Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging

Small interfering RNA (siRNA) molecules are potent effectors of post-transcriptional gene silencing. Using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function, the effects of target-specific and treatment-specific parameters on siRNA-mediated gene silencing are monitored in cells stably expressing the firefly luciferase protein. In vitro, luciferase protein levels recover to pre-treatment values within <1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting ~10 days in subcutaneous tumors in A/J mice and 3–4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. To demonstrate the practical use of the model in treatment design, model calculations are used to predict the dosing schedule required to maintain persistent silencing of target proteins with different half-lives in rapidly dividing or nondividing cells. The approach of bioluminescent imaging combined with mathematical modeling provides useful insights into siRNA function and may help expedite the translation of siRNA into clinically relevant therapeutics for disease treatment and management

IMPROVING THE SENSITIVITY OF ADVANCED LIGO THROUGH DETECTOR CHARACTERIZATION

This dissertation focuses on the impact of detector characterization work on searches for gravitational waves from compact binary coalescences (CBCs) in Ad- vanced LIGO’s second observing run (O2). This observing run started on November 30, 2016, and lasted until August 25, 2017, and resulted in the identification of 8 unambiguous gravitational-wave signals, including the first observation of a binary neutron star merger. The role of detector characterization is to leverage knowledge of both the interferometers and the data in order to improve aLIGO’s ability to observe gravitational-waves. I focus on the construction of the O2 noise subtracted data set that was searched as a part of the LIGO-Virgo Collaboration’s first gravitational-wave catalog, GWTC- 1. This data set was processed with a noise subtraction pipeline to remove the excess noise identified at each interferometer that resulted in a 30% improvement in the sensitive volume that aLIGO was able to probe. Equally important to the finalized data set is the inclusion of data quality vetoes that indicate periods of instrumental artifacts. I also examine how these instrumental artifacts can mimic gravitational-wave waveforms and reduce the sensitivity of searches for CBC signals, with particular emphasis on the PyCBC pipeline. Understanding this connection is one of the key ways that gravitational waves are differentiated from instrumental artifacts. Finally, I detail the final results presented in the GWTC-1 catalog from a detector charac- terization perspective, and discuss how the efforts highlighted in this dissertation allowed for the detection of new gravitational-wave events and improved analyses of previously identified events

Mitchell V Helms and The Modern Cultural Assault on the Separation of Church and State

This Article suggests that the Mitchell v. Helms decision, and the course on which its sets us—offering government aid to religion as a social good—is a blunder that will have serious adverse consequences for the vital role that religion plays in American society. The intention of aiding religion through the beneficent emasculation of traditional tests of government establishment observed in Helms is just the latest instance of our recurrent attempts to kill American religion with kindness. This process is spurred on by a perceived national crisis following tragedies like those in Paducah, Kentucky and Littleton, Colorado. This Article suggests that while the United States has largely resisted the temptation to alter the inherent wisdom of the system, recent political and judicial changes make the First Amendment and American religious groups that depend on it more vulnerable

Analogy-Based Assessment of Domain-Specific Word Embeddings

No abstract availabl

PyCBC Live: Rapid Detection of Gravitational Waves from Compact Binary Mergers

We introduce an efficient and straightforward technique for rapidly detecting gravitational waves from compact binary mergers. We show that this method achieves the low latencies required to alert electromagnetic partners of candidate binary mergers, aids in data monitoring, and makes use of multidetector networks for sky localization. This approach was instrumental to the analysis of gravitational-wave candidates during the second observing run of Advanced LIGO, including the period of coincident operation with Advanced Virgo, and in particular the analysis of the first observed binary neutron star merger GW170817, where it led to the first tightly localized sky map ($31~\mathrm{deg}^2$) used to identify AT 2017gfo. Operation of this analysis also enabled the initial discovery of GW170104 and GW170608 despite non-nominal observing of the instrument.Comment: 10 pages, 5 figures, submitted to Physical Review

