Doctor of Philosophy

dissertationEukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, and associate with each other in some of their biologically important contexts. Spt6 functions at several important regulatory steps in transcription, including nucleosome reassembly, transcription elongation, and mRNA processing and export. As a histone chaperone, Spt6 is important for reassembly of nucleosomes in the wake of elongating RNA polymerase II, a process that is required to regulate transcription initiation and prevent inappropriate transcription from repressed promoters as well as cryptic intragenic transcription start sites. In conjunction with Spt6, Spn1 coordinates the recruitment of mRNA processing and export factors, such as Yra1 and the exosome, thereby enabling biogenesis of mature and export competent mRNA molecules. The functional relevance of Spn1 and Spt6 in chromatin organization and mRNA maturation is well established, although mechanistic details of how these processes are performed are poorly understood. In order to enhance our understanding of the molecular details of Spn1 and Spt6 functions, this thesis has focused on structural, biochemical and functional studies of Spn1 and Spt6 from Saccharomyces cerevisiae. iv The work presented in this dissertation establishes the structures of the entire ordered region of the Spt6 protein, the ordered core of the Spn1 protein, and the Spn1 core in complex with the binding determinant of Spt6. Additionally, we demonstrate the capacity of Spt6 to interact with factors that very likely influence Spt6 function, including histones and nucleosomes. The structures and the functional data described in this dissertation have enhanced our understanding of how Spt6 binds and chaperones histones, and describes a novel role for Spn1 in regulating the histone chaperone activity of Spt6. The Spt6 and Spn1 structures and the biochemical assays developed in this work will aid in future functional and mechanistic studies that will aim to develop a complete molecular and mechanistic model for each Spt6 and Spn1 function

Herpes zoster risk factors in a national cohort of veterans with rheumatoid arthritis

BACKGROUND: Herpes zoster occurs more commonly in patients taking immunosuppressive medications, though the risk associated with different medications is poorly understood. METHODS: Retrospective cohort study including 20,357 patients who were followed in the Veterans Affairs healthcare system and treated for rheumatoid arthritis from October 1998 through June 2005. Cox proportional hazards regression was used to determine risk factors for herpes zoster, and herpes zoster-free survival. Chart review was performed to validate the diagnosis of herpes zoster. RESULTS: The incidence of herpes zoster was 9.96 per 1000 patient-years. In time-to-event analysis, patients receiving medications used to treat mild rheumatoid arthritis were less likely to have an episode of herpes zoster than patients receiving medications used to treat moderate and severe rheumatoid arthritis (p<0.001). Independent risk factors for herpes zoster included older age, prednisone use, medications used to treat moderate and severe rheumatoid arthritis, malignancy, chronic lung disease, renal failure, and liver disease. Among patients receiving tumor necrosis factor-alpha antagonists, etanercept (HR 0.62) and adalimumab (HR 0.53) were associated with lower risk of herpes zoster. There was excellent agreement between ICD-9-CM diagnosis of herpes zoster and diagnosis by chart review (kappa = 0.92). CONCLUSIONS: Risk factors for herpes zoster included older age, prednisone use, medications used to treat moderate and severe rheumatoid arthritis, and several comorbid medical conditions. These results demonstrate that the Department of Veterans Affairs’ national administrative databases can be used to study rare adverse drug events

Associations of Suboptimal Growth with All-Cause and Cause-Specific Mortality in Children under Five Years: A Pooled Analysis of Ten Prospective Studies

Background: Child undernutrition affects millions of children globally. We investigated associations between suboptimal growth and mortality by pooling large studies. Methods: Pooled analysis involving children 1 week to 59 months old in 10 prospective studies in Africa, Asia and South America. Utilizing most recent measurements, we calculated weight-for-age, height/length-for-age and weight-for-height/length Z scores, applying 2006 WHO Standards and the 1977 NCHS/WHO Reference. We estimated all-cause and cause-specific mortality hazard ratios (HR) using proportional hazards models comparing children with mild (−2≤Z<−1), moderate (−3≤Z<−2), or severe (Z<−3) anthropometric deficits with the reference category (Z≥−1). Results: 53 809 children were eligible for this re-analysis and contributed a total of 55 359 person-years, during which 1315 deaths were observed. All degrees of underweight, stunting and wasting were associated with significantly higher mortality. The strength of association increased monotonically as Z scores decreased. Pooled mortality HR was 1.52 (95% Confidence Interval 1.28, 1.81) for mild underweight; 2.63 (2.20, 3.14) for moderate underweight; and 9.40 (8.02, 11.03) for severe underweight. Wasting was a stronger determinant of mortality than stunting or underweight. Mortality HR for severe wasting was 11.63 (9.84, 13.76) compared with 5.48 (4.62, 6.50) for severe stunting. Using older NCHS standards resulted in larger HRs compared with WHO standards. In cause-specific analyses, all degrees of anthropometric deficits increased the hazards of dying from respiratory tract infections and diarrheal diseases. The study had insufficient power to precisely estimate effects of undernutrition on malaria mortality. Conclusions: All degrees of anthropometric deficits are associated with increased risk of under-five mortality using the 2006 WHO Standards. Even mild deficits substantially increase mortality, especially from infectious diseases

