Lipids are important for the structural and physiological functions of neuronal cell membranes. Alterations in their lipid composition may result in membrane dysfunction and subsequent neuronal deficits that characterise various disorders. This study focused on profiling lipids of aged and LPS-treated rat brain and liver tissue with a view to explore the effect of atorvastatin in neuroinflammation, and examining lipid changes in different areas of rat brain of the Flinders Sensitive Line (FSL) rats, a genetic model of depression. Lipids and other analytes extracted from tissue samples were analysed with proton nuclear magnetic resonance spectroscopy (1H-NMR), gas chromatography (GC) and liquid chromatography-tandem mass spectroscopy (LC/ESI-MS/MS). Changes in the lipid profiles suggested that brain and liver responded differently to ageing and LPS-induced neuroinflammation. In the aged animals, n-3 PUFA were reduced in the brain but were increased in the liver. However, following treatment with LPS, these effects were not observed. Nevertheless, in both models, brain concentration of monounsaturated fatty acids was increased while the liver was able to maintain its monounsaturated fatty acid concentration. Atorvastatin reversed the reduction in n-3 PUFA in the aged brain without reducing brain and liver concentration of cholesterol. These findings further highlight alterations in lipid metabolism in agerelated neuroinflammation and show that the anti-inflammatory actions of atorvastatin may include a modulation of fatty acid metabolism. When studying the FSL model, there were differences in the lipid profile of different brain areas of FSL rats compared to Sprague-Dawley controls. In all brain areas, arachidonic acid was increased in the FSL rats. Docosahexaenoic acid and ether lipids were reduced, while cholesterol and sphingolipids were increased in the hypothalamus of the FSL rats. Furthermore, total diacylglycerophospholipids were reduced in the prefrontal cortex and hypothalamus of the FSL rats. These results show differences in the lipid metabolism of the FSL rat brain and may be suggestive of changes occurring in the brain tissue in depression.

The aim of the research documented in this thesis was to explore issues associated with the development of instrumentation for life detection and characterisation in a planetary exploration context. Within this aim, the following objectives had to be achieved: 1. To consider current and near-future single molecule detection (ultra-low lower limit of detection) analytical techniques that would be compatible with development into a Space qualifiable in situ analytical instrument for the detection of biomarkers in a planetary exploration context. 2. To practically consider the consequences of Planetary Protection and Contamination Control on the development of a sample return instrumentation in a planetary exploration context. 3. To consider the implications of flying an in situ instrument on-board a stratospheric balloon platform in order to apply them into a specific planetary exploration mission: In order to achieve the objectives described above, the following work was pursued:  A desk-based European Space Agency (ESA) study was carried out which entailed producing a literature review on single molecule detection technologies that had to be validated by the expert community. This was done by organising an International Workshop on Single Molecule Detection Technologies for Space Applications in March 2009 at Cranfield University, UK. The approved technologies then had to be analysed with standard analytical techniques (i.e., tradeoffs) in order to propose a specific technology for development and present its breadboard implementation and test plans at the end of the study.  A sample return experiment implementing PP&CC constraints and protocols was designed, built, tested and flown on-board the ESA, Swedish Space Corporation (SSC), Swedish National Space Board (SNSB) and German Space Agency (DLR) BEXUS stratospheric balloon platform. The biological and engineering results obtained from the sample return flight were then analysed and lessons learnt obtained for future flights.  Another desk-based study was performed to research future stratospheric balloon platforms for the exploration of Venus’ cloud layer. The in situ instrument previously proposed for the detection of biomarkers for planetary exploration missions was then put forward as a possible payload for a Venusian stratospheric balloon platform and approved by experts during the Venus Exploration Analysis Group (VEXAG) conference held in August 2011 in Washington D.C, USA. The first part of the research involved studying ultra-low lower limit of detection technologies as these have the potential to impact significantly on the technological and scientific requirements of future Space missions. Two systems were proposed: one based on Tandem Mass Spectrometry (with Cylindrical Ion Trap analysers) followed by Surface Enhanced Raman Scattering spectroscopy to create an MS/MS-SERS instrument for the detection of astrobiology biomarkers in Martian regolith, Europan ice and samples from Titan’s hydrocarbon lakes; and a second one as a Stand-Alone SERS system for the detection of biomarkers in Enceladean plumes, Venusian clouds and cometary coma. The second part of the research practically explored the design of instrumentation for stratospheric balloon platforms. CASS•E, the Cranfield Astrobiological Stratospheric Sampling Experiment, was a life detection experiment that aimed to be capable of detecting stratospheric microorganisms. The experiment consisted of a pump which drew air from the Stratosphere through a 0.2 μm collection filter which retained any microorganisms and >0.2 μm particulates present in the pumped air. Due to the expected rarity of microbes in the Stratosphere compared to the known levels of contamination at ground level, Planetary Protection and Contamination Control (PP&CC)constraints were introduced. Therefore PP&CC protocols were followed to implement Space qualified cleaning and sterilisation techniques; biobarrier technology was implemented to prevent re-contamination of the instrument after sterilisation; and cleanliness and contamination was monitored throughout assembly, integration and testing. The third part of the research demonstrated how an instrument from the first part of the study could be proposed as a payload on-board a stratospheric balloon platform with a focused mission context, i.e., a life detection mission for Venus. Therefore, the research concluded with the proposal of a payload for a Venus mission based on SERS technology on-board a stratospheric balloon platform to search for life above or in the mid Venusian cloud cover.

