Mass spectrometry is a widely used technique for the performance of elemental analysis: not only does it provides excellent limits of detection for a large number of elements, but it is also able to provide information about the isotopic distribution of the analyte. The radio-frequency quadrupole ion trap is a relatively new design of mass spectrometer, which offers the ability to confine charged particles for extended periods of time in a well defined volume by applying a radio-frequency oscillating voltage to an arrangement of three electrodes. A mass analysis of the trapped ions can be obtained by selectively extracting the ions from the cavity of the trap, where they can be detected by an electron multiplier. Despite its unique capabilities, to date the applications of the ion trap mass spectrometer have mostly been restricted to gas chromatography detection. Until recently, there have been very few attempts to use it for any other types of routine analysis. Our interest lies in the development of an instrument capable of performing a complete mass spectrometric elemental analysis of small volume liquid samples (a few (μL) at trace or ultra-trace concentration levels. The ability of the ion trap to accumulate ions in its cavity and to provide an entire mass spectrum of these ions in a single scan of the radio-frequency oscillating voltage applied between the electrodes, makes it a very interesting candidate for the ultra-trace analysis of small size samples. However, to perform an analysis on a sample with the ion trap the sample must first be vaporized; and if an elemental analysis is required, the sample will also have to be atomized. The graphite furnace atomizer used in atomic absorption spectroscopy offers a number of advantages which make it potentially useful for this purpose: it has a high transport efficiency of the analyte from liquid or solid state to the vapour phase, the ionization of the analyte in the furnace is very low (as required by the ion trap) and it handles small volume samples very well. A graphite furnace ion trap mass spectrometer was designed to fulfil the need of having instrumentation capable of multielemental mass spectrometric analysis of small volume samples containing traces of the analytes of interest. This document contains a description of the principles of operation of the ion trap as well as a detailed description of the instrument actually built. Data are presented in order to assess the capabilities of the instrument, as well as some of the problems encountered with it. The results obtained with the graphite furnace ion trap mass spectrometer allow us to conclude that the proposed design is not appropriate for the performance of elemental analysis, but is appropriate for mass spectrometric study of low boiling point compounds which can interfere with atomic absorption analysis: it is calculated that these compounds could be analysed at the ppm level. Promising results obtained with a set up in which the analyte is vaporized directly into the cavity of the ion trap through laser ablation are also presented. These limited results show the potential of this methodology for direct elemental analysis of solid samples. / Science, Faculty of / Chemistry, Department of / Graduate

Cancer is one of the most devastating human diseases that cause a vast number of mortalities worldwide each year. Cancer research is one of the largest fields in the life sciences and despite many astounding breakthroughs and contributions over the past few decades, there is still a considerable amount to unveil on the function of cancer that would improve diagnostics, prognostics and therapy. Since cancer is known to involve a wide range of processes, applying methods to study it from a systems perspective could reveal new properties of cancer. Systems biology is becoming an increasingly popular tool in the life sciences. The approach has been applied to many biological and biomedical analyses drawing upon recent advancements in technology that make high throughput analyses of samples and computational modelling possible. In this thesis, the effect of hypoxia inducible factor-1 (HIF-1) on cancer metabolism, the entity considered most closely related to phenotype has been investigated. This transcription factor is known to regulate a multitude of genes and proteins to promote survival in a low oxygen environment that is prevalent in solid tumours. However its effect on the metabolome is less well characterised. By revealing the effect of HIF-1 on the metabolome as a system it is hoped that phenotypic signatures, key metabolic pathways indicative of cancer function and potential targets for future cancer therapy, can be revealed.The system has been studied using two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby metabolism has been profiled using a range of analytical platforms. In each model, wild type cells have been compared to cells deficient in HIF-1 to reveal its effect on cellular metabolism. Gas chromatography mass spectrometry (GC MS) and ultra high performance liquid chromatography - mass spectrometry (UHPLC MS) have been employed for metabolic profiling of cells exposed to a range of oxygen conditions. Additionally, time-of-flight secondary ion mass spectrometry (ToF SIMS) has been employed for imaging mass spectrometric analysis of multicellular tumour spheroids cultured from wild type cells and cells with dysfunctional HIF-1 to represent small initiating tumours. Using these techniques in metabolic profiling it has been possible to reveal metabolites associated with the effect of oxygen and HIF-1 on cancer metabolism along with key pathways and hubs that could be targeted in future therapy. Using imaging mass spectrometry it has been possible to localise metabolites in situ revealing how tumour structure relates to function. Finally, a novel approach to consider how metabolites are correlated with one another in the response to oxygen level or presence or absence of functional HIF-1 has been undertaken to better understand the systems properties of cancer metabolism. Metabolites found to be differently correlated with respect to oxygen and/or HIF-1 have been mapped onto a human metabolic network to determine their network-based origins. This allowed the simulation of sub-networks of metabolism most affected by oxygen and HIF-1, highlighting the key mechanisms in HIF 1 mediated cancer cell survival.

