<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>

Philosophiae Doctor - PhD / This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. / South Africa

Philosophiae Doctor - PhD / Sorghum (Sorghum bicolorï, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotie stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI- TOF and MALDI- TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germ in proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. Following 200 mM NaCl and 400 mM sorbitol stress treatments, the expression/abundance of a protein spot similar to a rice wall-associated protein kinase was upregulated in the sorghum secretome in response to both stresses. Amino acid sequence alignment of the matching peptides between these two proteins showed that the sorghum CF spot possesses a protein kinase domain. Therefore, this protein could possibly participate in cell signalling functions, which link the external environment with the cell's cytoplasm. Using whole plant systems, a comparative study of leaf protein expression between two sorghum varieties, AS6 (salt sensitive) and MN1618 (salt tolerant) was conducted. Forty well resolved spots of varying abundances were picked for MS analysis. Of these, 28 were positively identified, representing proteins with functions in carbohydrate metabolism (60.7%), proton transport (17.9%), protein synthesis (7.1%), hydrolytic functions (7.1%), nucleotide metabolism (3.6%) and detoxification (3.6%). Using PDQuest™ Advanced 2D Analysis Software version 8.0.1 (BIO-RAD), a comparative analysis of leaf proteome expression patterns between the two sorghum varieties was conducted. The results indicated proteins with similar expression patterns as well as qualitative and quantitative differences between the two leaf proteomes. The effect of 100 mM NaCI on leaf proteome expression between the two sorghum varieties was also studied. Western blotting analysis of leaf, sheath and root tissues using Hsp70 antibodies showed that this treatment induced Hsp70 expression, a known stress protein, in both varieties. Thereafter, the partially annotated leaf proteome map was used to landmark other salt responsive proteins. Examples of differential expression patterns included glutathione S transferase and hydroxynitrile lyase proteins whose abundances were upregulated in both varieties, while the large subunit of RuBisCo was downregulated in AS6 but upregulated in MN1618. Qualitative spot expression differences in response to salt stress were also observed between the two sorghum varieties but these remained unidentified after both MALDI-TOF and MALDI-TOF-TOF MS, possibly indicating novel and previously uncharacterised sorghum proteins. The results of this study can be used as reference tools by proteomics researchers worldwide as well as a foundation for future studies.