Bayesian modelling of scattered light in the LIGO interferometers

Excess noise from scattered light poses a persistent challenge in the analysis of data from gravitational wave detectors such as LIGO. We integrate a physically motivated model for the behavior of these "glitches" into a standard Bayesian analysis pipeline used in gravitational wave science. This allows for the inference of the free parameters in this model, and subtraction of these models to produce glitch-free versions of the data. We show that this inference is an effective discriminator of the presence of the features of these glitches, even when those features may not be discernible in standard visualizations of the data.Comment: This article has been submitted to Applied Physics Letter

Physicochemical and Biological Characterization of Targeted, Nucleic Acid-Containing Nanoparticles

Nucleic acid-based therapeutics have the potential to provide potent and highly specific treatments for a variety of human ailments. However, systemic delivery continues to be a significant hurdle to success. Multifunctional nanoparticles are being investigated as systemic, nonviral delivery systems, and here, we describe the physicochemical and biological characterization of cyclodextrin-containing polycations (CDP) and their nanoparticles formed with nucleic acids including plasmid DNA (pDNA) and small interfering RNA (siRNA). These polycation/nucleic acid complexes can be tuned by formulation conditions to yield particles with sizes ranging from 60 to 150 nm, ζ potentials from 10 to 30 mV, and molecular weights from ∼7 × 10^7 to 1 × 10^9 g mol^(-1) as determined by light scattering techniques. Inclusion complexes formed between adamantane (AD)-containing molecules and the β-cyclodextrin molecules enable the modular attachment of poly(ethylene glycol) (AD−PEG) conjugates for steric stabilization and targeting ligands (AD−PEG−transferrin) for cell-specific targeting. A 70 nm particle can contain ∼10 000 CDP polymer chains, ∼2000 siRNA molecules, ∼4000 AD−PEG_(5000) molecules, and ∼100 AD−PEG_(5000)−Tf molecules; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The particles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. Uptake of the nucleic acid-containing particles by HeLa cells is measured by flow cytometry and visualized by confocal microscopy. Competitive uptake experiments show that the transferrin-targeted particles display enhanced affinity for the transferrin receptor through avidity effects (multiligand binding). Functional efficacy of the delivered pDNA and siRNA is demonstrated through luciferase reporter protein expression and knockdown, respectively. The analysis of the CDP delivery vehicle provides insights that can be applied to the design of targeted nucleic acid delivery vehicles in general

Too good to be true: when overwhelming evidence fails to convince

Is it possible for a large sequence of measurements or observations, which support a hypothesis, to counterintuitively decrease our confidence? Can unanimous support be too good to be true? The assumption of independence is often made in good faith, however rarely is consideration given to whether a systemic failure has occurred. Taking this into account can cause certainty in a hypothesis to decrease as the evidence for it becomes apparently stronger. We perform a probabilistic Bayesian analysis of this effect with examples based on (i) archaeological evidence, (ii) weighing of legal evidence, and (iii) cryptographic primality testing. We find that even with surprisingly low systemic failure rates high confidence is very difficult to achieve and in particular we find that certain analyses of cryptographically-important numerical tests are highly optimistic, underestimating their false-negative rate by as much as a factor of $2^{80}$

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by {approx}50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics

Virtual Learning in New Zealand: Achieving Maturity

This proposal describes a study into the development of virtual learning in New Zealand, specifically the obstacles that e-learning clusters face or have faced in their journey to sustainability and maturity through the lens of the Learning Communities Online Handbook. Using a variety of data collection methods, the researchers identified three common barriers, including a lack of a coherent vision, difficulty in securing the necessary funding and resources, and a lack of collaboration and cooperation within and between clusters. Based on these findings, it is recommended that individual e-learning clusters develop specific strategies to encourage greater collaboration between clusters and work towards greater consistency between their activities, including professional and organizational development and also of the approaches to virtual learning