Molecular and structural insights into Chironex fleckeri venom

This thesis focuses on the characterization of the bioactivity, composition, molecular pathways and structural aspects of Chironex fleckeri venom proteins. C. fleckeri is a box jellyfish that recurrently causes minor to fatal envenomations on the beaches of the northern half of Australia. While there is an antivenom available, its effectiveness is subject to controversy and a rapidly acting treatment has not yet been found. One essential aspect in developing such a treatment is to further the current knowledge regarding the venom components and their effects. C. fleckeri venom is composed of a complex mixture of proteins which can cause rapid cardiovascular collapse in humans and animals. Two highly cardiotoxic, haemolytic and potentially pore-forming toxins have been previously identified, CfTX-1 and -2, and are thought to be the underlying cause for the cardiovascular collapse. Each of my chapters focussed on different toxinological aspects with the overall aim of shedding some light on the complex nature of C. fleckeri venom. Chapter 2 focussed on the intracellular effects of C. fleckeri venom on human cardiomyocytes. While the chosen method, namely fluorescence microscopy, proved inadequate for the intended analysis of C. fleckeri venom, the study provided some interesting results. All cells consistently displayed loss of adherence, nuclear condensation and loss of membrane integrity. The nuclear condensation, an event often observed in apoptosis, has not been previously reported in relation to C. fleckeri venom. Chapter 3 focussed on the characterisation of three previously reported bioactive fractions in the venom (CTF-α, CTF-β and CTF-γ). The previously reported cardiotoxic activity of CTF-α and CTF-β, but not CTF-γ, suggested the presence of the toxins CfTX-1 and -2 in the former two fractions. Interestingly, the mass spectrometric analysis revealed the presence of these toxins in all three fractions, and further these toxins were most abundant in CTF-α, which in the present analysis displayed the least cardiotoxicity. Overall the fractions all contained CfTX-1 and -2, CfTX-A and -B as well as three other cubozoan toxins, CqTX-1, CaTX-A and CrTX-A, in differing proportions. This was reflected in the distinct bioactivity and activated molecular pathways of each of the fractions. Flow cytometry analyses revealed that neither C. fleckeri venom, nor CTF-α (top two hits: CfTX-1 and -2), induced apoptosis, whereas CTF-β (CaTX-A, CfTX-A and CfTX-B) and CTF-γ (CrTX-A and CfTX-A) treated cardiomyocytes were in early and late apoptotic stages, respectively. Overall, there was no apparent difference in bioactivity between cardiomyocytes and fibroblasts, whereas the effects of the venom on mouse erythrocytes was significantly higher than on human erythrocytes. This higher potency on mouse cells might explain why haemolysis is a symptom in laboratory animals but not in humans. Chapter 4 represents the first structural analysis of a C. fleckeri toxin. Two predicted helical regions of CfTX-1 were synthesised to assess experimentally whether they had helical structure that may have some relevance in the putatively pore-forming activity of the venom. While complications were encountered in aqueous solutions, both peptides formed a helical structure in the membrane-mimicking solvent SDS. This data represents the first experimental structural data in favour of a pore-forming mode of action. Overall this thesis has provided insight into the bioactivity of C. fleckeri proteins and their mechanisms of action, highlighted the complexity and the difficulty of working with animal venoms and provided some valuable insight for future studies, including those of a structural nature

Proceedings, MSVSCC 2014

Proceedings of the 8th Annual Modeling, Simulation & Visualization Student Capstone Conference held on April 17, 2014 at VMASC in Suffolk, Virginia

Deep Model for Improved Operator Function State Assessment

A deep learning framework is presented for engagement assessment using EEG signals. Deep learning is a recently developed machine learning technique and has been applied to many applications. In this paper, we proposed a deep learning strategy for operator function state (OFS) assessment. Fifteen pilots participated in a flight simulation from Seattle to Chicago. During the four-hour simulation, EEG signals were recorded for each pilot. We labeled 20- minute data as engaged and disengaged to fine-tune the deep network and utilized the remaining vast amount of unlabeled data to initialize the network. The trained deep network was then used to assess if a pilot was engaged during the four-hour simulation