Le remodelage ventriculaire gauche (VG) en post-infarctus du myocarde (IDM) est associé à une augmentation du risque d’insuffisance cardiaque et de décès, mais il demeure difficile à prédire en pratique clinique.L’objectif principal de ma thèse était la recherche de biomarqueurs circulants du remodelage VG par l’approche protéine candidate et par protéomique différentielle dans la population REVE-2.Par l’approche protéine candidate, nous avons confirmé que le peptide natriurétique detype B (BNP) était un puissant facteur prédictif du remodelage VG en post-IDM. La métalloprotéase matricielle-8 (MMP-8), la MMP-9, l’hepatocyte growth factor (HGF), la Créactive protéine (CRP), la troponine I ont également fait la preuve de leur association.Par l’approche protéomique différentielle, en électrophorèse 2D différentielle en fluorescence (2D-DIGE), la clusterine a été identifiée comme biomarqueur potentiel,positivement associée au remodelage VG, nécessitant toutefois des travaux de confirmation.Par SELDI TOF MS, nous avons sélectionné 26 pics définis par leur rapport m/z, commebiomarqueurs potentiels du remodelage VG, dont 12 ont pu être identifiés et devrontdésormais être validés : le pic de m/z 2777 a été identifié comme le peptide N-terminal issu del’albumine après clivage par la pepsine. Les autres pics correspondraient à des fragments protéolytiques de protéines que sont le fibrinogène, le complément C3, C4 et C1q.La découverte de nouveaux biomarqueurs du remodelage VG devrait permettre d’améliorer la stratification du risque en post-IDM afin d’identifier les patients devant bénéficier d’un suivi plus rapproché et peut-être d’une prise en charge thérapeutique plus agressive / Left ventricular (LV) remodelling after myocardial infarction (MI) indicates a high risk of heart failure and death but remains difficult to predict in clinical practice. Biomarkers may help to refine risk stratification. The main purpose was to find circulating biomarkers of LV remodelling after MI, using two strategies : candidate protein approach and differential proteomic approach, working on a population with a clearly defined phenotype, the REVE-2 study, a prospective multicenter study including 246 patients with a first anterior Q-wave MI. Blood samples were obtained at hospital discharge, at 1 month, 3 months and 1 year. An echocardiography was performed at the same time except for the 1st month to assess LVR.By candidate protein approach, we confirmed that B-type natriuretic peptide (BNP) was a powerful predictor of LV remodelling after MI. Additional biomarkers, such as matrix metalloproteinase-8 (MMP-8), MMP-9, hepatocyte growth factor (HGF), C-reactive protein (CRP) and cardiac troponin I were found to be associated with LV remodelling, highlighting several pathways implicated in pathophysiology of LV remodelling. We have also shown that biomarkers in association (BNP and cardiac troponin I, BNP and MMP-8, BNP and MMP-9) could improve risk stratification in post-MI by selecting groups of patients at higher risk.As the ideal biomarker was still not identified, we applied a differential proteomic approach, with no a priori hypothesis, in order to characterize proteomic signature of LV remodelling. The use of a protein enrichment kit, consisting of a library of combinatorial hexapeptide ligands, compressed the protein concentration range of plasma and serum, through the simultaneous onestep dilution of high-abundance and concentration of lowabundance proteins. Protein enrichment kit prior to two-dimensional (2D) electrophoresis or SELDI TOF MS (surface-enhanced laser desorption–ionization time of flight) analysis enabled the detection of proteins that were not detected in native blood sample and the accessibility to proteolytic fragments obtained from major proteins. Clusterin (apolipoprotein J) was identified as a potential biomarker of LV remodelling by 2D-DIfferential Gel Electrophoresis (2D-DIGE). Clusterin was quantified by Western blot and ELISA and was found to be positively associated with LV remodelling. However, this association was not found with all LV remodelling parameters nor at each time during the year following MI, requiring further analysis. Differential proteomic approach by SELDI TOF MS selected 26 m/z peaks, as potential biomarkers of LV remodelling. Of them, 12 were identified by mass spectrometry. The 2777 m/z peak was identified directly from the ProteinChip array as being the N-terminal peptide (24–48 aa) generated from albumin by pepsin cleavage. Other peaks were identified after purification using chromatographic columns or liquid-phase isoelectric focusing : most of them were found to be proteolytic fragments of proteins like fibrinogen, C3, C4 and C1q complement. Identifications have now to be validated with specific techniques, usually by immmunoprecipitation and Western blot analysis.Finding new biomarkers of LV remodelling could help refine risk stratification and identify patients in whom more aggressive therapy and/or more frequent follow-up could be needed.

