PART I The basic helix-loop-helix (bHLH) family of transcription factors are involved in a variety of developmental processes. MyoR is the mouse homologue of the human transcription factor ABF-1 . MyoR is classified as a class II basic-helix-loop-helix transcription factor. In order to better understand the relationship between MyoR and muscle cell differentiation, we analyzed the temporal expression at both the mRNA and protein level. Unlike previous studies, we have utilized reverse transcriptase quantitative PCR to analyze mRNA expression. This allows quantitative analysis of MyoR mRNA levels during muscle cell differentiation. We have also analyzed MyoR expression at the protein level. Our studies suggest that the temporal expression of MyoR at the mRNA level is similar to the expression profile seen at the protein level. To ascertain differences in the MyoR DNA-binding activity during myogenesis we performed EMSA. Results suggest that changes in MyoR expression fail to account for differences in the DNAbinding complexes to an E-box site. Part II Members of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including lymphopoiesis, myogenesis, neurogenesis and sex determination. Screening a eDNA library prepared from silk-producing glands of the black widow spider, we have identified a new bHLH transcription factor named BW6. Within the bHLH region, BW6 shows considerable conservation with other HLH proteins, including Drosophila melanogaster achaete and scute, as well as three HLH proteins identified by gene prediction programs. The expression pattern of bw6 is restricted to a subset of silk producing glands, which includes the tubuliform and major ampullate glands. BW6 is capable of binding an E-box element as a heterodimer with E2A, but was unable to bind this motif as a homodimer. BW6 is also capable of inhibiting the transactivation of rE47 in mammalian cells. BW6 represents the first example of a silk-gland-restricted bHLH protein, and its expression pattern suggests that BW6 may play a role in regulating differentiation of cells in the spider that control silk gland formation or egg case silk gene expression.

While development in flies is well understood, pattem formation and the evolution thereof in arachnids have yet to be clarified. Flies and other metazoans primarily use two families of genes called Hox genes and Pax genes to regulate embryogenesis. Because of the high evolutionary conservation of Hox and Pax proteins, I hypothesize that arachnids also use this system to organize their body pattern. To enable studies of the Westem black widow spider, Latrodectus hesperus, an embryonic eDNA library and a fixation protocol were developed for L. hesperus embryos. The generation of these tools will allow comprehensive analysis of black widow spider development and give insight into whether, and how, spiders use Hox and Pax genes to organize their bodies. Finally, it will provide a more thorough understanding of how different developmental mechanisms have evolved and ultimately how changes in gene expression can lead to a change in overall body plan.

Belonging to the diverse order Araneae, the black widow spider Latrodectus 4 hesperus produces high-performance silks with a broad range ofbiological functions and mechanical properties. The cob weaver spider spins different fibers by using seven specialized glands located in its abdomen. Egg case silk originates from the tubuliforrn gland and to date, no proteins that participate in the assembly process of egg case silk proteins have been identified. The goal of this project was the expression, purification, and characterization of such protein products. De novo sequencing of peptides from in-solution tryptic digestion of black widow spider dragline silk, the most studied type of silk, identified a novel cysteine-rich nonfibroin- like peptide that we named cysteine-rich component or CRC- 1. Further analysis of a large pool of nucleic acid sequences deposited in our custom eDNA database revealed 4 additional sequences with similarities to each other at the amino acid level called CRC-2, CRC-3, CRC-4, and CRC-5, suggesting a new family of proteins. Specifically, Q-PCR analysis revealed that the CRC-5 mRNAs were predominantly expressed in the tubuliform and aggregate glands. Since the aggregate gland manufactures a more complex aqueous solution compared to the tubuliforrn gland, we focused these studies on the tubuliform gland and resultant egg case fibers. Westem blot analysis using a cross-reactive polyclonal anti-CRC-1 antiserum conoborated the presence of CRC-5 in the tubulifmm gland and egg case silk, supporting the colocalization ofTuSpl, a tubuliform gland-specific protein, and CRC-5. Thus, we have demonstrated that these two proteins are present within tubuliform silks. In vitro studies suggested that recombinant CRC-5 displayed enzymatic activity similar to a sulfhydryl oxidase. Collectively, our findings provide new insights into novel proteins that have a potential role in the silk assembly and extrusion pathway of egg case silk fibers.

