Visualizing Protease Activation, Retrochmp3 Activity, and VPR Recruitment During HIV-1 Assembly

This thesis covers my contributions to the field of HIV-1 assembly and the field of science communication. Over the course of my studies, I have determined when the HIV-1 protease becomes active during the assembly of new virions, elucidated the mechanism by which the protein retroCHMP3 has an antiviral function, and examined the kinetics of recruitment of the HIV-1 accessory protein Vpr. I have also added significantly to science communication research through a study which evaluated the effectiveness of science summary methods. HIV-1 virions assemble at the plasma membrane of infected cells. The assembly of new viruses is driven by the HIV-1 Gag structural polyprotein and involves a number of viral and host proteins to produce infectious virions. Although many of these factors have been studied extensively, there are still steps during viral assembly that have yet to be fully characterized. In Chapter 2 of this thesis, I will discuss my study of the activation of the HIV-1 protease which is necessary for infectious particle production. Previous research has suggested that protease becomes active prior to scission of the particle from the cell, but there was no study which specifically measured protease activation and its cleavage of Gag during the assembly process. The timing of protease activation and Gag cleavage directly affects particle morphology and infectivity. Using polarization TIRF microscopy, I have directly measured protease activation and Gag cleavage. My results suggest that protease becomes active prior to recruitment of the host ESCRT proteins and scission from the cell. Chapter 3 of this thesis describes my work on the protein retroCHMP3, which was performed in collaboration with Sundquist lab at the University of Utah. The retroCHMP3 protein was originally found in squirrel monkeys and is a truncated and mutated version of the endogenous CHMP3 ESCRT protein. Data from the Sundquist lab shows that retroCHMP3 inhibits the budding of many ESCRT-dependent viruses, including HIV-1, while not preventing essential ESCRT-dependent cell functions such as cytokinesis. To determine the mechanism of retroCHMP3, I used TIRF imaging to show that retroCHMP3 alters ESCRT recruitment to sites of HIV-1 assembly. This altered recruitment suggests that a delay in scission occurs when retroCHMP3 is present in cells. My results also suggest that there is a loss of HIV-1 proteins back into the cell due to protease activation as assayed by loss of fluorescence. As a confirmation of these results, I show that this loss of fluorescence was rescued by genetic inactivation of the protease. We suspect that the retroCHMP3 alteration of ESCRT function affects viral budding more than cellular events like cytokinesis due to the need for viruses to assemble quickly to avoid cellular defense mechanisms and loss of viral proteins from protease activation. My final contribution to the HIV-1 field is a study of the recruitment kinetics of the HIV-1 accessory protein Vpr. My study of Vpr will also be presented in Chapter 3 of this thesis. Vpr is a protein which increases infectivity in infected animals and humans. It is packaged specifically into virions through interactions with the Gag protein. Through simultaneous TIRF imaging of Gag and Vpr, I was able to show that Vpr has a delayed accumulation compared to Gag, suggesting that Vpr is not bound to Gag before coming to the membrane. This work hints at possible interactions between Vpr and the host ESCRT proteins which have recruitment sites close to the Vpr recruitment site. Finally, Chapter 4 of this thesis will show my findings regarding the effectiveness of different science summaries. Science summaries like video abstracts, graphical abstracts, and plain language summaries all help with accessibility of research papers. To study the efficacy of each of those summaries, I created a survey which showed participants video abstracts, graphical abstracts, plain language summaries, and academic abstracts from two HIV-1 research papers. My findings suggested that video abstracts and plain language summaries are both effective ways to summarize scientific research while graphical abstracts and academic abstracts are not as effective. After presenting all of my work in Chapters 2-4, I will discuss the implications and immediate future directions of each contribution I have made. These discussions will be in Chapter 5 of this thesis

A case study in wetlands conservation: Identifying best management practices for landowner stewardship