The anti-cancer agent 4, 4-dihydroxybenzophenone-2, 4- dinitrophenylhydrazone (A007) forms complexes with pdelocalized lymphangitic dyes that allow its penetration through the skin effectively delivering it to a meta-stable type cancerous site. Previous in vitro studies, combined with gas phase mass spectrometry studies, have shown that a stronger binding affinity equates to a greater efficacy of the drug. For the determination of drug:dye complex binding strength coefficients in solution, two methods have been developed by affinity capillary electrophoresis (ACE), and cation exchange liquid chromatography (CELC). The methods demonstrated that A007 non-covalent binding strength was greatest for methylene green, followed by methylene blue, and lastly toluidine blue. Bond dissociation energies and apparent reaction enthalpies for the fragmentation pathways of lithiated acylglycerols were experimentally determined by collision activation in a triple quadrupole mass spectrometer. A developed novel derived effective path length approach for predicting bond dissociation energies (BDE) for electrostatic complex's alkali metal adducts (Li+), and halide adducts (Cl-) of acylglycerols was applied to the major fragmentation product ions of a lithiated mono-acylglycerol, a 1, 2-diacylglycerol, and a 1, 3-diacylglycerol, to predict the covalent bond dissociation energies involved in fragmentation pathways. The model's calculated apparent reaction enthalpies are used in conjunction with the energy-resolved mass spectrometry method of breakdown graphs to give a more complete quantitative aspect to the interpretation of the fragmentation processes. The dry eye condition affects millions of individuals world wide. The symptoms can be a result of simple irritation to the eye or a serious disease state. A dry eye model was developed using rabbits in order to study the compositional makeup of the tear components in hopes of identifying an underlying cause, or expressed effect of the dry eye condition. The major non-polar lipids of the tear were identified by mass spectrometry as mono and diacylglycerols, with a smaller extent of triacylglycerols. The major polar phosphorylated lipids were identified in the tear extract revealing that sphingomyelin based species were being expressed in the dry eye condition. The major proteins were determined to be the lower molecular weight lipid binding proteins where two specific species were found to increase in expression for the dry eye condition.

The possibilities of differentiating linkage positions and anomeric configurations of small oligosaccharides by negative ion mode MALDI using anion attachment followed by PSD are investigated. By careful initial adjustment of the focusing mirror ratios allowing acquisition of the peaks of interest within the same PSD segment, it is possible to obtain highly reproducible relative ion abundances. Discrimination of different linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of selected diagnostic fragment pairs make differentiation of anomeric configuration possible. F- and Ac- cannot form anionic adducts with the oligosaccharides in significant yields. However, Br-, I- and NO3- anionic adducts consistently appear in higher abundances relative to [M - H]-, just like Cl-. Mildly acidic saccharides form both deprotonated molecules and anionic adducts, making it possible to simultaneously detect neutral and acidic oligosaccharides via anion attachment. PSD of [oligosaccharide + Cl]- yields structurally-informative fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, whereas PSD of Br-, I- and NO3- adducts of oligosaccharides yield the respective anions as the main product ions without offering structural information concerning the sugar. PSD of the chloride adduct of saccharides containing 1-2 linkages also yields chlorine-containing fragment ions. MALDI-TOF-MS and LDI-TOF-MS are shown to be useful for characterization of ultra-small titania nanoparticles. Peak maxima in MALDI-TOF mass spectra are found to correlate with nanoparticle size. The size distributions of TiO2 nanoparticles, obtained from MALDI- and LDI-TOF-MS are in good agreement with parallel TEM observations. PSD analysis of inorganic x nanomaterials is performed and valuable information about the structure of analytes has been obtained. A group of inorganic nitrate and perchlorate salts of forensic and health interest are investigated by LDI- and MALDI-TOF MS. In each case, a series of characteristic cluster ions are predominant in the negative-ion mode. The number and identity of metal atoms and anions in the recorded cluster ions can be positively identified by their m/z values, distinctive isotopic patterns and characteristic PSD fragmentation patterns.

