Mass spectrometry (MS) as an analytical approach could provide comprehensive identification and quantitation of the biomolecules (proteins, peptides, nucleic acids, lipids) in a cell, tissue, or organism, from biomarker discovery to prediction of response to therapy or intervention. Inductively coupled plasma mass spectrometry (ICP-MS), can determine the elemental composition of materials and has been used for below ppt levels (ppq) and better in some cases (transuranics and non-metals) to detect metals and other elements in water, soil, and air or blood and urine samples. Mass cytometry is an implementation of ICP-MS to single-cell analysis; it is based on metal isotope-tagged antibodies to quantify these bioconjugates. Imaging mass cytometry (IMC), a commercially available immunohistochemistry laser ablation-inductively coupled plasma-time-of-flight-mass spectrometry (LA-ICP-TOF-MS) system, was designed for molecular biomarkers imaging in the tissue sections (e.g., brain) through metal-tagged antibodies (typically, lanthanides). This thesis highlights the contributions of ICP-TOF-MS-based approaches towards advanced developments of mass cytometry (CyTOF) and discusses its biomedical applications for investigating neurodegenerative diseases while comparing it to other imaging modalities such as PET, MRI, Allen brain, etc. In conclusion, CyTOF, as a high-dimensional imaging tool, provides information on many clinical applications, such as hematopoiesis, transplantation, cancer, and autoimmunity.

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role. Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost and skill requirements. Novel TB diagnostics are thus required that meet these requirements. Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior to current existing mycobacteriophage-based TB diagnostics. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde TB-diagnostiese toetse.

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En IT-styrd lager och orderhantering är idag vanligt, metoden för att ta emot leveranser har dock sett likadan ut länge. Man använder sig oftast av streckkoder/QR-koder eller rent manuell hantering med penna och papper. Radio frequency identification (RFID) möjliggör en trådlös och autonom avläsning av gods som är taggade. Tekniken har funnits länge men har under senaste år visats sig få en mer positiv trend, där mer och mer objekt blir taggade. Samarbetspartnern i denna studie Fiskarhedenvillan använder sig idag av ett helt manuellt system för inleveranser av gods på deras distributionscentral. De vill nu börja deras digitalisering av distributionscentralen genom att undersöka möjligheten att införa ett RFID-system som metod för hantering av gods vid inleveranser. Denna studie har i samarbete med Sogeti och Fiskarhedenvillan genomförts för att identifiera möjligheter och faktorer och undersöka ifall en implementation är möjlig. Intervjuer har genomförts med nyckelpersoner inom vad som kallats ”världens bästa RFID-projekt”. Från slutsatserna identifieras ett antal utmaningar kring processer av flöden som måste uppdateras för att kunna dra full nytta utav ett RFID-system. Vidare så blir RFID teknik som metod för inleverans av gods sällan mer effektivt, man måste också börja kolla på arbetssätt och börja utveckla de nuvarande processerna för att kunna utnyttja fördelarna med RFID. Då möjligheten att gardera sig mot en icke fulländad logistik-kedja med kombinerad tagg/streckkods-etikett samtidigt som mer och mer blir taggat gör det att RFID-system blir mer direkt användbara. Implementeringar underlättas av befintliga beprövade standarder. Kritiska faktorer att beakta inför implementering innebär bland annat omgivningsanalys och att implementeringar utav RFID-system ofta har stor inverkan på hela kedjan. Vidare identifieras möjligheter som tidigare beprövade system och en positiv trend för implementeringar inom RFID-tekniken. / An IT-controlled warehouse and order management is today common, however, the method for receiving deliveries has looked the same for a long time. You usually use bar codes / QR codes or purely manual handling with pen and paper. Radio frequency identification (RFID) enables a wireless and autonomous reading of goods that have been tagged. The technology has been aorund for a long time but has been shown to have a more positive trend in recent years, where more and more objects are being tagged. The partner in this study Fiskarhedenvillan today uses a completely manual system for deliveries of goods at their distibution center. They now want to start their digitalization of the distribution center by examining the posibility of introducing an RFID-system as a method for hondling goods at deliveries. This study, in collaboration with Sogeti and Fiskarhedenvillan, has been carried out to identify opportunities and factors and to investigate wether an implemantation is possible. Interviews have been conducted with key personell within what has been called ”the world’s best RFID project”. From the conclusions, a number of challenges are identified around processes of flows that must be updated to be able to take full advantage of an RFID system. Futhermore, RFID technology as a method for receiving goods rarely becomes more efficient unless the processes between flows are changed or updated, and the working methods should also be looked upon to be able to utilize the advantages of RFID. Since the possibility of gourding against an uncompleted logistics chain with a combined tag / barcode label at the same time as more and more objects are being tagged, it makes RFID systems more directly usable. Implementations are facilitated by existing proven standards. Critical factors to consider prior to implementation include environmental analysis and that implementations of RFID systems often have a great impact on the entire chain. Furthermore, opportunities are identified as previously proven systems and a positive trend for implementations in the RFID field.