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Orientador: Marco-Aurelio De Paoli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T18:06:53Z (GMT). No. of bitstreams: 1 Franco_MarcosFernando_M.pdf: 4184515 bytes, checksum: a8d548fc3ec83db853e6ccc3169b252e (MD5) Previous issue date: 2015 / Resumo: Focamos no desenvolvimento de técnicas em análise de termoplásticos para determinar os efeitos causados pela corrosão dos equipamentos de processamento nos polímeros durante o uso e o efeito da contaminação do poli(tereftalato de etileno), PET, com poli(cloreto de vinila), PVC, durante a sua degradação. O estudo de migração dos metais do equipamento de processamento para a resina polimérica foi realizado com fluorescência de Raios-X e tratamento estatístico de dados. Para analisar qualquer composto por espectrometria de massas, este deve ter carga formal diferente de zero e estar no estado gasoso, que é o grande empecilho na análise de termoplásticos, pois os mesmos são pouco solúveis, tem alta massa molar para serem volatilizados e se degradam termicamente antes de se volatilizar ou entrar em ebulição. Para volatilizar um polímero, são necessárias condições drásticas de pressão e temperatura. Foi desenvolvido um método de acoplamento entre a extração de analitos da superfície dos polímeros e a fonte de ionização. Os métodos acoplados foram: ablação por Laser e dessorção térmica. A funcionalidade do acoplamento foi testada, em comparação com outras técnicas já estabelecidas na literatura, com a análise de cafeína diretamente no grão de café. Depois de comprovada a eficiência do acoplamento entre os dois métodos de extração de analitos, o método foi aplicado no estudo do efeito da estabilidade térmica do PET contaminado com PVC, na identificação de aditivos em uma formulação comercial de polímeros e na interação entre duas espécies de aditivos distintas. A comparação dos resultados obtidos com as novas técnicas e os métodos tradicionais demonstrou que elas são mais sensíveis, precisas, rápidas e limpas / Abstract: I focused on the development of techniques for analysis of thermoplastics, which determine the effects of the corrosion of the processing equipment on the polymer during its use and the effect of contamination with poly(vinyl chloride), PVC on the degradation of poly(ethylene terephthalate), PET. The metal migration from the processing equipment to the polymer was studied by X-Ray fluorescence and statistical data treatment. To analyze any compound by mass spectrometry it must have a formal charge different from zero and must be in the gaseous state. This is a hindrance to analyze thermoplastics because they have very little solubility, have high molar mass to be volatilized and thermally degrade before volatilizing or boiling. Volatilizing a polymer would require drastic pressure and temperature conditions. A coupling method between the analyt extraction from the polymer surface and the ionization source was developed. The methods coupled are Laser ablation and thermal desorption. The coupling functionality was tested in comparison to other techniques of the literature, by the analysis of caffeine directly from a coffee bean. After this positive test, the coupling method was used to study the thermal stability of PET contaminated with PVC, to identify additives in a commercial polymer formulation and the interactions of two different types of additives. The comparison of the results obtained with the new techniques and the traditional methods demonstrated that they are sensitive, precise, quick and clean / Mestrado / Quimica Analitica / Mestre em Química