Neutron Scattering

This book brings suitable data concerning theory and experiments of neutron interactions with different materials. Since the neutron discovery by Chadwick in 1932, researchers of the entire world begin to make studies about it. It is well known that neutron have no charge, and their electric dipole moment is either zero or too small to measure, but theories and experiments show that neutron has spin (presence of magnetic moment), and polarization neutron scattering is plausible. The reader can obtain remarks about inelastic scattering cross sections for neutron; polarized neutron reflectivity; scattering methods; neutron reflectometry tool to probe the chemical structures; neutron scattering for amino acid crystals; and small-angle neutron scattering nanoemulsion heat transfer fluids in this book

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

Selection and characterisation of single-stranded DNA aptamers for triclosan

Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection

The Role of Schwann Cell Mitochondrial Metabolism in Schwann Cell Biology and Axonal Survival

Mitochondrial dysfunction has emerged as a common cause of peripheral neuropathies. While the role of neuronal and axonal mitochondria in peripheral nerve disease is well appreciated, whether Schwann cell: SC) mitochondrial deficits contribute to peripheral neuropathies is unclear. Greater insight into the biology and pathology of SC mitochondrial metabolism could be relevant to the treatment of peripheral neuropathies, particularly because SCs critically support axonal stability and function as well as promote peripheral nerve regeneration. The present thesis investigates the contribution of SC mitochondrial deficits to disease progression in peripheral neuropathies as well as the gene regulatory network that drives the SC regenerative response after injury and in disease states. We describe the generation and characterization of the first mouse model useful in directly interrogating the contribution of SC mitochondrial dysfunction to peripheral neuropathy. These mice: Tfam-SCKOs) were produced through the tissue-specific deletion of the mitochondrial transcription factor A gene: Tfam), which is required for mtDNA transcription and replication. Interestingly, induction of SC-specific mitochondrial dysfunction did not affect SC survival; instead, these deficits resulted in a severe, progressive peripheral neuropathy characterized by extensive axonal degeneration that recapitulated critical features of human neuropathy. Mechanistically, we demonstrated that SC mitochondrial dysfunction activates a maladaptive integrated stress response and causes a shift in lipid metabolism away from new lipid biosynthesis towards increased lipid oxidation. These alterations in lipid metabolism caused the early depletion of key myelin lipid components as well as a dramatic accumulation of acylcarnitine lipid intermediates. Importantly, release of acylcarnitines from SCs was toxic to axons and induced their degeneration. Our results show that normal mitochondrial function in SCs is essential for maintenance of axonal survival and normal peripheral nerve function, suggesting that SC mitochondrial dysfunction contributes to human peripheral neuropathies. Moreover, our work identifies alterations in SC lipid metabolism and the accumulation of toxic lipid intermediates as novel mechanisms driving some of the pathology in peripheral neuropathies associated with mitochondrial dysfunction. Tfam-SCKO mice showed a severe deficiency in their ability to remyelinate peripheral nerve axons after injury. To gain insight into the highly orchestrated process of SC-mediated support of axonal regeneration, we also investigated the transcriptional and post-transcriptional gene regulatory program that drives the SC regenerative response. We profiled the expression of SC microRNAs: miRNAs) after peripheral nerve lesions as well as characterized the injury response of SCs with disrupted miRNA processing and showed that SC miRNAs modulated the injury response largely by targeting positive regulators of SC dedifferentiation/proliferation. SC miRNAs cooperated with transcriptional regulators to promote rapid and robust transitions between the distinct differentiation states necessary to support nerve regeneration. Moreover, we identified miR-34a and miR-140 as regulators of SC proliferation and myelination. We then used a novel computational approach to infer the gene regulatory network involved in this SC injury response and gain insight on cooperative regulation of this process by transcription factors and miRNAs. Together, the results described in the present thesis represent a significant increase in our understanding of how mitochondrial abnormalities specifically in SCs contribute to clinical impairment in patients with peripheral neuropathy. Moreover, the mechanistic characterization of lipid metabolism abnormalities in SCs following mitochondrial dysfunction elucidates potentially important therapeutic targets. Finally, our analysis of the transcriptional and post-transcriptional gene regulatory network involved in the SC regenerative response also provides valuable insight that could be harnessed to help restore normal nerve function in patients with peripheral neuropathy