Mass spectrometry is a versatile, sensitive and fast technique with which to probe biophysical properties in biological systems and one of the most important analytical tools in the multidisciplinary field of proteomics. The study of nativestate proteins and their complexes in the gas-phase is well established and direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques are becoming increasingly popular as a tool for screening and determining quantitative information on protein-protein and protein-ligand interactions. However, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting purely gas-phase results. This thesis details modification and advancement of solution phase techniques devised by Gross et al. utilising ESI-MS and Fitzgerald et al. applying matrix assisted laser desorption ionisation (MALDI)-MS termed PLIMSTEX (protein-ligand interactions by mass spectrometry, titration and hydrogen-deuterium-exchange)[1] and SUPREX (Stability of unpurified proteins from rates of H/D exchange)[2] to quantify these interactions with regards to high throughput analysis. The first part of this thesis describes the different developmental stages of the devised HPLC-front ends and their optimisation with myoglobin and insulin. The successfully developed HPLC-front end in conjunction with PLIMSTEX and SUPREX and ESI-MS then gets tested with self expressed and purified cyclophilin A(CypA)- cyclosporin A (CsA) system, followed by a test screen with potential CypA binding ligands. Dissociation constants (Kd’s) within one order of magnitude to reported values are determined. In the third part of this thesis the application of the devised ESI-SUPREX methodology has been applied to anterior gradient 2 (AGr2) and the factor H complement control proteins module 19-20 (fH19-20) exhibiting binding potential to a taggedhexapeptide and a synthetic pentasaccharide, respectively, resulting in thermodynamical data for these protein-ligand interactions. For the AGr2 system another dimension of investigation has been added by temperature controlling the devised ESI-SUPREX approach, revealing a phase transition in the protein at higher temperatures. The final part of this thesis describes the application of the ESI-SUPREX methodology to probe folding properties of CypA in the presence of the self expressed and purified E. coli chaperonin groEL. Thereby the denaturing properties of groEL have been emphasised along with the stabilisation of a denatured CypA species.

How are neuromodulatory networks organised to adapt sensory discrimination for different contexts? I hypothesised that neurons within a sensory circuit express different neuromodulatory receptors for differential modulation. Here I aimed to use the simple and genetically amenable Drosophila larval Mushroom Body (MB) calyx, a higher order processing area involved in learned odour discrimination, as a model to map octopamine (OA) neuromodulatory circuitry. I first identified olfactory projection neurons (PNs), a GABAergic feedback neuron and cholinergic extrinsic neurons as putative postsynaptic partners to OA neurons in the MB calyx using GFP reconstitution across synaptic partners. Next, I used novel EGFP-tagged OA receptors generated from recombination-mediated cassette exchange with MiMIC insertions in receptor genes to visualise endogenous expression patterns of OA receptors. Most notably, this is the first report of α2-adrenergic-like OA receptor localisation in any insect. For the first time, I showed that the α1-adrenergic-like OAMB localised to PN presynaptic terminals in the calyx; while Octβ1R localised diffusely in the calyx, resembling the innervation pattern of MB neuron dendrites. I detected EGFP-tagged Octα2R and Octβ2R in some PN cell bodies but not in neuron terminals - suggesting that Octα2R and Octβ2R may be expressed in some PNs, provided the misfolded fusion proteins are retained in the cell bodies of the neurons they are normally expressed in. Furthermore, I found that Octα2R and GABAAR fusion proteins localised to OA cell bodies but not to neuronal terminals, suggesting that OA neurons are subjected to inhibition, again given that these are not artefacts of the fusion proteins. To obtain tools to study OA modulation in the larval calyx, I then confirmed the expression patterns of driver lines that more specifically labelled calyx-innervating OA and extrinsic neurons, and tested the efficacy of three OAMB receptor knockdown lines. This initial attempt of mapping OA receptors, while subjected to further verification and development, is consistent with my hypothesis that a single neuromodulatory source can regulate multiple neuronal types in the same circuit through the distribution of different types of neuromodulatory receptors. This provides a new perspective in how the anatomical organisation of neuromodulation within a sensory network may translate to flexible outputs.