abstract: Short-lived radionuclides (SLRs) once present in the solar nebula can be used to probe the Solar System’s galactic formation environment. Isotopic analyses reveal that the first solids formed in the Solar System, calcium- and aluminum-rich inclusions (CAIs) in chondritic meteorites, formed with the live SLRs 10Be (t1/2 = 1.4 Ma) and 26Al (t1/2 = 0.7 Ma). Beryllium-10 is produced when high-energy ions, solar energetic particles or galactic cosmic rays (GCRs), spall nuclei in gas or dust. The most likely source of Solar System 10Be is inheritance of GCR-irradiated protosolar molecular cloud material, but only if all CAIs recorded the same initial 10Be abundance. The goal of this dissertation is to assess the homogeneity of 10Be by measuring CAIs for 10Be–10B isotope systematics, correlated to 26Al–26Mg and oxygen isotopes. I synthesized appropriate standards for secondary ion mass spectrometry (SIMS) measurements of 10Be–10B, necessary for accurate determination of the 10Be/9Be ratio. I then analyzed 32 CAIs for 10Be–10B as well as 6 CAIs for 26Al–26Mg and 5 CAIs for oxygen isotopes within this sample set using SIMS. Previous studies analyzed CAIs primarily from CV3 chondrites, which are known to have experienced thermal metamorphism and aqueous alteration. My work included a variety of CAIs (Type A, B, fine-grained, igneous) from CV3oxidized, CV3reduced, CO3, CR2, and CH/CB chondrites. Finally, after evaluating my data and literature data consistently, I statistically tested whether all CAIs belong to a single 10Be population. I find that the majority (~85%) of the normal (i.e., without large isotopic fractionations or anomalies), 26Al-bearing CAIs recorded a single value, 10Be/9Be = (7.0 ± 0.2) × 10-4. Although 6 CAIs recorded higher or lower values, these are plausibly explained by secondary alteration processes. The galaxy-wide average value of 10Be/9Be from GCR interactions 4.56 billion years ago is predicted to be <2 × 10-4; the value I measured is more than 3 times higher. Because GCRs trace supernovae and star formation, my results suggest a similarly enhanced star formation rate in the molecular cloud within ~1 kpc of the Sun, in the ~15 Ma prior to the Sun’s birth. / Dissertation/Thesis / Doctoral Dissertation Geological Sciences 2020

The introduction of mass spectrometry techniques to the field of biology has made possible the exploration of the proteome as a whole system as opposed to prior techniques, such as anti-body based assays or yeast two-hybrid studies, which were strictly limited to the study of a few proteins at a time. This practice has allowed for a systems biology approach of exploring the proteome, with the possibility of viewing entire pathways over increments of time. In this study, the effect of treating Arabidopsis thaliana suspension culture cells with 3’,5’-cyclic guanosine monophosphate (cGMP), which is a native second messenger, was examined. Samples were collected at four time points and proteins were extracted and enriched for both oxidation and phosphorylation before analysis via mass spectrometry. Preliminary results suggest a tendency towards an increased number of phosphorylated proteins as a result of cGMP treatment. The data also showed a sharp increase in methionine oxidation in response to the treatment, occurring within the first ten minutes. This finding suggests that cGMP may utilize methionine oxidation as a mechanism of signal transduction. As such, this study corroborates a growing body of evidence supporting the inclusion of methionine oxidation in intracellular signaling pathways.

