MA

thesisThis thesis examines the Narcissus theme and narcissism in the literature of the nineteenth century, focusing on the theme as it is reflected in Mary Shelley's Frankenstein and Nietzsche's Zur Genealogie der Moral, especially the dramatic shift in perspective towards narcissism reflected in these two works. Historical and cultural changes over the course of the nineteenth century that may have led to this reversal are discussed, and it is suggested that the earlier negative view of narcissism is bound with a theological concept of the "self" that is no longer compatible with the understanding of humanity's radically animal nature. A positive view of narcissism in Nietzsche's writing may reflect a vacuum in the traditional understanding of the "self" that demands "self-creation." Narcissistic expression may also represent a liberation from the superstitious awe surrounding the former, more theological concept of the "self" and from the moral constraints of the theology from which this derived. Analysis of the Narcissus theme, beginning with Ovid's version of the tale in the Metamorphoses then moving into the nineteenth-century works, reveals common strands in all the depictions of narcissism discussed. The analysis concludes that while Nietzsche assumes a positive stance towards a narcissistic pose, the self-idealization reflected in his work is as illusory as the negatively reflected narcissism of the earlier versions, and that below the surface of Nietzsche's jubilant tone the philosophical voice of his work seems as haunted by the idealized self-image he has created as the hero of Shelley's novel

Exploring biomolecules, metallodrugs, and their interactions via the use of UHR-FT-ICR mass spectrometry