Tip60 is a 60 kDa nuclear protein which exists in three isoforms, belongs to the MYST/HAT family of proteins and was discovered after its interaction with the Human HIV-1 Tat. As a nuclear protein, Tip60 can act as a coactivator or repressor. To understand the HAT action of Tip60, two possible catalytic models exist; the ping-pong and the ternary complex formation models. In correlation with the exploration of HAT catalytic action, mutations of a Cys to Ala and a Glu to Gln on Esa1 (yeast homolog of Tip60 and MYST/HAT prototype), was reported to show wild type-like and decreased acetylating properties, respectively. In this work, Tip60 HAT action was explored. In Tip60, the Cys in the active site is important for acetylation of the H4(1-20) substrate and the Glu showed semi loss in acetylating the H4(1-20) peptide substrate. These data highlight a unique mechanism of Tip60 catalysis.

To aid diagnosis, treatment planning, and patient education, clinicians require tools to anal- yse and explore the increasingly large three-dimensional (3-D) datasets generated by modern medical scanners. Direct volume rendering is one such tool finding favour with radiologists and surgeons for its photorealistic representation. More recently, volume illustration — or non-photorealistic rendering (NPR) — has begun to move beyond the mere depiction of data, borrowing concepts from illustrators to visually enhance desired information and suppress un- wanted clutter. Direct volume rendering generates images by accumulating pixel values along rays cast into a 3-D image. Transfer functions allow users to interactively assign material properties such as colour and opacity (a process known as classification). To achieve real-time framerates, the rendering must be accelerated using a technique such as 3-D texture mapping on commod- ity graphics processing units (GPUs). Unfortunately, current methods do not allow users to intuitively enhance regions of interest or suppress occluding structures. Furthermore, addi- tional scalar images describing clinically relevant measures have not been integrated into the direct rendering method. These tasks are essential for the effective exploration, analysis, and presentation of 3-D images. This body of work seeks to address the aforementioned limitations. First, to facilitate the research program, a flexible architecture for prototyping volume illustration methods is pro- posed. This program unifies a number of existing techniques into a single framework based on 3-D texture mapping, while also providing for the rapid experimentation of novel methods. Next, the prototyping environment is employed to improve an existing method—called tagged volume rendering — which restricts transfer functions to given spatial regions using a number of binary segmentations (tags). An efficient method for implementing binary tagged volume rendering is presented, along with various technical considerations for improving the classifi- cation. Finally, the concept of greyscale tags is proposed, leading to a number of novel volume visualisation techniques including position modulated classification and dynamic exploration. The novel methods proposed in this work are generic and can be employed to solve a wide range of problems. However, to demonstrate their usefulness, they are applied to a specific case study. Ischaemic heart disease, caused by narrowed coronary arteries, is a leading healthconcern in many countries including Australia. Computed tomography angiography (CTA) is an imaging modality which has the potential to allow clinicians to visualise diseased coronary arteries in their natural 3-D environment. To apply tagged volume rendering for this case study, an active contour method and minimal path extraction technique are proposed to segment the heart and arteries respectively. The resultant images provide new insight and possibilities for diagnosing and treating ischaemic heart disease.

