The subunit exchange rate of the cyanobacterial circadian clock component kaic is independent of phosphorylation state

The study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock’s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator

DOWN THE RABBIT HOLE: UNRAVELING THE PATHOGENESIS OF PULMONARY CAVITATION DURING MYCOBACTERIUM TUBERCULOSIS INFECTION

Tuberculosis infects an estimated one-third of the world’s population, and is responsible for more deaths than any other single infectious agent. The continued success of Mycobacterium tuberculosis (MTB) in the post-antibiotic area can be attributed principally to pulmonary cavities: pathologic air spaces surrounded by scar tissue that have replaced healthy lung tissue. Cavities are the primary source of bacterial transmission during infection and contribute significantly to antibiotic resistance and treatment failure, yet the pathogenesis of cavitation is poorly understood. Proposed contributing factors include mechanical stress and enzymatic tissue destruction. To investigate these phenomena, we use a novel repetitive aerosol infection protocol in rabbits to produce a reliable model of tuberculous cavitation in which cavities are monitored by serial computed tomography. Using this model, we demonstrate that pharmacologic inhibition of collagenases does not reduce cavitation, contrary to their speculated role as drivers of cavitation. Using high-resolution 4D cavity maps to track cavity dynamics over time, we show that mechanical stress contributes significantly to cavity formation and persistence dynamics. We also establish that central necrosis of the granuloma is a necessary precursor lesion, but is not sufficient in itself to cause cavitation. Finally, we examine the role of necrosis during infection and cavitation in C3HeB/FeJ mice – specifically, we probe the involvement of the RIP-kinase mediated programmed necrosis pathway. We demonstrate robust necroptosis activation in infected macrophages within and around granulomas in mice – the first in vivo demonstration of necroptosis induction during MTB infection. However, pharmacologic inhibition of RIP1 – the key decision checkpoint in the necroptosis pathway - does not alter outcomes in this model, suggesting alternative activation by one of several RIP1 bypass pathways. In this thesis, we establish two optimized animal models for investigating the pathogenesis of cavitation, and show that these models are well-suited for screening of novel therapeutics. It is our hope that these models will be used in the future not only to further our understanding of the disease, but also to advance novel host-directed therapies to improve patient outcomes and decrease the worldwide burden of tuberculosis

Classification of polyethylene cling films by attenuated total reflectance-Fourier transform infrared spectroscopy and chemometrics

Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was utilised to analyse nine differently branded cling films. Principal component analysis (PCA) was used to assess the intra-sample variability, i.e. the variation within individual cling film rolls; as well as the inter-sample variability, which explores the variability between different rolls of cling film. Linear discriminant analysis (LDA) was then employed to develop a predictive classification model which gave 100% correct differentiation between three brand groupings of cling film, and accurately classified all of the validation samples obtained from different rolls from the same manufacturers

Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS)

The capabilities of current computer simulations provide a unique opportunity to model small-angle scattering (SAS) data at the atomistic level, and to include other structural constraints ranging from molecular and atomistic energetics to crystallography, electron microscopy and NMR. This extends the capabilities of solution scattering and provides deeper insights into the physics and chemistry of the systems studied. Realizing this potential, however, requires integrating the experimental data with a new generation of modelling software. To achieve this, the CCP-SAS collaboration (http://www.ccpsas.org/) is developing open-source, high-throughput and user-friendly software for the atomistic and coarse-grained molecular modelling of scattering data. Robust state-of-the-art molecular simulation engines and molecular dynamics and Monte Carlo force fields provide constraints to the solution structure inferred from the small-angle scattering data, which incorporates the known physical chemistry of the system. The implementation of this software suite involves a tiered approach in which GenApp provides the deployment infrastructure for running applications on both standard and high-performance computing hardware, and SASSIE provides a workflow framework into which modules can be plugged to prepare structures, carry out simulations, calculate theoretical scattering data and compare results with experimental data. GenApp produces the accessible web-based front end termed SASSIE-web, and GenApp and SASSIE also make community SAS codes available. Applications are illustrated by case studies: (i) inter-domain flexibility in two- to six-domain proteins as exemplified by HIV-1 Gag, MASP and ubiquitin; (ii) the hinge conformation in human IgG2 and IgA1 antibodies; (iii) the complex formed between a hexameric protein Hfq and mRNA; and (iv) synthetic 'bottlebrush' polymers

The in vitro selection and biochemical characterization of metalloDNAzymes

DNAzymes are strands of catalytic DNA. First discovered in 1994, they have proved themselves capable of catalyzing many different types of reactions with significant rate enhancements. Because they often require divalent metal-ion cofactors, DNAzymes have readily been developed into metal-ion sensors, in some cases with part-per-trillion sensitivity. These enzymes are currently isolated through in vitro selection. With little to base a DNAzyme selection’s sequence upon, in vitro selections typically begin with randomized DNA pools. As more is learned about the properties of DNAzymes, more efficient means of isolation involving rational design will become more feasible. Fundamental inquiries into the properties of heavy-metal-ion-dependent DNAzymes was the theme of this work. Heavy metal ions have significant health impacts, and thus are an active area of research in bioinorganic chemistry. Additionally, DNAzymes have proven their ability to distinguish between various metal ions with as high as million-fold selectivities. Such selectivities between metal ions with similar charge, ionic radii, and other properties are fundamentally intriguing. Co2+ and Zn2+ are two closely related metal ions, and the factors governing one DNAzyme family’s ability to distinguish between them were examined. During the course of a DNAzyme selection, it is customary to truncate the selected sequence to transform a cis-cleaving construct into a trans-cleaving construct. This general method was found to be ineffective in the case of this family, because peripheral sequences enhanced these DNAzymes’ selectivity for Co2+ over Zn2+ and Pb2+. While DNAzymes have been successfully selected against Mg2+, Zn2+, Hg22+, Mn2+/Mg3+, and other divalent cations, Cd2+-, Fe2+-, and Fe3+-dependent DNAzymes have not yet been isolated. A DNAzyme pair selective for Fe2+ and Fe3+ is of particular interest, because of their interconversion in an biological environment and the fundamental understanding a comparison of the DNAzymes selective for each would provide about DNAzymes’ abilities to distinguish between metal ions. Finally, the Pb2+--dependent DNAzyme 17E was mutated at the G1.1 position with the guanine analogs inosine, diaminopurine, and 2-aminopurine to analyze its catalytic mechanism. 17E contains the 8-17 motif that has dominated selections carried out by multiple labs under a multiplicity of conditions. By investigating the basic properties of DNAzymes, more light can be shed on the structure-function of these molecules, and expand the library of catalytic DNA ready to be used in new applications

Effect of Catalysts on the Kinetics of the Water-toluene Diisocyanate Reaction

The water-isocyanate reaction is important in the production of polyurethane-polyurea foams and films. This reaction is usually investigated with emphasis upon the kinetics of foam formation or film curing and the final properties of the polymer product. The reaction involves a carbamic acid intermediate which decomposes into carbon dioxide and an amine which may further react with isocyanate. This paper describes the evolution of carbon dioxide from the reaction of the diisocyanate with water conducted in an organic solvent under a variety of conditions and catalytic influences