Advances in Molecular Simulation

Molecular simulations are commonly used in physics, chemistry, biology, material science, engineering, and even medicine. This book provides a wide range of molecular simulation methods and their applications in various fields. It reflects the power of molecular simulation as an effective research tool. We hope that the presented results can provide an impetus for further fruitful studies

59th Annual Rocky Mountain Conference on Magnetic Resonance

Final program, abstracts, and information about the 59th annual meeting of the Rocky Mountain Conference on Magnetic Resonance, co-endorsed by the Colorado Section of the American Chemical Society and the Society for Applied Spectroscopy. Held in Snowbird, Utah, July 22-27, 2018

NMR studies of ion transport and diffusion in liposomes and human erythrocytes

This thesis describes investigations of the transport and diffusion of ions in liposomes and human erythrocytes which were carried out using nuclear magnetic resonance (NMR) spectroscopy. The thesis is composed of 8 chapters and 3 appendices. Chapter 1 contains a brief summary of the basic physicochemical and biophysical concepts that were used in the membrane transport studies. Chapter 2 contains the baCkground theory that was used for the analysis of the NMR-derived data and for.the interpretation of the action of the reconstituted plasma membrane Ca2+-A’I'Pase. Chapter 3 describes the materials and general methods that were used. Chapters 4-7 contain the results that were obtained. The experimental findings are preceded by an Introduction and any specific experimental techniques that were used in the experiments, and these are followed by a Discussion. Chapter 4 describes NMR experiments of rapid ionophore-mediated equilibrium exchange of sodium ions between different internal compartments in a suspension of liposomes. The derivation of equations and the results of the numerical calculations that were carried out for the analysis of the experiments described in this Chapter, are presented in Appendix 1. Some of the results that are presented in Chapter 4 are discussed in more detail in Appendix 2. Chapter 5 is concerned with NMR diffusion measurements of ionophore-mediated lithium and sodium ion transport in suspensions of liposomes. Chapter 6 discusses the putative high substrate inhibition kinetics of fluoride self-exchange of fluoride ions in human erythrocytes, as investigated by NMR diffusion measurements. Chapter 7 describes an NMR investigation into the passive, ionophore-mediated, and active transport of calcium ions across the membranes of liposomes and proteoliposomes. A model that describes the action of the reconstituted Ca2+-ATPase is given in Appendix 3A. Appendix 3B discusses the putative electrogenicity of the transport in more detail. The implications of the complete work with respect to an understanding of the physical phenomena involved with membrane transport and the NMR measurement of these phenomena, as well as directions for future research, are discussed in Chapter 8

The evolution of AroA and MurA enzymes from Bacillus

MurA and AroA are important antibacterial targets due to their essentiality in microorganisms and the absence of their respective pathways within mammals. Although much research has focussed on these enzymes, little is known about the evolution of AroA and MurA. Ancestral sequence reconstruction (ASR), a technique used to infer sequences of ancestral proteins and study their properties in the laboratory, was used to study the evolution of AroA and MurA enzymes from Bacillus. The ancestral AroA and MurA enzymes were functionally and structurally characterised and their properties compared to those of contemporary AroA and MurA. AroA and MurA from the last common ancestor (LCA) of Bacillus along with three intermediate Bacillus MurA ancestors were inferred using ASR. The reconstructed AroA and MurA ancestral enzymes show comparable kinetic properties to the contemporary enzymes. The thermal properties of Bacillus AroA LCA, Bacillus MurA LCA and the remaining three MurA ancestors were found to be moderately thermophilic. However, the contrasting thermal profiles observed for AroA, MurA and LeuB ancestral enzymes from Bacillus at the same period of time led to the hypothesis that reconstructed ancestral enzymes provide a snapshot of the evolving host at a given point in time. The exclusive inhibition of AroA and MurA by their inhibitors glyphosate and fosfomycin respectively remains unchanged for AroA LCA and MurA LCA. On the other hand, the high affinity for PEP and increased glyphosate sensitivity exhibited by AroA LCA indicate AroA LCA to be intermediate between class I (glyphosate sensitive) and class II (glyphosate insensitive) AroA. Characterisation of AroA LCA in vivo resulted in AroA LCA posing a fitness cost to the cells carrying this enzyme on a plasmid in a ΔaroA background. AroA LCA and MurA LCA remain highly structurally similar to the contemporary enzymes, with minor differences predominantly located on the protein surface