In recent years, endocrine disruption of corticosteroid signaling pathways in wildlife and humans by environmental chemicals have attracted increasing attention. The integrated potential of chemicals in the aquatic environment that disrupt corticosteroid actions have been evaluated using in vitro glucocorticoid receptor (GR) mediated bioassays. Exogenous natural and synthetic corticosteroids (CSs), which are widely used in human and animal therapeutic applications, were demonstrated to be the most important GR agonists, that can potentially cause adverse effects, especially on aquatic organisms. To date, only a few studies have investigated the occurrence and behavior of GR agonists in the aquatic environment and their removal in conventional wastewater treatment plants. Furthermore, there are hardly any data reported on the removal of GR agonists by advanced water and wastewater treatment, especially those synthetic CSs with high potency. To further understand the fate of GR agonists in water and wastewater treatment processes, a sensitive and robust LC-MS/MS method was successfully developed for analyzing a wide range of GR agonists in various environmental waters. The occurrence of GR agonists in surface water and groundwater was monitored along the Lower Santa Cruz River (SCR). Several GR agonists were detected, and a trend of degradation was observed downstream the two WWTP outfalls for both surface water and groundwater. The fate of GR agonists in a local wastewater treatment plant (WWTP) was investigated, and up to 14 GR agonists were detected at different stages. Highly potent synthetic CSs, including clobetasol propionate (CBP), fluticasone propionate (FTP), fluocinolone acetonide (FCA), and triamcinolone acetonide (TCA), were poorly removed in WWTP. Negative removal of some CSs was observed in primary treatment, which may due to the deconjugation of CS conjugates. Removal of GR agonists in secondary effluent during various advanced water treatment processes, including UV, ozonation, MF, RO and chlorination, were studied. UV and RO appeared to be the most efficient treatment process for the attenuation of GR agonists, followed by ozone, while chlorination had little effects on GR agonists in water. Bench-scale experiments were then carried out to investigate the removal of GR agonists by ultraviolet based advanced oxidation processes (UV/AOPs), and powder activated carbon (PAC). UV/chlorine and UV/H2O2 were demonstrated to be effective in removal GR agonists in wastewater, and UV photolysis would be the predominant mechanism in UV/AOP processes. Four types of PACs were tested for removing GR agonists in wastewater effluent, and Cabot HDB carbon was suggested, while Calgon PWA carbon was not recommended due to its low removal efficiency.