Spider silk is a natural high-performance biopolymer with superior mechanical propetiies. Although these fibers out perfmm several man-made and natural biomaterials, there are cha llenges to be circumvented before commercialization. One of the silkproducing glands warranting further study is the pyrifonn gland, which produces gluelike threads functioning to cement dragline silk to substrates. We focused on the molecular properties of PySp 1, the major component of pyrifonn silk from Latrodectus hersperus, and its putative Oiiholog, PySp2, from Nephi/a clavipes. To date, there are no reports describing the secondary structure of PySp internal block repeats. Moreover, because the PySp C-terminus amino acid residues are distinct from MaSp C-terminus and the morphology of these glands is different, we hypothesized that PySp C-terminal domains form distinct secondary structures. The MaSp C-terminus has been shown to regulate the silk assembly process and whether the PySp C-terminus performs a similar function is unknown. In order to test this supposition, we used the following experimental approaches: I) we developed a series of PySp prokaryotic expression constructs carrying various block repeat modules representative of the internal iterations found within the protein chain; 2) we constructed prokaryotic expression vectors coding for the PySp C-terminal domains; 3) we expressed and purified the PySp C-terminal domains from bacteria; 4) we performed structural analyses of the purified PySp C-terminal domains using cd spectroscopy and atomic force microscopy. After expression and purification of the PySp C-tennini proteins, our studies support that this domain displays a predominantly ~-sheet structure, distinctive from the NMR-determined ahelical nature of MaSp C-tennini. The difference in secondary structure implies the MA and pyriform glands use different biochemical mechanisms during fiber extrusion to control protein folding and assembly. By investigating protein folding and fiber formation for different spider silk types, its characteristics can be customized for spinning different materials for industrial applications.

Spider silk is a material that both has impressive mechanical properties and is also environmentally friendly. Though there are limitless potential engineering applications for such materials, industrial production of spider silk has proven to be challenging. Farming silk from spiders, as is done with silkworms, is not a viable option for large-scale production of spider silk due to the venomous and predatory nature of spiders. Here, an attempt is made to express synthetic spider silk minifibroins heterologously in Escherichia coli, to purify the recombinant spidroins from cell lysate, and to spin them into artificial fibers through a biomimetic process. Silk minifibroins were designed to be similar to Major Ampullate Spidroin 1 from Latrodectus hesperus. Synthetic fibers were examined by scanning electron and light microscopy, and their mechanical properties were tested by a tensometer. Properties of synthetic silk were compared to those of native dragline silk from the same species from which their design was inspired, revealing synthetic silk fibers with lower breaking stress and breaking strain.

This thesis explores the roles of widowhood and sympathy in Henry James's short and long fiction. By the time James established himself as a writer of fiction, the culture of sentiment and its formation of sympathetic identification had become central to American and British writers. Critically, however, sympathy in James's fiction has been overlooked because he chose to write about rich expatriates and European nobility. James's pervasive use of widowed characters in his fiction suggests the he too participated in the same aesthetic agenda as William Dean Howells and George Eliot to evoke sympathy in their readers as a means of promoting class unity. In this thesis I show how James's use of widowed characters places him in the same sympathetic tradition as Howells and Eliot not by eliciting sympathy for themselves, but, rather, by awakening a sympathetic response from his readers for his protagonists seeking love. In chapter one I explore why James may have used so many widowed characters in his fiction. I cite the death of his cousin Minny Temple as a defining moment in his literary career and argue that he may have experienced an "emotional widowhood" after her early death. I also discuss the role of widows in his short fiction, which I suggest, is different from the role of widows in his novels. This chapter is biographical, yet provides important background for understanding why, more than any other author, James's fiction is replete with widowed characters. Chapter two explains the culture of sentiment of which James has been excluded. It explores the theories of David Hume and Adam Smith and their influence on the aesthetic principles defining Howells and Eliot's work. In this chapter I contend that James is indeed part of this sentimental tradition despite his renunciation of sentiment in his fiction because he tried to promote sympathy among his readers through his widowed characters. In chapter three I do close readings of The Portrait of a Lady (1881) and The Wings of the Dove (1902) and argue that these two texts best represent James's attempt at sympathetic writing.

Thesis advisor: Celeste Wells / This paper analyzes the rights and privileges Black Widow and Captain Marvel are afforded in their respective films coupled with audience perception to understand how that might inform the future of the action heroine genre. There is a stark contrast between the reception of Black Widow and Captain Marvel that is best understood through the release timing of the films along with the character’s plotline within the Marvel Cinematic Universe. Regardless, the rights and privileges each character is afforded in both films demonstrate a dimension of the female experience that had not been fully realized in the MCU prior to their release. Captain Marvel and Black Widow indicate a progression of female representation within the action heroine genre– both in frequency and quality. / Thesis (BA) — Boston College, 2022. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Communication.

This study examined mental health outcomes for widowed Tamil women in Sri Lanka to identify any associations between type of spousal loss and several outcomes, including internalized stigma as a result of widowhood, anxiety symptoms, depressive symptoms, and posttraumatic stress symptoms. A sample of 381 Tamil female widows living in Eastern Sri Lanka were surveyed in 2016 to understand their experiences in a post-disaster and post-war context. Type of spousal loss was separated into seven categories: war-related death, death as a result of tsunami, illness-related deaths, accidental death, suicide, disappearance, and other. Path analysis was used to assess whether type of spousal loss predicts variations in symptom outcomes, controlling for time they have been bereaved, number of children, social problems, and perceived sense of community. The Conservation of Resources (COR) theory (Hobfoll, 1989) was used to conceptualize how spousal loss is connected to distress symptoms and to explain the findings. Analysis revealed that the only types of spousal loss which associated with significant variation in symptom distress were spousal loss as a result of accident and "other" causes. Specifically, accidental causes of spousal death were associated with lower levels of depression, and "other" causes of death were associated with lower levels of depression and anxiety as compared to all other causes of death. In addition, the control variables of sense of community and social problems predicted significant variation in symptom distress such that higher levels of sense of community were associated with lower levels of depression, anxiety, and posttraumatic stress symptoms, and social problems were associated with higher levels of all measured types of mental health distress symptoms.

Nightjars are a group of nocturnal and aerial insectivorous birds that have experienced long-term decline likely driven primarily by habitat loss and declines in prey populations. Eastern Whip-poor-will (Antrostomus vociferus) and Chuck-will’s-widow (Antrostomus carolinensis), two nightjar species native to Illinois, declined 69% and 58% since 1966, respectively. Although previous survey efforts have documented presence of Chuck-will’s-widow and Whip-poor-will, their current distribution in the state is not well known. Using Autonomous Recording Units (ARUs) deployed in a uniform, systematic grid, I surveyed 142 locations from May – July 2022 on public and private lands across the southern eleven counties of Illinois to assess Whip-poor-will and Chuck-will’s-widow distribution and estimate species occupancy. I estimated species relationships with proportion of landcover types, forest patch configuration, and proximity to other landcover types. Additionally, I quantified disturbances from the past 15 years to estimate species relationships to the severity and duration of disturbances. I deployed ARUs for 710 survey days collecting 170,400 minutes or 3,000 hours of recordings. Acoustic bird call identification software, BirdNet, was highly accurate at detecting focal species and greatly reduced the time spent manually reviewing acoustic data. BirdNet identified 43,922 calls of Whip-poor-will and 31,447 calls of Chuck-will’s-widow. I detected Whip-poor-will on 78 surveys with 100% accuracy and Chuck-will’s-widow on 75 surveys with 76% accuracy. Whip-poor-will were positively associated with forest patches with large core areas that neighbored pastures. Additionally, Whip-poor-will were likely to occupy landscapes that had experienced low to moderate disturbance within the previous 15 years. Covariates used to model Chuck-will’s-widow occupancy explained little variation in detection or occupancy and there were no significant relationships with any covariate. However, examining non-significant trends suggest similar relationships as Whip-poor-will in the area. Results highlight the efficiency of passive acoustic monitoring for these birds and the need for further investigation into Chuck-will’s-widow species-environmental relationships. In southern Illinois, Chuck-will’s-widow populations appears to be consistent with previous estimates from the 1990s while Whip-poor-will follow the broader trend of decline.

Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow (Latrodectus hesperus) MaSp1 spidroins in bacteria that incorporate the NTD and CTD, along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Nano-liquid chromatography with tandem mass spectrometry (nLC-MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis and oxidative stress. Collectively, this study unveils new bacterial genes to target by genetic engineering to overcome bottlenecks that throttle spidroin overexpression in microorganisms.