The Protein Naming Utility: a rules database for protein nomenclature

Generation of syntactically correct and unambiguous names for proteins is a challenging, yet vital task for functional annotation processes. Proteins are often named based on homology to known proteins, many of which have problematic names. To address the need to generate high-quality protein names, and capture our significant experience correcting protein names manually, we have developed the Protein Naming Utility (PNU, http://www.jcvi.org/pn-utility). The PNU is a web-based database for storing and applying naming rules to identify and correct syntactically incorrect protein names, or to replace synonyms with their preferred name. The PNU allows users to generate and manage collections of naming rules, optionally building upon the growing body of rules generated at the J. Craig Venter Institute (JCVI). Since communities often enforce disparate conventions for naming proteins, the PNU supports grouping rules into user-managed collections. Users can check their protein names against a selected PNU rule collection, generating both statistics and corrected names. The PNU can also be used to correct GenBank table files prior to submission to GenBank. Currently, the database features 3080 manual rules that have been entered by JCVI Bioinformatics Analysts as well as 7458 automatically imported names

A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation

Induction of Bcl6 (B cell lymphoma 6) is essential for T follicular helper (Tfh) cell differentiation of antigen-stimulated CD4(+) T cells. Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins. DP stage-specific deletion of the E3 ligase Cul3, or of Bcl6, induced the derepression of the Bcl6 target genes Batf (basic leucine zipper transcription factor, ATF-like) and Bcl6, in part through epigenetic modifications of CD4(+) single-positive thymocytes. Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter. Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses. Thus, although previous studies have emphasized the essential role of Bcl6 in inducing Tfh responses, our findings reveal that Bcl6-Cul3 complexes also provide essential negative feedback regulation during both thymocyte development and T cell activation to restrain excessive Tfh responses

Restoring shellfish reefs: Global guidelines for practitioners and scientists

Widespread global declines in shellfish reefs (ecosystem-forming bivalves such as oysters and mussels) have led to growing interest in their restoration and protection. With restoration projects now occurring on four continents and in at least seven countries, global restoration guidelines for these ecosystems have been developed based on experience over the past two decades. The following key elements of the guidelines are outlined: (a) the case for shellfish reef resto- ration and securing financial resources; (b) planning, feasibility, and goal set- ting; (c) biosecurity and permitting; (d) restoration in practice; (e) scaling up from pilot to larger scale restoration, (f) monitoring, (g) restoration beyond oyster reefs (specifically mussels), and (h) successful communication for shell- fish reef restoration projects

Natural climate solutions for the United States

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Science Advances 4 (2018): eaat1869, doi:10.1126/sciadv.aat1869.Limiting climate warming to <2°C requires increased mitigation efforts, including land stewardship, whose potential in the United States is poorly understood. We quantified the potential of natural climate solutions (NCS)—21 conservation, restoration, and improved land management interventions on natural and agricultural lands—to increase carbon storage and avoid greenhouse gas emissions in the United States. We found a maximum potential of 1.2 (0.9 to 1.6) Pg CO2e year−1, the equivalent of 21% of current net annual emissions of the United States. At current carbon market prices (USD 10 per Mg CO2e), 299 Tg CO2e year−1 could be achieved. NCS would also provide air and water filtration, flood control, soil health, wildlife habitat, and climate resilience benefits.This study was made possible by funding from the Doris Duke Charitable Foundation. C.A.W. and H.G. acknowledge financial support from NASA’s Carbon Monitoring System program (NNH14ZDA001N-CMS) under award NNX14AR39G. S.D.B. acknowledges support from the DOE’s Office of Biological and Environmental Research Program under the award DE-SC0014416. J.W.F. acknowledges financial support from the Florida Coastal Everglades Long-Term Ecological Research program under National Science Foundation grant no. DEB-1237517

PLZF induces an intravascular surveillance program mediated by long-lived LFA-1–ICAM-1 interactions

PLZF-expressing NKT cells establish residence at intravascular locations, failing to enter the circulation because of constitutive interactions with LFA-1 and ICAM-1