The human gut microbiome exists as a community of microorganisms in symbiosis with its host. Prebiotics are functional compounds that modulate this microbial community, promoting the growth and activity of bacteria that are beneficial to human health. Resistant starches (RS), a subclass of prebiotics, are compounds linked to a number of host-beneficial effects when included in human diets. Understanding how RS shapes gut flora composition and function is crucial to understanding these effects; however, these effects are clouded by the complexity of the microbiome’s interactions. Comprehensively characterizing microbiome shifts as the result of prebiotics is an intriguing bioanalytical problem. In the thesis project, I hypothesize that: RS changes microbiome biochemical pathway expression community-wide and at different taxonomic levels; that RS forms will affect microbiome bacterial taxonomic distribution; and that a linear programming optimization approach can parsimoniously distribute ambiguous peptide abundances amongst their constituent species, leading to different interpretations of functional and structural characteristics in microbiome metaproteomics data. To address these hypotheses, the thesis project utilizes a combined metaproteomics and bioinformatics approach. The Figeys lab-developed RapidAIM bioanalytical assay is deployed to generate label-free mass spectrometry metaproteomics data, testing for these effects experimentally. Further, Cerberus, a bioinformatics platform for microbiome metaproteomics analyses, was developed to integrate workflows from different software sources into a unified pipeline. Cerberus also implements a novel linear optimization approach addressing the shared-peptide problem. Through experimental data analyses using Cerberus, microbiomes encountering RS showed concerted taxonomic shifts, general and specific functional modulations linked to these taxonomic changes, and a significantly variable pathway expression profile for host-beneficial microbiome processes. The peptide-species linear optimization procedure demonstrates how naïve approaches to the shared-peptide problem greatly skew downstream taxonomic and functional analyses in metaproteomics experiments, marking an important consideration for microbiome studies seeking to resolve taxon-specific alterations.

Please read the abstract in the dissertation. / Dissertation (MSc)--University of Pretoria, 1991. / gm2014 / Chemistry / unrestricted

Interstellar chemistry has been a growing field over the last several decades. There is particular interest on the nature and reactivity of interstellar molecules; most notably that of polycyclic aromatic hydrocarbons (PAHs). My thesis focused on the kinetics of unimolecular dissociation of small PAH and PAH-like molecules under interstellar conditions. PAHs (naphthalene (NAP), anthracene (ANT) and pyrene (PYR)), some dihydro- equivalents (1,2-dihydronapthalene (DHN) and 9,10-dihydrophenanthrene (DHP)) and a few other small aromatic organic molecules (indene (IND), ethynylbenzene (EB), propynylbenzene (PB) and benzocyclobutene (BCB)) were studied using imaging photo-electron photo-ion coincidence spectroscopy (iPEPICO) and electron impact mass spectrometry (MS); both mass analyzed ion kinetic energy spectrometry (MIKES) and collision induced dissociation (CID). Experiments were performed at different ionization energies to produce breakdown diagrams for the various fragments. These diagrams are then fit using RRKM theory to determine the zero Kelvin activation energy (E0) and the entropy of activation (Δ‡S); these results are then compared and discussed. All these molecules were compared in order to try and find any overlying trends which could be applied to their role in the interstellar medium (ISM). It was determined that H loss was the dominant fragmentation channel, as it was the only dissociation channel common to the majority of molecules studied. It was also seen that organic fragment loss (C2H2, CH3 and C4H2) was only observed in smaller molecules which indicates that PAHs are not likely a source of these molecules. The small fragment molecules gave insight into the stability of closed ring structures, such as PAHs, through the comparison of the dissociation of closed and open structures. The dihydro-PAHs, selected as a probe to investigate the proposed catalytic role of PAHs in the formation of molecular hydrogen, yielded very interesting results. It was seen that these molecules would readily undergo isomerisation prior to dissociation. This added an unexpected level of difficulty to the calculations but quickly demonstrated how the presence of additional hydrogen atoms could greatly disrupt the dissociations, as it was not the simple process of removing them as it was originally believed. The overall trend observed was that it is the structure, not the size, which has the dominating effect on the dissociation. Ions of similar structure behaved similarly, regardless of a change in mass; isomers, however, had radically different behaviours which can only be attributed to their differing molecular conformations. This observation could aid in the understanding of larger PAHs, those which are believed to exist in the ISM, and what role they may play in the chemistry of the universe.

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