The work presented herein focuses on the study of novel metallodrugs and their interaction with various possible targets and off-targets in the form of biomolecules such as peptides, proteins, DNA, and small molecules via the use of ultra-high resolution Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. Beyond traditional platinum(II) metallodrug such as cisplatin and oxaliplatin, new designs of metallodrugs are being conceived to attack and kill cancerous cells via new mechanisms of action in an effort to exceed the potency, selectivity, and effectiveness of metallodrug treatments. These new metallodrugs range from various activation strategies, to specific target binding, and even catalysis of cell-disrupting processes. As a result these novel drugs can have a wide array of effects on various biomolecule species the may encounter in the body. It is the aim of this thesis to show that mass spectrometry, specifically FT-ICR Mass Spectrometry, is uniquely suited to studying the wide array of metal-based drugs, their biomolecule targets/off-targets, and the numerous reaction products produced from their interactions. Though the study of metallodrug-modified biomolecules via mass spectrometry was shown to be challenging in many cases, mass spectrometry is currently the only analytical technique viable for studying the complex systems involved to a useful level of detail. The majority of the thesis focuses on study of isolated biomolecules interacting with novel metallodrugs and the MS and tandem-MS based study of the resulting observable components. A great range of metallodrugs and biomolecule interactions were observed. A photoactivated platinum(IV) metallodrug (trans, trans, trans[Pt(py)2(OH)2(N3)2]) was shown to produce a variety of platinum(II) based modifications to the peptides studied when activated with blue (463nm) visible light, with the ligand configurations varying depending on whether a histidine amino acid residue was present, allowing retention of both pyridine ligands, or not, allowing release of any of the bound ligands. Tandem MS studies using electron based dissociations showed the primary preference of binding to be at the Histidine residues, and when not available the complexes could bind to lysing functional groups, distinctly different behaviour to previously studied Pt(II) complexes. Oxidation of peptide species was also found to be a significant product of these reactions. Tandem MS studies located the oxidation sites to methionine and tryptophan residues, the latter of which provided insights into the oxidation mechanism. The oxidation process was found to be due to a hydroxyl radical process, not a singlet oxygen mechanism. UV/vis and EPR measurements were also undertaken and supported the results found. Studies into the interaction of a potent osmium(III) compound with isolated DNA strands showed that the metallodrug could bind to both guanine and cytosine sites along the biomolecule. Analysis of these species via CAD MS/MS proved challenging due to damage and eventual dissociation of the metal complex modifications. Whereas electron detachment dissociation enabled the elucidation of the two distinct binding locations. CAD MS/MS was found to be useful for studying hydrogen bonded/pi-stacking stabilised structures. Further investigations into DNA MS and MS/MS lead to the study of the platinum(IV) compound interactions with DNA stands, showing a vastly improved rate of reaction for the compound when compared to the previous peptide reactions. In addition uniform retention of pyridine ligands and no oxidation was observed, providing further evidence of the biomolecule playing a key role in the activation process of the Pt(IV) drugs. MS/MS studies of platinated DNA resulted in similar findings to the osmium-based metallodrugs. Experiments with native MS of DNA showed that observation and interrogation of duplex-DNA strands is possible, even with DNA strands which are not stabilised easily in ammonium acetate solutions, and without annealing. MS, MS/MS, and MS3 was achieved on duplex DNA strands using large concentrations of ammonium acetate and potassium chloride solutions, along with extremely carefully tuned MS source and transfer parameters. The effects of metals on tandem-MS techniques was uniquely apparent during the study of functionalised Iridium-based metallodrug modified peptides. The iridium complexes were shown to effectively quench many electrons used during ECD MS/MS. Though the species were still able to be studied using optimised ECD and CAD MS/MS parameters. The reactivity of metal centres was also shown to affect their own bound ligands, as observed herein with rhodium(II) compounds. The rhodium piano-stool complexes were shown to vastly accelerate and enable room temperature activation of C-H bonds within arene ligands towards hydrogen-deuterium exchange experiments, with some compounds achieving full exchange of available groups within just 1 hour. The process was studied using FT-ICR MS to track the exchange process and observe sequential exchange for 10 different rhodium compounds. An iridium analogue was also studied, which was ineffective, displaying the Rh metal centre’s unique chemistry for this reaction. A novel method for the enhancement of the electrospray ionisation process was conceived and developed in order to achieve improvement of analyte charging, so-called “supercharging”. Using a combination of standard electrospray ionisation and atmospheric chemical ionisation, CH5 +“superacid” ions were introduced into the ESI plume and enabled solution-additive free supercharging of analytes under a variety of conditions. The achievement of higher charge states/enhancement of charge is of uniform benefit to biomolecule characterisation and could help reduce the need for solution phase additives which can disrupt many chemical processes and biomolecule structure. The final section of the thesis is concerned with the escalation of metallodrug-biomolecule interaction from isolated biomolecule up to full-cell proteomes via FT-ICR MS. The great array of challenges faced in previous studies is addressed and strategies for accurate and reliable studies of large metallodrug-modified systems are outlined and tested. Two major strategies are proposed, one based on liquid-chromatography mass spectrometry with modified data processing techniques. The other using a niche MS/MS technique known as two-dimensional mass spectrometry, which would enable whole proteome characterisation without chromatographic separation. Preliminary result using both approaches and future outlook are presented

Transverse centroid oscillations in solenoidially focused beam transport lattices

Linear equations of motion are derived that describe small-amplitude centroid oscillations induced by displacement and rotational misalignments of the focusing solenoids in the transport lattice, dipole steering elements, and initial centroid offset errors. These equations are analyzed in a local rotating Larmor frame to derive complex-variable"alignment functions" and"bending functions" that efficiently describe the characteristics of the centroid oscillations induced by mechanical misalignments of the solenoids and dipole steering elements. The alignment and bending functions depend only on properties of the ideal lattice in the absence of errors and steering and have associated expansion amplitudes set by the misalignments and steering fields. Applications of this formulation are presented for statistical analysis of centroid deviations, calculation of actual lattice misalignments from centroid measurements, and optimal beam steering

Comparison of risk factors for, and prevalence of, antibiotic resistance in contaminating and pathogenic urinary Escherichia coli in children in primary care: prospective cohort study