Sialic acids are involved in many biological processes. In glycoproteins and glycolipids they are essential for signalling and mediate molecular interactions as well as being targets for many pathogens such as influenza virus. The synthesis of sialylated glycoconjugates is of great importance. The incorporation of sialic acid through chemical synthesis carries several difficulties, enzymatic strategies using glycosyltransferases are very attractive alternative strategy, and have been used on a broad range of substrates forming glycosidic linkages with regio-and stereo-specificity. The work presented herein shows the study and application of two enzymes, Photobacteriumdamselae alpha2,6-sialyltransferase (Pd2,6ST) and Trypanosoma cruzi trans-sialidase (TcTS) which are used in the synthesis of sialyloligosaccharides. Both enzymes were expressed in E.coli and purified for biotransformations. In the first application new sialylated chromogenic compounds were generated through this enzymatically by using TcTS and a Pd2,6ST. These compounds were used for the detection of neuraminidase activity in a number of biological samples and led to the discovery of neuraminidase activity from Bacillus pumilus and Arthrobacter aurescens, two different bacteria in which the presence of neuraminidases had never been described. Secondly, TcTS was used to study lipid glycosylations. Glycans in biological systems can be associated to complex lipidic microdomains and the presence of these microdomains can affect the activity of some enzymes. In case of Trypanosoma cruzi trans-sialidase, a decreased activity was detected when the acceptor substrate was part of the aggregated lipid rafts compared to activity observed when the reaction was performed using fully dispersed substrate. Thirdly, the sialylation of glycoarrays using Pd2,6ST was studied. For the first time, sialylated glycans with alpha2,6- glycosidic linkages were successfully incorporated into a gold glycoarray platform, which had been previously developed for the label-free detection of carbohydrate-protein interactions. Successful enzymatic incorporation of sialic acids onto the arrays was confirmed with commercial available lectins. Finally, by using the gold glycoarray platform containing both 2,3 and 2,6 linked sialic acids as well as other common glycans, the carbohydrate-binding properties of the surface proteins of the bacterium Lactobacillus reuteri was studied using MALDI-ToF MS techniques. For first time, strong interactions were observed between a mucus binding protein and Neu5Ac alpha2,6-linked glycans, with much weaker binding to 2,3-linked analogues. Such glycan structures have been identified in abundant manner in colon mucins and this study contributes to the understanding of complex interactions between mucins and probiotic organisms as well as pathogenic bacteria. These studies show that glycan arrays can contribute both to the understanding of probiotics as well as to the identification of glycan binding proteins as targets for new drugs.

Cellulosic biomass is the major organic matter produced in the biosphere. The biodegradation of this cellulosic material is achieved by enzymatic activities of the cellulose degrading microorganisms. These organisms usually express a complex extracellular or a membrane bound cellulolytic system comprising combination of several cellulase enzymes. Cellulases are the group of hydrolytic enzymes capable of hydrolysing insoluble cellulose to glucose. Phlebia gigantea is an aggressive white rot basidiomycete with ability to tolerate resinous extracts on freshly cut wood and higher growth rate. This helps the fungus to colonise the sapwood preventing other fungi from becoming established. Early research on the cellulase system of this organism reported the presence of a cellulase system composed of P-glucosidase, endoglucanase and a cellobiohydrolase. Based on these unpublished studies, our aim was to obtain a complete sequence of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and isolate the cellulase gene resulted in an incomplete cDNA sequence of I 154 bp. To understand the cellulase system, expression and regulation of the cellulase enzymatic activity was examined for incubation of P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose and cellobiose. The pH, total protein and biomass production results indicated that the capacity of P. gigantea to degrade cellulose is dependent upon the nature of the carbon source and the regulation of the cellulase synthesis is repressed in the presence of simple sugars like glucose and xylose. The study employed the highly effective method of purification by affinity adsorption and purified cellulase complex in large quantity. Characterisation of the kinetic properties of this cellulase complex revealed that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50GC. The purified complex was comprised of multiple proteins and demonstrated significant CMCase and CBHase activity on zymogram analysis. The purified cellulase complex was characterised by 2D gel electrophoresis and by peptide mass finger printing using MALDI-TOF massspectrometry analysis. The 2D gel analysis of the purified cellulase complex showed 15 spots within the range of pI 3.5 to pI 7 and the molecular weight between 20KDa to 100KDa. Three protein spots were selected based on the IEF and SDS zymogram and identified using MALDI-TOF MS analysis. These proteins were identified based on the peptide mass data belonging to the 6-phospho-a-glucosidase, p-glucosidase and glycosyl hydrolase family 13 a-amylase or pullulanases, suggesting the divergent evolution of specific cellulase proteins. This study showed P. gigantea as a potential cellulase source and the cellulase complex secreted by the induction of substrate, comprises a variety of enzymes related to hydrolysis of cellulose biomass. It is evident from this and previous studies that P. gigantea cellulase complex comprises of a specific set of enzymes that possess the ability to degrade crystalline cellulose and is one of the first organisms to colonise freshly cut wood. Further studies on the cellulase system of this primary colonist may open up the prospects to utilise this organism as the potential onsite bioreactor agent, pre-treating the biomass and increasing the economic feasibility of the industrial bioenergy processes.