This research explores wetlands conservation techniques employed by private landowners owning 1,000 or more acres who were recipients of an Environmental Law Institute National Wetlands Award. Study of private landowner stewardship is timely and important because in the United States, 75 percent of all wetlands are under private ownership. Given that wetlands provide a suite of valuable ecosystem services such as water filtration, flood abatement, and carbon storage, their conservation is critical to a healthy environment and productive economy. To accomplish this research, landowner files were processed into a digital archive and sub-categorized for research purposes. Telephone interviews were conducted with a study group of seven landowners. Interview results were studied with archival resources, and a comprehensive profile was generated for each individual. Standard case study methodology was employed to interpret and analyze the emergent results. Key findings of this research include the presence of a shared land ethic between land owners. That land ethic is an individualized sentiment, though landowners expressed similarity through a desire to share conservation success with others. Additionally, the majority of landowners reported using a suite of best management practices. These are grouped according to wildlife and wildlife habitat, wetland hydrology, technical assistance and conservation partnerships, and conservation management techniques. Other key findings include a set of site-specific techniques employed by a smaller subset of the study group. Landowners collectively reported other best practices, including participating in community outreach. Also uncovered during analysis was the range of historical factors that influence land management approach, such as agricultural drainage policies. In its entirety, this research seeks to provide a reference guide for both landowners and policy makers, presenting the best management practices for conserving wetlands on private lands

Food Allergies: Safe Food Handling to Prevent Triggering an Allergic Reaction

The prevalence of food allergies is on the rise, especially among children. Safe food handling is important for preventing triggering a food allergic reaction. Guidelines for safe food handling for food allergies are scarce; therefore, data from key-informant interviews with health professionals, food professionals, and the public were used to develop educational factsheets for food service and child caretakers. These factsheets can be used by Extension professionals for both self-education and outreach education on this increasingly important health topic

Establishing Criterion Validity for the Revised Critical Nutrition Literacy Tool in U.S. College Students

BACKGROUND: Critical nutrition literacy (CNL) plays an important role in how college students make everyday decisions about nutrition choices. Increasing CNL is an aim of many introductory nutrition courses, but there are limited instruments measuring this construct. This study aimed to assess the changeability of CNL and the relationship between CNL and markers of diet quality in young adults. DESIGN: This was a two-phase research project consisting of a nonexperimental, pre–post study and a cross-sectional assessment from 2018 to 2019. Participants were U.S. college students, 18-24 years old, recruited from introductory-level courses from three participating universities, located in Rhode Island, West Virginia, and New Jersey. SUBJECTS AND METHODS: Interventions consisted of (1) a 4-credit, 13-week nutrition course and (2) a cross-sectional, online behavior, environment, and perception survey. CNL was measured using the Revised CNL Tool (CNLT-R) instrument across both phases. Measures for phases include: (1) the changeability of CNL and (2) the relationship between CNL and markers of diet quality. ANALYSIS: Paired t-tests and multivariate analysis of variance were utilized through SPSS version 25.0. RESULTS: CNL score significantly increased from baseline to postintervention from 3.38 ± 0.48 to 3.61 ± 0.55 (P = 0.014). There was an overall significant effect of CNL on markers of diet quality, such as cups of fruits and vegetables (F/V) and teaspoons of added sugar (F [2,1321] = 3.12, P \u3c 0.05; Wilks\u27 Λ = 0.99). CONCLUSIONS: This research found that an introduction to nutrition course was associated with an increase in CNL and that CNL is related to diet quality. The instrument could be used by nutrition educators as an outcome assessment. Future research should investigate other components of the CNL construct as well as predictive validity. Keywords: Decision-making, health behavior, health education, health literacy, young adul

“If my family is happy, then I am happy”: Quality-of-life determinants of parents of school-age children

Objective: Obesity is a public health concern for children and adults and effective obesity prevention programming is needed urgently. The effectiveness of health-related messaging and interventions is influenced by the way content is framed. HomeStyles is an obesity prevention program, which aims to promote health through the frame of improved quality of life. Methods: Thus, focus groups were conducted with English- and Spanish-speaking parents of school-aged children (ages 6–11) to identify key quality-of-life determinants as described by parents. Results: Parents (n = 158) reported that their quality of life was influenced by family happiness and parent and child health (e.g. adequate sleep, exercise, healthy diet). Many parents expressed that their busy schedules and lack of family time were detrimental to their quality of life. Work–life balance and financial stability were other factors commonly noted to impact quality of life. Spanish-speaking parents also reported being undocumented and feeling a lack of a sense of community negatively influenced their quality of life. Conclusion: Considering parent-defined quality-of-life determinants when framing health-related messaging and developing interventions may increase participant interest and ultimately improve health-related behaviors. Next steps in the HomeStyles project include using parent-reported quality-of-life determinants to guide the development of intervention materials

Sticky Decisions: Peanut Butter in a Time of Salmonella

We present a consumer-focused perspective on creating communications regarding potentially contaminated foods. It is illustrated with decisions that might have faced US consumers during the 2009 recalls of peanut and pistachio products. The example shows how knowledge about test results and regulatory processes might be made more useful to consumers