Background All-cause antibiotic prescribing affects bowel flora antimicrobial susceptibility, and may increase risk of urinary autoinoculation with antibiotic-resistant microbes. However, little is known about relative prevalence of, or risk factors for, antimicrobial resistance among potentially pathogenic microbes thought to be contaminating and infecting urine. Methods Secondary analysis of 824 children under 5 years of age consulting in primary care for an acute illness and their Escherichia coli isolates cultured at ≥103 cfu/mL fromthe Diagnosis of Urinary Tract infection in Young children (DUTY) study. Multivariable logistic regression investigating risk factors for resistance to amoxicillin, co-amoxiclav, cefalexin, ciprofloxacin, trimethoprim, nitrofurantoin and cefpodoxime in microbes meeting the laboratory criteria for urinary tract infection: ‘pathogens’ (>105 cfu/mL, n=79) and ‘contaminants’ (103 to 105 cfu/mL, n=745). Results Forty-three percent of E. coli were resistant to at least one tested antibiotic, with resistance highest to amoxicillin (49.37% pathogenic versus 37.32% contaminant, P=0.04), trimethoprim (27.85% versus 16.52%, P=0.01) and co-amoxiclav (16.46% versus 21.48%, P=0.30). Multidrug resistance (to ≥3 antibiotic groups) was present in 17.07% of pathogens and 30.13% of contaminants (P=0.04). No isolates were resistant to nitrofurantoin. Recent (0–3months) exposure to antibiotics was associated with resistance in both pathogens (aOR: 1.10, 95% CI: 1.01–4.39) and contaminants (1.69, 1.09–2.67). Conclusions Prevalence of resistance (including multidrug) was high, but there was no consistent relationship between isolate pathogen/contamination status and resistance. Recent all-cause antibiotic prescribing increased the probability of antimicrobial resistance in both pathogenic and contaminating urinary E. coli in children in primary care.</p

Coupling electron capture dissociation and the modified Kendrick mass defect for sequencing of a poly(2-ethyl-2-oxazoline) polymer

With increasing focus on the structural elucidation of polymers, advanced tandem mass spectrometry techniques will play a crucial role in the characterization of these compounds. In this contribution, synthesis and analysis of methyl initiated and xan-thate terminated poly (2-ethyl-2-oxazoline) using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was achieved. Electron capture dissociation (ECD) produced full end group characterization as well as back bone fragmentation including complete sequence coverage of the polymer. A method of fragment ion characterization is also presented with the use of the high resolution modified Kendrick mass defect plots as a means of grouping fragments from the same fragmentation pathways together. This type of data processing is applicable to all tandem mass spectrometry techniques for polymer analysis but is made more effective with high mass accuracy methods. ECD FT-ICR MS demonstrates its promising role as a structural characterization technique for polyoxazoline species

2D FT-ICR MS of Calmodulin : a top-down and bottom-up approach

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments

Metal ion binding to the amyloid beta monomer studied by native top-down FTICR mass spectrometry

Native top-down mass spectrometry is a fast, robust biophysical technique that can provide molecular-scale information on the interaction between proteins or peptides and ligands, including metal cations. Here we have analyzed complexes of the full-length amyloid β (1-42) monomer with a range of (patho)physiologically relevant metal cations using native Fourier transform ion cyclotron resonance mass spectrometry and three different fragmentation methods—collision-induced dissociation, electron capture dissociation, and infrared multiphoton dissociation—all yielding consistent results. Amyloid β is of particular interest as its oligomerization and aggregation are major events in the etiology of Alzheimer’s disease, and it is known that interactions between the peptide and bioavailable metal cations have the potential to significantly damage neurons. Those metals which exhibited the strongest binding to the peptide (Cu2+, Co2+, Ni2+) all shared a very similar binding region containing two of the histidine residues near the N-terminus (His6, His13). Notably, Fe3+ bound to the peptide only when stabilized toward hydrolysis, aggregation, and precipitation by a chelating ligand, binding in the region between Ser8 and Gly25. We also identified two additional binding regions near the flexible, hydrophobic C-terminus, where other metals (Mg2+, Ca2+, Mn2+, Na+, and K+) bound more weakly—one centered on Leu34, and one on Gly38. Unexpectedly, collisional activation of the complex formed between the peptide and [CoIII(NH3)6]3+ induced gas-phase reduction of the metal to CoII, allowing the peptide to fragment via radical-based dissociation pathways. This work demonstrates how native mass spectrometry can provide new insights into the interactions between amyloid β and metal cations