<p>Buildings, maps and objects et cetera, can be modeled using a computer or reconstructed in 3D by data from different kinds of cameras or laser scanners. This thesis concerns the latter. The recent improvements of Time-of-Flight cameras have brought a number of new interesting research areas to the surface. Registration of several ToF camera point clouds is such an area.</p><p>A literature study has been made to summarize the research done in the area over the last two decades. The most popular method for registering point clouds, namely the Iterative Closest Point (ICP), has been studied. In addition to this, an error relaxation algorithm was implemented to minimize the accumulated error of the sequential pairwise ICP.</p><p>A few different real-world test scenarios and one scenario with synthetic data were constructed. These data sets were registered with varying outcome. The obtained camera poses from the sequential ICP were improved by loop closing and error relaxation.</p><p>The results illustrate the importance of having good initial guesses on the relative transformations to obtain a correct model. Furthermore the strengths and weaknesses of the sequential ICP and the utilized error relaxation method are shown.</p>

The quantitative analysis of environmental and toxicological samples must be reliable, rapid, and in some cases field portable. In the United States, the employment of chemical weapons by rogue states and/or terrorist organizations is an ongoing concern. Nerve agent degradative products (methylphosphonic acid) as well as surrogates (glyphosate) must be detected at low quantities in various water matrices. Current methods involve tedious and time-consuming derivatizations for gas chromatography-mass spectrometry and liquid chromatography in tandem with mass spectrometry. Two solid phase extraction (SPE) techniques for the analysis of glyphosate and methylphosphonic acid are described with the utilization of isotopically enriched analytes for quantitation using atmospheric pressure chemical ionization-quadrupole-time of flight-mass spectrometry (APCI-Q-TOF-MS) that does not require derivatization. &lt;br&gt;The use of illicit drugs is also an increasing problem in the United States. Toxicological analysis of illicit drugs is important for death investigation as well as in the treatment of individuals who abuse and misuse drugs. This dissertation describes a newly developed analytical method for the simultaneous quantitative analysis of heroin, 6-acetylmorphine, morphine, cocaine, codeine, methadone, and fentanyl in synthetic urine. The resolution of an electrospray ionization-time of flight-mass spectrometer (ESI-TOF-MS) was utilized for simultaneous analysis of the drugs after extraction from urine using two newly developed SPE procedures. &lt;br&gt;The first SPE technique described in this dissertation is solid phase extraction-isotope dilution mass spectrometry (SPE-IDMS). It involves applying EPA Method 6800 in which a naturally occurring sample is pre-equilibrated with an isotopically enriched standard prior to SPE. The second extraction method, i-Spike, involves loading an isotopically enriched standard onto a SPE column independently from the naturally occurring sample. The sample and the spike are then co-eluted from the column enabling precise and accurate quantitation by molecular IDMS calculations. The SPE methods, in conjunction with IDMS, eliminate concerns of incomplete elution, matrix and sorbent effects, and MS drift. For accurate quantitation with IDMS, the isotopic contribution of all atoms in the target molecule must be statistically taken into account. This dissertation describes two newly developed sample preparation techniques for the analysis of environmental and toxicological samples as well as statistical probability analysis for accurate molecular IDMS. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation