link activist strategies of the 1977 - 1983 Mothers of the Plaza de Mayo with the Catamarcazo movement of 1990 - 1991. I discuss how the Catamarcazo drew and built upon the strategies of Las Madres and introduced consciousness of gendered violence to the Argentinian public in the first nationally acknowledged protest movement centered on violence against women. The purpose of this study is to establish and recognize the continual development of feminist activism within Argentinian history and shed light on the subversive, revolutionary tactics used to combat state repression and gendered violence. By analyzing histories of feminist activism in Argentina, we can gain a greater understanding of how the strategies of feminist movements are built upon and expanded over time, and how the strategies of a past movement can be modified to serve a current movement.

Traditional vaccines use inactivated or live attenuated pathogens to elicit an effective adaptive immune response, but these vaccines can lack safety for immunocompromised individuals. Subunit vaccines–which display characteristic components of pathogens–are safe, stable, and readily engineered, but struggle to elicit a strong immune response. These next-generation vaccines require adjuvants (substances that stimulate the immune system) to increase efficacy. However, many currently used adjuvants lack well-understood mechanisms or wide applicability across vaccines. There is a need for new adjuvant platforms, and protein-based adjuvants are appealing because they are stable, readily engineered, and can be co-delivered with antigens on subunit vaccines. Toll-like Receptor (TLR) proteins are promising adjuvant targets that bind pathogen-associated molecules to activate the innate immune system. Of this family, TLR5 binds the bacterial protein flagellin, but flagellin is not a suitable adjuvant candidate because it is degradation- and aggregation-prone. Therefore, this project aims to design, test, and characterize novel protein-based adjuvants that can bind TLR5 and activate the immune system. Here, I test and characterize de novo mini-proteins that I have computationally designed to bind mouse TLR5 (mTLR5). I use cell-based binding and signaling assays to identify and characterize successful binders. Preliminary results show de novo mini-proteins bind and activate mTLR5 and key knockout mutations at the binder interface decrease signaling. Ultimately, this work hopes to provide a mouse model for these novel protein-based vaccine adjuvants with clinical aims. This project has wide-reaching public health implications, as vaccines offer the potential to improve the health and lives of countless individuals.

In place of an adaptive immune system, pattern recognition receptors (PRRs) that perceive host-derived herbivore-associated molecular patterns (HAMPs) induce immune signaling cascades in plants. Inceptins are a class of proteolytic peptides that originate from chloroplastic ATP synthase that are produced in the oral secretions of all studied species of caterpillars in the order Lepidoptera. Upon introduction to the plant, inceptin-11 (+ICDINGVCVDA−) binds to Inceptin Receptor (INR), triggering a signaling cascade to up-regulate defensive hormones, specialized metabolite toxins, and other direct resistance mechanisms against herbivores. However, the full ecological extent of the effects of the INR signaling cascade is poorly understood. Here, we show that inceptin-11 signaling in common bean (Phaseolus vulgaris) can mediate the attraction of predatory wasps (Polybia sp., Polistes sp.) as an added, indirect line of defense against herbivore threats. Using a near-isogenic line of P. vulgaris containing a 103 base pair deletion in the INR locus, we found that Polybia and Polistes preferentially forage on sibling lines with wild-type INR that respond to herbivore threats. Siblings with fully functioning INR produce nearly three-fold more (E)-4,8-Dimethyl-1,3,7-nonatriene (DMNT), an established volatile wasp attractant, than the deletion line. Our results demonstrate that INR can leverage cross-kingdom predator-prey relationships to aid in defense of the plant. Moreover, our near-isogenic line of P. vulgaris provides a genetic resource for studying the role of pattern-triggered immunity in indirect defenses.

 The human influenza A Virus (IAV) is a contagious respiratory pathogen that can lead to severe maternal illness during pregnancy. IAV infections during pregnancy impart a higher risk of preterm birth, but the mechanism is unknown. We used a nonhuman primate (NHP; pigtail macaque, Macaca nemestrina) model to investigate the immunopathogenesis of IAV infection during pregnancy with a focus on the link between lung infection and placental injury. We hypothesized that a greater IAV infection in the lungs of a pregnant macaque would result in immune injury of the placenta. Pregnant macaques (n=8) were inoculated with 1 x 10^7 plaque forming units (PFU) of the IAV strain A/California/07/2009(H1N1) or media. Blood and bronchoalveolar lavage fluid (BAL) was collected at baseline and at several time points until Day 5, the study endpoint, when Cesarean section and necropsy was performed. I used a tissue culture infectious dose (TCID50) assay to quantify IAV in maternal lung tissues. The TCID50 titer, determined at the dilution where 50% of wells exhibit cytopathic effects, provides a quantitative assessment of actively replicating virus. Further, the laboratory quantified cytokine and chemokine concentrations from the placenta providing an opportunity to correlate viral loads in the lung with placental immune response. All lungs had evidence of replicating virus by TCID50 with a median PFU of 8 x 10^3. Higher viral loads were significantly correlated with higher concentrations of placental interleukin-1 beta (R= 0.94, p<0.0016) and interleukin-4 (R= 0.97, p<0.005). These results are the first to link the IAV viral load in the lungs with the cytokine response in the placenta. In summary, the data suggests that a higher load of replicating virus in the lungs correlates with a placental inflammatory response that may increase preterm birth risk.

Chemical communication is the oldest and most widespread form of communication across the tree of life, and markedly present among lizards. However, the drivers of chemical profile variations in this group remain for the most part uninvestigated. In South American lizards of the Tropiduridae family, semiochemicals are produced by epidermal gland organs called α-glands, exclusively found on the ventral side of male individuals of at least 40 species from four genera. The chemicals produced by these glands are hypothesized to interact with their environments in different ways since chemical species are naturally reactive and tend towards their lowest energetic state. Thus, the intrinsic properties of a semiochemical impact its survival and efficacy for communication. Given the diverse ecology and broad geographical distribution of tropidurids, we investigated whether variation in the chemical composition of α-gland secretions correlates with temperature, humidity, and habitat openness. We performed liquid chromatography-mass spectrometry (LCMS) to obtain the metabolomes of three different sample types. We sampled male skin containing the α-glands, undifferentiated male skin, and female skin. Environmental and chemical property data were extracted from online databases, literature, and field observations. Preliminary tests were done by making Venn diagrams comparing the metabolomes of each sample type. These revealed differences in metabolite compositions, notably between males and females as well as between glandular and undifferentiated skin. From the metabolomes of α-glands, we expect to see chemical species with properties that confer greater survival given the specificities of the environment. For example, given a lizard from a hot and humid environment, we expect the metabolome of the α-glands to contain higher molecular weight species with less functional group complexity. Understanding how environmental parameters drive the chemical composition of α-glands is expected to provide a deeper understanding of the evolutionary history of chemical signaling in terrestrial vertebrates.

Mycobacterium abscessus are non-motile bacilli that cause soft-tissue and pulmonary infections, commonly in healthcare settings or patients with cystic fibrosis. Though it is considered an opportunistic pathogen, its many virulence factors signal its potential for evolution into a true pathogen. Upon infection, the bacilli are internalized by macrophages, forming granulomas to contain the infection. Macrophages can harbor bacilli during infection stages and induce drug resistance by expelling toxins through ABC transporters. Treatment is often challenging as M. abscessus is intrinsically resistant to many antibiotics. Current treatment uses a combination of two or more intravenous drugs and one or more oral antibiotics over several months. Treatment success is challenged by patient adherence and may also be impacted by drug efflux by macrophage ABC transporters. Transporters identify certain drugs as toxic to the body and try to flush them out of the cell. Since Mycobacteria infect macrophages, these channels pose a significant disadvantage to treatment since the cell will actively efflux the drug, preventing the drug's intracellular concentration from increasing to an effective level against the bacilli inside. Certain drugs are known to inhibit ABC transporters, and the addition of these inhibitors in treatment could increase bacteriocidal activity and reduce the development of drug tolerance. First, we will determine the drug's minimum inhibitory concentrations to each inhibitor to see if there is an intrinsic activity on M. abscessus. Next, we will use the Human THP-1 cell line infect with Mycobacterium abscessus and treat with known ABC transport inhibitors in concert with a current therapeutic drug, Clarithromycin. They will then be plated at various time points to determine the colony-forming units. If efflux by macrophage transporters reduces the efficacy of Clarithromycin, bacteriocidal activity will increase between the combination therapy and the clarithromycin-only treatment. These results may improve the current treatment regimens for M. abscessus.

Lizards in the family Tropiduridae have ventral epidermal gland organs that are involved in chemical signaling and whose secretory mechanism is entirely unknown. This is because, like other epidermal generation glands, 'alpha-glands' lack a pore through which their secretion can be exerted. Chemical signaling is a valuable aspect of tropidurid lizards' social and ecological interactions, and some have been observed territorially scraping their alpha-glands against the substrate. This process has been hypothesized to facilitate the release of chemical signals via abrasion. To investigate this abrasion hypothesis, we analyzed 74 skin samples from 27 tropidurid species, using light microscopy and scanning electron microscopy (SEM). The SEM revealed incredible surface variability in epidermal glands, providing morphological insight. We found that the exposed glandular mass of each gland scale rests atop the oberhautchen layer of the skin's subjacent generation, which indicates that the secretion of chemicals involves exposing a mostly solid glandular material on the outside of the scales. Histological sectioning of gland scales revealed morphological consistency, indicating that the same secretory mechanism is shared across the tropidurid phylogeny. Imaging of histology samples also revealed that the shedding process which exposes the glandular material may be facilitated by the clear layer, found directly above the glandular mass during development. Characterization of morphological patterns in the formatted SEM images and comparison with histological data should provide evidence for or against taxon-specific or ecology-specific alpha-gland structures, and further support the idea of chemical secretion requiring epidermal exposal of glandular material. Investigations of the morphology and functional mechanism of this unique organ provide insight into the behavior and evolution of tropidurid lizards and shed light on factors influencing the evolution of chemical signaling in terrestrial organisms. 

Pregnant individuals infected with influenza A viruses (IAV) have higher risks of mortality, hospitalization, preterm birth, and stillbirth. The objective was to determine how the transcriptional program induced by IAV infection in the lung differs between pregnant and non-pregnant states. I hypothesized that a cluster of genes linked to aggravation of influenza disease would be upregulated in the pregnant lung early in IAV infection versus the non-pregnant lung. We used a non-human primate model [NHP; pregnant (N=10), non-pregnant (N=10); Macaca nemestrina, pigtail macaque] to investigate the transcriptional response in the lung of pregnant versus non-pregnant NHPs infected with the IAV CA/04/2009 (H1N1) strain. Maternal lung tissues were collected from the animals at necropsy 5 days after infection. mRNA-Seq was performed by first extracting mRNA from tissues, preparing mRNA libraries, and aligning raw sequencing data, using Spliced Transcripts Alignment to a Reference (STAR), to the macaque genome. I performed normalization of the raw gene count matrix using EdgeR in R Studio and alignment to the macaque reference genome. Next, I performed a single gene analysis using Limma-voom to determine differentially expressed genes (DEG). A total of 115 genes were significantly differentially expressed (>2-fold change, p<0.05) with 77 upregulated and 38 downregulated. Remarkably, genes linked to aggravation of influenza A viral disease, tissue injury, or acidification were upregulated in the infected pregnant versus non-pregnant lung 5 days after infection (MMP8, ATP12A, LGR4, NUP58, KBTBD6; log2fold change 1.28 - 2.8, all p<0.05). Next steps include gene set enrichment analysis and ingenuity pathway analysis to further investigate the gene networks linked to these upregulated genes. In summary, pregnancy was associated with upregulation of genes in the lungs 5 days after IAV infection that may predispose to greater tissue injury versus the non-pregnant lung.