Insights from modeling metabolism and amoeboid cell motility in the immune system

This thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome scale metabolic reconstruction for zebrafish, that we used to study zebrafish metabolism upon infection with Mycobacterium marinum integrating gene expression data from control and infected zebrafish larvae. The chapters focusing on cell motility in response to the environment, revolve around the question of how the environmental inputs of cell-matrix interactions, cell-sized obstacles and cell-signalling upon wounding shape and guide cell motility.Analysis and Stochastic

Controlling mitochondrial dynamics: population genetics and networks

Mitochondria form an essential component of nearly all eukaryotic cells, are implicated in numerous diseases and may play important roles in ageing. Mitochondrial populations are dynamic, controlled and heterogeneous, with different types -- both mutant and wildtype -- potentially coexisting in single cells. This thesis will study the dynamics of both mitochondria and their genetic material (mtDNA) to improve our understanding of the role of these dynamics in pathology and ageing. This study suggests, as well as critically evaluates, reasons for the existence of complex continuous mitochondrial networks using coarse-grained mathematical models, underlining a nonlinear relation between functionality and network structure. Understanding the link between morphology and function is important as disruption of the former is directly implicated in cellular dysfunction. We perform experiments in which we measure the influence of mitochondrial fusion and division events on integrated mitochondrial membrane potential, an indicator of functionality, and find evidence for its conservation. The cellular homeostatic control acting on a mitochondrial population is poorly understood; to address this, we study the influence of general feedback control strategies on mutant and wildtype mtDNA dynamics. We introduce a simple linear control mechanism that captures a wide variety of biologically observed dynamics, and study optimal parameterisations through the construction of an energy-based mitochondrial cost function. Not only cellular control, but also gene-therapeutic control of mtDNA is studied, allowing us to investigate optimal treatment strategies to reduce mutant loads. The cellular proportion of mutant mtDNA molecules, known as heteroplasmy, is crucial in mitochondrial disease and we study the influence of cellular mtDNA exchange on heteroplasmy dynamics and mutant expansion during ageing. We find that this exchange of genetic material can induce preferential mutant expansion during ageing (even in the face of selection against mutants) through a stochastically driven increase in cellular mean heteroplasmy levels.Open Acces

Development of a non-invasive method to detect pericellular spatial oxygen gradients using FLIM

PhDExtracellular oxygen concentrations affect cellular metabolism and influence tissue function. Detection methods for these extracellular oxygen concentrations currently have poor spatial resolution and are frequently invasive. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing FLIM methods also show limited spatial resolution >1 μm and low time-resolved accuracy and precision, due to widefield time-gate. This study describes a new optimised approach using FLIM to quantity extracellular oxygen concentration with high accuracy (±7 μmol/kg) and spatial resolution ( ≅ 0.3 μm). An oxygen sensitive fluorescent dye, tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]+2, was excited with a multi-photon laser and fluorescence lifetime was measured using time-correlated single photon counting (TCSPC). The system was fully calibrated with optimised techniques developed for avoiding artefacts associated with photon pile-up and phototoxicity, whilst maximising spatial and temporal resolution. An extended imaging protocol (1800 sec) showed no phototoxic effects on cells at dye concentrations of <0.4 mM. Extracellular spatial oxygen gradients were identified around isolated chondrocytes, seeded in three-dimensional agarose gel. The technique was validated by regulating oxygen cellular consumption and thus confirming that the oxygen gradient was governed by cellular consumption. The technique identified a subpopulation of cells exhibiting statistically significant spatial oxygen gradients at the cell perihery. The subpopulation was shown to be significantly larger in cell diameter correlating with what that expected from chondrocytes in the deep zone. This technique provides an exciting opportunity to non-invasively quantify pericellular spatial oxygen gradients from within three-dimensional cellular constructs without prior manipulation of the cells. Thus by examining cellular metabolisms it will advance our understanding of the optimal cellular environment for tissue engineering and regenerative medicine