Synthetic cannabinoids have become a growing concern in society. The extensive list of synthetic cannabinoids and the abuse rate has drawn the attention by government agencies throughout the world. These synthetic cannabinoids can adopt a number of different structures, while still acting on endogenous cannabinoid (CB1 and CB2) receptors. In addition, due to structural modifications of these synthetic cannabinoids, many of these compounds can bind to CB1 and CB2 receptors with greater affinity causing severe adverse and life-threatening effects. Because of their structural dissimilarity to the phytocannabinoid Δ9-THC, combating the rapid growth and emergence of synthetic cannabinoids with conventional THC-based methods has become an ongoing struggle. The purpose of this research was to develop and validate a robust and reliable method to accurately identify and quantify 17 synthetic cannabinoids in human whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated in accordance to SWGTOX guidelines for quantitative analysis using the following analytes: 4-cyano-CUMYL-BUTINACA, 5F-3,5-ABPFUPPYCA, 5F-ADB-PINACA, 5F- PY-PINACA, ADB-PINACA, APP-PICA, CUMYL-THPINACA, EMB-FUNICACA, JWH-250, MDMB-FUBICA, MEP-CHMICA, MO-CHMINACA, NM2201, PB-22, RCS-8, UR144, and XLR11. With this developed method, total analysis time was 8 minutes with samples eluting from 3.8 to 5.8 minutes. Calibration curves for each analyte had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes, except for APP-PICA and NM2201 which were quantifiable at 0.1 ng/mL and PB-22 which used a quadratic model. Extraction of analytes using supported liquid extraction (SLE) cartridge improved sample-prep time by more than half, compared to traditional solid phase extraction (SPE) methods. Percent recovery of analytes using SLE was determined to be from 54.92 to 83.36%. Bias and Precision was assessed at 1, 3, 7, and 20 ng/mL for all analytes. All samples had acceptable calculated percent bias and percent coefficient of variation (%CV) within ±20%. No carryover was observed with this method. Matrix effect, using 10 different sources, did not have any interfering effects on detection and quantification of analytes. Ionization suppression and enhancement was observed at various levels, from -4.47 to 76.67%, but had little effect on other validation parameters. Analysis of other commonly encountered drugs (clonazepam, diazepam, (+) methadone, morphine, fentanyl, cocaine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 25I-NBOMe, and phencyclidine (PCP)) does not show any source of interference. The overall development and validation of this method demonstrates a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood in rapid time. / 2020-01-31

In attempts to explore the usefulness of the newly introduced electron capture dissociation (ECD) mass spectrometry for structural analysis of peptides/proteins, different fundamental aspects of the ECD method were investigated using a combination of controlled experiments and high-level theoretical calculations. The relative propensity for dissociation (RPD) of different amino acid residues were extracted from a series of ECD experiments using a common peptide model of RGGGXGGGR, where X was varied systematically among 20 common amino acid residues. Although polar and aromatic amino acid residues were found to behave differently, there exists a fair correlation between the experimental RPD values of aliphatic amino acid residues and the corresponding calculated hydrogen atom affinities of the nearby carbonyl groups. The existence of this correlation reinforces the importance of "hot hydrogen-atom model". From the same set of experiments, the side chain loss reactions of the reduced precursor ions and the zn+• species were extracted. To account for the observed secondary fragments, several generalized dissociation pathways were proposed. The energetics of these dissociation pathways were evaluated theoretically with truncated peptide models using ab initio and DFT calculations; and the kinetics of several competitive reactions were evaluated using Rice-Ramsberger-Kassel-Marcus (RRKM) calculations. / The effect of charge carriers on ECD of peptides/proteins was also studied. Peptides charged through protonation of different basic amino acid residues were found to give ECD spectra of different complexities. The formation of b-/y- and atypical internal fragment ions in peptides with histidines (and lysine, to a lesser extent) as proton carriers was attributed to the higher electron-proton recombination energy as revealed from the energy cycle diagram. Peptides charged through attachment of divalent metal ions were found to give very different ECD spectra. It was believed that typical c/z • fragments were formed from neutralization reactions involving electron-proton recombination; whereas a/b/y fragments were formed from reaction involving electron-metal ion recombination. The preference of recombination channels was somehow related to the electronic configurations of the divalent metal ions. / Fung Yi Man. / "July 2006." / Adviser: T. W. Chan. / Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5254. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 180-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The potentially oncogenic human pathogen BK Polyomavirus (BKPyV) was first identified in 1971 and has since been associated with a number of diseases primarily in immunosuppressed patients. Infection is established in early life and by adulthood up to 90% of populations show seroconversion for the major capsid protein VP1. Despite this infections are rarely cleared, maintaining a silent asymptomatic persistence punctuated with periods of viral shedding in the urine. The virus is non-enveloped and comprises a simple ~5.2 Kb dsDNA genome which expresses just seven known proteins, necessitating a heavy reliance on, and interactions with, host mechanisms in order to efficiently replicate and disseminate within a population. The poorly understood lifelong persistence and failure to clear infection highlights our lack of understanding of the viral life cycle and viral interactions with host processes and responses to infection. Indeed, non-enveloped viruses are thought to spread solely through infected cell lysis but such large-scale lysis should trigger an acute inflammatory response, which is rarely seen in healthy immunocompetent individuals. The research conducted for this thesis first investigates the egress of BKPyV in a non-lytic manner, presenting evidence for an active non-lytic method of viral egress that is dependent on cellular anion homeostasis. Moreover, data generated for this thesis suggests that virions egress via an unconventional secretion pathway which traffics directly from the endoplasmic reticulum (ER) to the plasma membrane in single-membraned vesicles. Further research undertook a whole cell quantitative temporal viromic (QTV) approach, post-experimentally tagging whole cell lysate peptides with isobaric labels (Tandem Mass Tagging, TMT) to provide a greater understanding of host cell proteomic changes throughout BKPyV infection in two primary human cell types over 72 hours of infection. Such an approach identified ~9000 cellular proteins, of which a surprisingly small number changed significantly in abundance in response to BKPyV infection. Of those that were changed in abundance a large proportion were related to cell cycle, revealing that BKPyV infection induces a pseudo-G2 arrest, similar to the G2/M checkpoint. Validation of TMT results in both cell types provided confidence in this robust data set, and further studies highlighted the importance of not only cell cycle status, but the activity of CDK1 for efficient viral infection and replication. Additionally, TMT generated data emphasised the lack of innate immune induction in response to BKPyV infection, suggesting BKPyV exhibits a sophisticated evasion of pathogen recognition.

Li, Gang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 102-108). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.v / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.xi / ABBREVIATIONS --- p.xii / Chapter Chapter One --- Introduction / Chapter 1.1 --- Matrix-assisted Laser Desorption/Ionization (MALDI) --- p.2 / Chapter 1.1.1 --- Laser Desorption --- p.2 / Chapter 1.1.2 --- Matrix-assisted Laser Desorption/Ionization --- p.2 / Chapter 1.2 --- Time-of-flight Mass Spectrometry --- p.6 / Chapter 1.2.1 --- Linear Time-of-flight Mass Spectrometer --- p.6 / Chapter 1.2.2 --- Reflectron Time-of-flight Mass Spectrometer --- p.7 / Chapter 1.2.2.1 --- Linear-field Reflectron --- p.9 / Chapter 1.2.2.2 --- Nonlinear-field Reflectron --- p.12 / Chapter 1.3 --- Structural Analysis Using Time-of-flight Mass Spectrometer --- p.13 / Chapter 1.4 --- Project Objectives --- p.17 / Chapter Chapter Two --- Instrumentation and Experimental / Chapter 2.1 --- Instrumentation --- p.20 / Chapter 2.1.1 --- Laser system --- p.20 / Chapter 2.1.2 --- Flight Tube and Vacuum System --- p.20 / Chapter 2.1.3 --- Ion source --- p.22 / Chapter 2.1.4 --- Deflector and Time Ion Selector --- p.24 / Chapter 2.1.5 --- Two-stage Gridless Reflectron --- p.28 / Chapter 2.1.6 --- "Detectors, Digitizer and Computer System" --- p.28 / Chapter 2.2 --- Experimental --- p.31 / Chapter 2.2.1 --- Sample preparation --- p.32 / Chapter 2.2.2 --- PSD calibration --- p.32 / Chapter Chapter Three --- "Simulation Studies of Time Ion Selector, Collision cells and Curved-field Reflectron" / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Time Ion selector --- p.37 / Chapter 3.3 --- Collision cell --- p.46 / Chapter 3.3.1 --- Simulation of Collision Induced Dissociation (CID) Conditions --- p.46 / Chapter 3.3.2 --- Design and Performance Evaluation of Different Collision Cells --- p.48 / Chapter 3.4 --- Curved-field reflectron (CFR) --- p.58 / Chapter 3.4.1 --- Introduction --- p.58 / Chapter 3.4.2 --- Derivation of Analytical Equations --- p.58 / Chapter 3.4.3 --- Effect of Floating Potential of the Collision Cell --- p.65 / Chapter 3.4.4 --- Effect of R and θ Parameters --- p.65 / Chapter 3.4.5 --- Effect of Length of the Reflectron --- p.70 / Chapter 3.5 --- Conclusions --- p.73 / Chapter Chapter Four --- Construction and Performance Evaluation of Modified Time-of-flight Mass Spectrometer / Chapter 4.1 --- Benchmark Results for the Origin Reflectron Time-of-flight Mass Spectrometer --- p.75 / Chapter 4.2 --- Hardware Modifications of Reflectron Time-of-flight Mass Spectrometer --- p.75 / Chapter 4.2.1 --- Collision Cell --- p.75 / Chapter 4.2.2 --- Curved-field Reflectron --- p.79 / Chapter 4.3 --- Evaluation of the Curved-field Reflectron --- p.81 / Chapter 4.4 --- Evaluation of the field-shaped cylindrical collision cell --- p.85 / Chapter 4.5 --- Conclusions --- p.95 / Chapter Chapter Five --- Concluding Remarks / Chapter 5.1 --- Concluding Remarks --- p.100 / References --- p.101 / Appendix / Appendix 1 User program for time ion selection --- p.108 / Appendix 2 User program for gas collision --- p.111 / Appendix 3 MATHEMATICA program used in calculation for curved-fleld reflectron --- p.114

Mitochondrial fatty acid �-oxidation disorders are a group of clinically and biochemically heterogeneous defects mainly associated with intolerance to catabolic stress. The diseases are potentially fatal, but treatable and the prognosis for most diagnosed disorders is generally favourable. Early diagnosis is thus important to prevent morbidity and mortality. This project describes an improved and validated quantitative fibroblast acylcarnitine profile assay for the investigation of suspected fatty acid �-oxidation disorders. Intact cells were incubated with deuterium-labelled hexadecanoate and L-carnitine, and the accumulated acylcarnitines in the medium analysed using electrospray tandem mass spectrometry. This modified procedure is less demanding technically, requires fewer cells and better reflects the in vivo acylcarnitine status than previously published methods. Mitochondrial fatty acid �-oxidation is coupled to the respiratory chain. Functional defects of one pathway may lead to secondary alterations in flux through the other. The diagnostic specificity and the prognostic potential of the in vitro acylcarnitine profile assay were investigated in fibroblasts from 14 normal controls, 38 patients with eight enzyme deficiencies of fatty acid �-oxidation presenting with various phenotypes, and 16 patients with primary respiratory chain defects including both isolated and multiple enzyme complex defects. All fatty acid �-oxidation deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects irrespective of the severity of symptoms or of different mutation. Preliminary studies suggested a correlation between severity of symptoms and higher concentrations of long-chain acylcarnitine species. However, the fibroblast acylcarnitine profiles from some patients with respiratory chain defects were similar to those of controls, whereas others had abnormal profiles resembling those found in fatty acid �-oxidation disorders. In vitro acylcarnitine profiling is useful for the detection of fatty acid �-oxidation deficiencies, and perhaps the prediction of disease severity and prognostic evaluation facilitating decisions of therapeutic intervention and genetic counselling. However, abnormal profiles do not exclusively indicate these disorders, and primary defects of the respiratory chain remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 149-167).

<p>A new method for analysis of acidic herbicides, mostly phenoxy acids and their esters, in cereals with liquid chromatography-tandem quadrupole mass spectrometry (LS-MS/MS) has been developed. Samples were hydrolyzed with sodium hydroxide in order to release covalently bound compounds followed by neutralization and finally extraction with acidified ethyl acetate. The extraction efficiency for both ester formulations and acids were studied. Acceptable results (70-120 %) were obtained for 2,4-D, dichlorprop, MCPA and mecoprop for both esters and acids. However, low recoveries were observed for ester formulations of dicamba, fluroxypyr, fluazifop and haloxyfop, possibly due to the complex structure of the compounds in combination with the matrix and/or incomplete hydrolysis step. The limit of quantification (LOQ) for targeted pesticides was 0.01 mg/kg. The method has been tested in the EU Proficiency Test for cereals with good results.</p>