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THE ROLE OF TAF1 AND CTCF IN HIV LATENCY
Get PDFDespite the effectiveness of combination antiretroviral therapy (cART) in suppressing HIV-1 replication, it cannot eliminate the virus due to the persistence of a latent reservoir in long-lived CD4⁺ T cells. These transcriptionally silent proviruses evade immune clearance and represent a major barrier to HIV cure strategies. This dissertation investigates host-cell mechanisms that regulate latency, with the goal of identifying novel molecular targets that could be leveraged for therapeutic latency reversal.In Chapter 2, I examine the epigenomic landscape of latency using a primary CD4⁺ T cell model. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) revealed a significant loss of chromatin accessibility across the HIV-1 provirus in latent cells, alongside a marked shift in transcription factor motif usage—namely increased Forkhead and KLF activity and reduced AP-1, RUNX, and GATA motifs indicating that lineage-specific transcriptional programs contribute to the maintenance of latency. Notably, chromatin insulator protein CTCF was enriched at differentially accessible sites, and its knockdown resulted in a moderate reduction of latent infection, implicating CTCF in the regulation of HIV-1 chromatin topology and gene silencing.Chapter 3 builds upon this work by identifying the transcription factor TAF1 as a selective regulator of HIV-1 latency through an epigenetic compound screen using the 2D10 cell line model. The TAF1 bromodomain inhibitor BAY-299 was found to induce HIV-1 expression in latently infected cells. Genetic depletion of TAF1 led to robust HIV-1 reactivation with minimal global transcriptional perturbation, supported by RNA-seq and CUT&RUN analyses. We hypothesize that TAF1 sequesters HIV-1 Tat, limiting its availability to initiate transcription from the viral LTR. Depletion of TAF1 increases Tat availability, driving viral gene expression. These findings suggest TAF1 as a promising target for latency reversal strategies. Together, these studies highlight two distinct, but complementary host regulatory pathways of chromatin insulation and core transcription factors contribute to HIV-1 latency. Both represent viable targets for novel therapeutic intervention.Doctor of Philosoph
Sequential trial emulation for observational vaccine effectiveness studies
Get PDFVaccines are a cornerstone of public health. Because vaccine effects can depend on complex biological, epidemiological, and social processes which unfold over time, statistical considerations for measuring vaccine effectiveness (VE) are often context dependent (Halloran et al., 2010, Ch. 3). Evidence from randomized controlled trials is used to establish safety and efficacy of vaccines and forms the basis for vaccine approval. Once efficacious vaccines are available, it may be infeasible or unethical to conduct additional randomized controlled trials, and in turn, vaccine studies often rely on surveillance data or other routinely collected data (Halloran and Hudgens, 2018). Recent decades have seen rapid development of principled approaches for causal inference in the observational setting. This dissertation considers methods for assessing COVID-19 and pertussis VE via target trial emulation (TTE; Hernán and Robins, 2016; Hernán et al., 2016). The TTE approach involves conceptualizing hypothetical randomized trials, then mimicking the design and analysis of these trials using observational data. The first project describes a nested trial emulation (NTE; Hernán et al., 2005, 2008) method for estimating COVID-19 VE among elders in the Abruzzo region of Italy in 2021. COVID-19 vaccine effects may vary over calendar time (as new viral variants emerge), time since vaccination, or both. A series of weekly trials are emulated and trial-specific effect estimates are used to assess VE trends across both time scales. A NTE inverse probability weighted estimator and a test for heterogeneity in VE across trials are proposed. The second project extends the two-time-scale NTE method described above to accommodate competing risks. In the context of COVID-19, competing risks commonly occur. For example, if an individual dies from causes unrelated to COVID-19, they cannot subsequently die from COVID-19, so non-COVID-19-related death is a competing event for death from COVID-19. Common approaches, such as treating the competing event as a censoring event, may lead to misleading conclusions. In the second project, results obtained using a NTE analysis that accommodates competing events are compared with those obtained from a NTE analysis which treats competing events as censoring events. Causal inference methods often rely on the assumption of no interference between individuals, i.e., an individual’s potential outcomes do not depend on other individuals’ treatment. In many VE studies, this assumption is unlikely to hold because widespread vaccination can have indirect effects that reduce the risk of disease for both the vaccinated and the unvaccinated. The third project proposes a sequential trial emulation method for settings with a time-varying interference structure. A parametric g-formula approach is considered for estimating population-level vaccine effects. The proposed methods are motivated by a large observational database containing diphtheria, tetanus toxoids, and acellular pertussis vaccination records and pertussis diagnoses for children in King County, Washington. BIBLIOGRAPHYHalloran, M.E., Longini, I.M., and Struchiner, C.J. (2010). Design and Analysis of Vaccine Studies. Springer, New York, NY.Halloran, M.E. and Hudgens, M.G. (2018). Estimating population effects of vaccination using large, routinely collected data. Statistics in Medicine, 37(2):294–301. Hernán, M.A., Robins, J., and Garcia Rodríguez, L. (2005). Discussion on ‘Statistical issues arising in the women’s health initiative’ by Prentice, Ross L. et al. Biometrics, 61(4):922–930. Hernán, M.A., Alonso, A., Logan, R., Grodstein, F., Michels, K.B., Stampfer, M.J., Willett, W.C., Manson, J.E., and Robins, J.M. (2008). Observational studies analyzed like randomized experiments: An application to postmenopausal hormone therapy and coronary heart disease. Epidemiology, 19(6):766–779. Hernán, M.A. and Robins, J. M. (2016). Using big data to emulate a target trial when a randomized trial Is not available. American Journal of Epidemiology, 183(8):758–764. Hernán, M.A., Sauer, B.C., Hernández-Díaz, S., Platt, R., and Shrier, I. (2016). Specifying a target trial prevents immortal time bias and other self-inflicted injuries in observational analyses. Journal of Clinical Epidemiology, 79:70–75.Doctor of Philosoph
Improvements and Applications of Scale Space and Image Analysis
Get PDFHi-C technology has been developed to profile genome-wide chromosome conformation capture. In the first project, we propose SSSHiC, a new loop calling algorithm based on Significance in Scale Space (SSS), which can be used to understand Hi-C data at different levels of resolution. By applying SSSHiC to neuronal and glial Hi-C data, we detected loops that are potentially engaged in cell type specific gene regulation. In order to improve SSSHiC, in the second project, we propose advanced distribution theory for significance in scale space. The classical SSS was developed for image data, enabling the detection of both slopes and curvatures across multiple spatial scales. However, fully valid inference for 2-D SSS has remained unavailable, largely due to the more complex dependence structure of random fields. In this project, we use a completely different probability methodology which gives an advanced distribution theory for SSS, establishing a valid hypothesis testing procedure for both slope and curvature detection. Both classical and advanced SSS methods analyze curvature structures in 2-D images in an unsupervised manner; however, they are not directly applicable to supervised learning tasks, such as predicting specific labels from images. In the third project, we developed a Convolutional Neural Network (CNN) and Multiple Instance Learning (MIL) framework to classify estrogen receptor (ER) status and PAM50 subtypes from breast cancer histopathology images. We assessed performance using both within-dataset and cross-dataset validation across five independent datasets. In the fourth project, we develop a new assessment method to evaluate the quality of reported Covid-19 data. Throughout the previous Covid-19 period, numerous models and methods were applied to pandemic data. However, the abnormal results obtained from modeling state-level data in the United States prompted us to place greater emphasis on testing the data quality before employing any methods to model it.Doctor of Philosoph
EPIGENETIC REGULATION BY LONG NONCODING RNAS AND THEIR PROTEIN COFACTORS
Get PDFLong noncoding RNAs (lncRNAs) silence genes across megabase-scale chromosomal domainsthrough recruitment of Polycomb Repressive Complexes (PRCs). The lncRNA Xist silences theentire X chromosome, while the imprinted lncRNAs Airn and Kcnq1ot1 repress multi-megabasedomains in the mouse placenta. Despite shared requirements for PRCs and the RNA-binding protein(RBP) HNRNPK, fundamental questions remain about how lncRNAs recruit PRCs to chromatin.Additionally, while RBP associations with Xist are well characterized, the RBPs that associate withAirn and Kcnq1ot1 and their functional roles remain largely unknown.In this dissertation, we profiled RBP and PRC associations across all three lncRNAs to uncovershared mechanisms of lncRNA-mediated gene silencing. Through formaldehyde crosslinked RNAimmunoprecipitation, we found that PRC1—not PRC2—components were consistently enrichedand colocalized with HNRNPK. HNRNPK depletion reduced PRC1 associations, supporting aprotein-bridged recruitment model.Using an isogenic ectopic expression system, we demonstrated that Airn recruits PRCs withpotency rivaling Xist. Yet strikingly, Airn failed to silence genes unless fused to Xist’s Repeat Adomain, indicating that Repeat A provides a silencing function distinct from PRC recruitment.We next systematically profiled the RBP landscape of Airn and Kcnq1ot1 through RIP-seq of27 RBPs, revealing novel highly enriched protein associations. Remarkably, Airn and Kcnq1ot1exhibited shared RBP enrichment patterns with Xist, suggesting conserved mechanisms acrosslncRNAs. Most importantly, we identified HNRNPU as universally required for PRC-directed chromatin modifications by all three lncRNAs, yet dispensable for Airn and Kcnq1ot1 chromatinlocalization.Together, this work establishes that lncRNAs recruit PRCs through protein bridges, revealsRBP associations mediating Airn and Kcnq1ot1 function, and identifies HNRNPU as essential forPolycomb deposition across all three lncRNAs.Doctor of Philosoph
AN ENVIRONMENTAL ORIGIN FOR DIVERSITY IN GAMMA-RAY BURST AFTERGLOWS
Get PDFThe most powerful and energetic explosions in the known universe – gamma-ray bursts (GRBs) – are thought to originate from the collapse of massive stars or the merger of compact objects. Often marking the birth of a black hole, GRBs produce broadband emission via synchrotron radiation as the resulting ultra-relativistic jet interacts with the surrounding medium. This emission, known as the afterglow, carries rich information about the nature of the burst and its environment. To better understand these cataclysmic events and the environments that surround them, we developed a Python-based modeling and inference framework, AMPy, capable of modeling afterglows in arbitrary and smoothly varying power-law density profiles, extending beyond the standard assumption of either a constant-density interstellar medium (k = 0) or an idealized stellar wind profile (k = 2). This dissertation presents detailed results from modeling 15 events with AMPy which reveal a wide diversity of environmental structures, including steep “superwinds”, shallow “subwinds,” stratified media with smooth transitions between regimes, and cavity-like regions consistent with effective density gradients of k < 0. The recovered stratified and cavity-like profiles are consistent with evolving progenitor winds, shocked regions, termination shocks, or the presence of shells ejected prior to the burst. These findings also reveal that radiative cooling plays a significant role in many events, that synchrotron self-absorption can influence modeling even when unobserved, and that calibration systematics and unaccounted-for statistical uncertainties (“slop”) must be incorporated to avoid biased fits, underestimated uncertainties, and consequently overstated conclusions. AMPy handles all of this self-consistently and efficiently. These results demonstrate that GRB afterglow environments are far more diverse than has been traditionally assumed for the past three decades and that oversimplified models likely misrepresent the physical nature of these explosions.Doctor of Philosoph
FIDELITY OF DNA DOUBLE-STRAND BREAK REPAIR BY NONHOMOLOGOUS END JOINING
Get PDFDNA stores the genetic information essential for all life as we know it, but it is constantly assaulted with damage. To survive, cells must maintain myriad DNA repair pathways addressing this damage. Chromosomal double-strand breaks are the most toxic of all damage types and can lead to mutations, cancer, and cell death if left unrepaired. Cells rely on three pathways to combat double-strand breaks: nonhomologous end joining, homologous recombination, and polymerase theta-mediated end joining. This dissertation broadly studies double-strand break repair, observing all pathways in collaboration (or competition) when a cell encounters chromosome breaks. It further describes key mechanistic features of nonhomologous end joining that confer fidelity and adaptability during the processing of diverse double-strand break ends. First, I analyze the independent contributions of double-strand break repair pathways at in a time-resolved approach, including the kinetics of chromosome breakage, the capture of transient repair intermediates, and the mutation patterns arising from the individual pathways. I further analyze repair spectra in contexts of genetic perturbation and small molecule inhibition of key players in the end joining pathways. Next, I study ribonucleotide incorporation by nonhomologous end joining polymerases. Using extrachromosomal substrates and chromosome breaks through CRISPR-Cas9 or an inducible V(D)J recombination system, I dissect the specific contributions of ribonucleotide incorporation to the efficiency and accuracy of double-strand break repair and what factors regulate their use. Finally, I address the mechanisms by which nonhomologous end joining processes and repairs the individual strands of a double-strand break. Using novel assay designs, I show that the nonhomologous end joining machinery adapts to a variety of end structures and sequences, not just between distinct double-strand breaks, but also between the two strands of a single broken DNA molecule. Throughout this dissertation, I develop a comprehensive picture of double-strand break processing. I define mechanisms of nonhomologous end joining by which it addresses diverse double-strand break end structures to promote high fidelity repair. These findings help further define processes that promote the genome maintenance essential for cell survival.Doctor of Philosoph
Functional super-resolution microscopy of fibers and polymers: convergence of artificial and biological systems at the nanoscale
Get PDFFluorescence nanoscopy has opened a new frontier for visualizing and understanding polymeric and fibrous materials with molecular precision. Building on advances in single molecule localization microscopy (SMLM), researchers are now extending beyond structure to probe dynamic and functional properties that govern material behavior. This Focus article highlights recent progress in functional SMLM for mapping polarity, viscosity and molecular motion within polymers and fibers, revealing how these nanoscale parameters influence macroscopic performance. Examples include tracking polymerization and phase evolution, resolving nanofiber organization, and correlating structural heterogeneity with local chemical environments. We further discuss the growing convergence between artificial and biological systems with shared principles of hierarchical organization. By integrating structural, dynamic, and functional imaging, fluorescence nanoscopy provides a unifying framework for studying and engineering complex molecular assemblies across living and synthetic matter
Symmetry and substituent electronics dictate electronic structure of low spin, mixed-ring rhenocene complexes
Get PDFIdentifying the impact of molecular structure and symmetry on excited state character and energetics is vital to enable and control photochemical reactions. In this work, density functional theory (DFT) and time-dependent DFT (TD-DFT) were used to determine the electronic structure and excited state reduction potentials of six heteroleptic rhenocene complexes, each bearing a cyclopentadienyl ligand and a functionalized tetramethylated cyclopentadienyl ligand (ReCp(CpMe4R), where Me = CH3 and R = Me, CF3, tBu, CHCH2, CHO, or OMe). Calculations were performed using the B3LYP and BP86 functionals, utilizing SDD + f effective core potential and its associated basis set for Re, and 6-311G* basis set for all other atoms. All six complexes exhibit eclipsed geometries with similar Re-ring bond distances and angles. Despite their structural similarities, the electronic structure of these complexes varies with ligand functionalization. ReCp(Cp*), 1, (where Cp* = pentamethylcyclopentadienyl) has a 2A1 ground state with a dz2-based LUMOβ orbital, whereas functionalized tetramethylated derivatives, 2–6, have 2A″ ground states with dx2−y2-based LUMOβ orbitals due to their lower molecular symmetries (C5v vs. Cs, respectively). Regardless, complementary TD-DFT and fragment orbital analyses show that low-energy LMCT excited states are retained across all six complexes (where LMCT character > 90%). Furthermore, hypsochromic shifts and higher oscillator strengths are observed for 2–6 compared to 1, resulting from the changes in HOMOβ–LUMOβ gaps and excitation into the dx2−y2 orbital for 2–6 rather than dz2 orbital for 1, which increases the orbital overlap between the hole–particle pairs that describe the lowest-energy LMCT excitations. Appending electron donating and withdrawing groups to these mixed-ring rhenocene derivatives also tunes ground state and excited state reduction potentials over a 400 mV and 700 mV range, respectively, enabling access to more oxidizing LMCT excited states. Collectively, these results showcase design strategies to control acceptor orbital character, orbital energetics, excited state energies, and reduction potentials, while simultaneously retaining low-energy LMCT excited states across a series of rhenocene derivatives. In result, this work establishes approaches to design and tailor next-generation mixed-ring rhenocenes with low-energy LMCT excited states for photochemical applications
Longitudinal correlates of quitting e-cigarettes in the United States
Get PDFObjective E-cigarette use has risen markedly among young adults, despite efforts to curb this trend. To inform future programs and policies, we sought to identify longitudinal correlates of quitting e-cigarette use in the United States (US). Methods The study design was longitudinal. A nationally representative sample of 1138 US adults and adolescents who used e-cigarettes took a baseline online survey between Nov 2022 and Jan 2023. Six months later, 844 respondents completed a follow-up survey. Analyses used weighted simultaneous multivariable logistic regression that included demographic and vaping characteristics assessed at baseline to predict quitting e-cigarettes at 6-month follow-up. Results At 6-month follow up, 15 % of respondents had quit e-cigarettes. Quitting was associated with having stronger quit intentions (adjusted odds ratio [aOR] = 1.49, 95 % CI = 1.09, 2.04), occasional use (aOR = 5.93, 95 % CI = 3.11, 11.30), and lower nicotine dependence (aOR = 2.56, 95 % CI = 1.38, 4.76). Quitting was also more common among gay, lesbian, or bisexual respondents than straight respondents (22 % vs. 13 %, aOR = 2.20, 95 % CI = 1.10, 4.38) and females than males (18 % vs. 11 %, OR 1.88; 95 % CI 1.06, 3.34). Conclusions Greater motivation to quit and lower markers of nicotine dependence were associated with quitting vaping at 6 months. Interventions focused on reducing nicotine dependence and increasing quit intentions, especially among daily users, could support e-cigarette cessation
Perceptions of a Menthol Cigarette Ban: Focus Group Study With US Adults who Smoke Menthol Cigarettes.
Get PDFPurpose This study examined perceptions of a proposed US menthol cigarette ban among adults who smoke menthol cigarettes. Design Focus group discussions. Setting Virtual focus groups with 7-9 participants each. Participants 50 US adults (age 21+) who currently smoke menthol cigarettes participated in six focus groups: two with Black participants; two lesbian, gay, or bisexual participants; and two general population groups. Methods Each 90-minute session was audio-recorded and transcribed. We used qualitative thematic analyses to examine participants' views on reasons for the ban and its potential impact on tobacco use, with a focus on differences across the three identity-based groups. Results Many participants, across all group types, believed the ban aimed to protect youth and future generations due to menthol's appeal and higher addictiveness. Some viewed the ban as government overreach, racially targeted, and economically or politically motivated. Several indicated they would seek menthol cigarettes through illicit markets that might emerge post-ban. Some considered switching to non-menthol cigarettes, vapes, or marijuana. However, several felt the ban could help them reduce smoking and quit entirely, citing non-menthol cigarettes' lower appeal and concerns about vaping's addictiveness and harms. Black participants expressed specific concerns about over-policing, racial profiling, community safety, and potential increases in crime related to menthol access. Conclusions Concerns about over-policing and targeted enforcement align with tobacco industry narratives, illustrating the pervasiveness of negative industry messaging. Findings underscore the importance of proactive communication about the ban's public health goals, while addressing community concerns about policing and racial equity