Orientadores: Marcos Nogueira Eberlin, Fernando Antonio Santos Coelho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T16:01:36Z (GMT). No. of bitstreams: 1 Galaverna_RenanSouza_M.pdf: 4687299 bytes, checksum: d06d242cdf07c9f8f7f1abfb050e76ba (MD5) Previous issue date: 2014 / Resumo: Essa dissertação de Mestrado visa estudar o mecanismo da reação de Morita-Baylis-Hillman (MBH) em três versões (Clássica, Aza e Assimétrica) utilizando a técnica de espectrometria de massas com ionização por eletrospray (ESI-MS). Para o monitoramento mecanístico das versões clássica e Aza (MBH/Aza-MBH) foi introduzida uma nova abordagem, que é a utilização de reagentes marcados com etiquetas de carga (líquidos iônicos), a fim de facilitar a detecção dos intermediários e maximizar a aquisição de dados obtidos pelo monitoramento destas reações. Entre os reagentes de partida, (eletrófilo, catalisador e alceno ativado) o alceno ativado foi o reagente marcado, utilizou-se o íon metil imidazólio como etiqueta de carga. Além do uso de um reagente marcado, ambas as versões foram monitoradas utilizando reagentes neutros, com o propósito de avaliar em nível de comparação as metodologias abordadas, marcada e não-marcada. Para versão assimétrica da reação de MBH, foram utilizadas duas aminas hidroxiladas quirais derivadas de alcalóides cinchona, quinidina e ?-isocupreidina. O monitoramento mecanístico da versão assimétrica foi realizado utilizando um espectrômetro de massas com mobilidade iônica IM-MS. O tipo de mobilidade utilizada nesse estudo foi a Traveling Wave, comercialmente conhecida como TWIM-MS (Traveling Wave Ion Mobility Mass Spectrometry). A utilização desta possibilitou a separação e caracterização de intermediários estereoisoméricos formados no percurso da reação. Cálculos teóricos dos estados de transição para formação desses intermediários foram realizados a fim de suportar os dados obtidos por TWIM-MS. A utilização de um reagente marcado no monitoramento completo da reação de MBH/Aza-MBH mostrou melhores resultados em relação à reação não-marcada. Intermediários não detectados pela metodologia não-marcada foram detectados e caracterizados pela metodologia marcada, além do fato que a intensidade desses intermediários foi maior quando comparado à versão não-marcada. Para versão assimétrica, os dados obtidos por TWIM-MS e cálculos teóricos possibilitaram um melhor entendimento acerca da enantiosseletividade da reação de MBH quando diferentes catalisadores quirais são utilizados / Abstract: This dissertation aims to study the mechanism of the Morita-Baylis-Hillman reaction in three versions (Classical, Aza and Asymmetric) using Electrospray Ionization Mass Spectrometry (ESI-MS). For the mechanistic monitoring of the classical and Aza versions (MBH/Aza-MBH) a new approach was introduced, that is the use of marked reagents with charge tag (ionic liquid) in order to facilitate the detection of the reaction intermediates and maximize the data acquisition when monitoring these reactions. Among the start material, (electrophile, catalyst and activated alkene) the activated alkene was the marked reagent, using imidazolium ion as charge tag. Besides the use of a marked reagent, both versions (MBH/Aza-MBH) were monitored using neutral reagents, in order to compare the methodologies used, with and without charge tag. For the asymmetric MBH reaction, were used two hidroxylated chiral amines derived from cinchona alkaloids, quinidine and ?-isocupreidine. The mechanistic monitoring of the asymmetric reaction was performed using an ion mobility mass spectrometry IM-MS. The type of mobility used in this study was "traveling wave", commercially known as TWIM-MS (Traveling Wave Ion Mobility Mass Spectrometry). Using this instrument was possible to separate and characterize stereoisomerics intermediates formed in the course of the reaction. Theoretical calculations of the transition states for the formation of these intermediates were performed in order to support the data obtained by TWIM-MS. The use of a marked reagent in complete monitoring of the MBH/Aza-MBH reaction showed better results than non-tag reaction. Intermediates not detected by the non-tag methodology were detected and characterized by the charge tag methodology, furthermore, the intensity of these intermediates were higher than non-tag reaction. For the asymmetric version, the data obtained by TWIM-MS and theoretical calculations enabled a better understanding of the enantioselectivity of the MBH reaction when different chiral catalysts are used / Mestrado / Quimica Organica / Mestre em Química