Pioneering work performed by Rosalind Franklin over half a century ago provided the first structural models of two distinct carbon types: those that become graphitic during carbonization at high temperatures, and those that do not. Moreover it is known that certain properties of carbonaceous materials including combustion, surface area, electrical resistivity, and catalytic properties are influenced by mineral impurities. The nature of this division in biocarbon structure and the known effects of minerals on carbon properties have led to this work; three principal topics were addressed; (1) the investigation of the solid state structure of biocarbons derived from various biomass feedstocks, (2) the removal of inorganic minerals from biomass, and (3) the investigation of biocarbon electronic structure subsequent to doping with select inorganic minerals. Charcoals and carbonized charcoals (i.e. biocarbons) were prepared from a wide variety of biomass substrates, including pure sugars containing 5- and 6-membered rings with furanose and pyranose configurations, lignin, agricultural residues (corncob and nut shells) and a hard wood. These biocarbons were subject to proximate and elemental analysis, gas sorption analysis, and analysis by ICP-MS, SEM, XRD, ESR, 13C CPMAS NMR, and MALDI-TOF MS. All the carbonized charcoals contained oxygen heteroatoms, had high surface areas, and were excellent conductors of electricity. Doping the biocarbon with boron or phosphorus resulted in a slight improvement in its electrical conductivity. The XRD analysis indicated that the carbonized charcoals possess an aromaticity of about 71% that results from graphite crystallites with an average size of about 20 . The NMR analysis confirmed the highly aromatic content of the carbonized charcoals. The ESR signals indicated two major types of carbon-centered organic radicals. A number of techniques employed highlighted differences between carbonized charcoals and synthetic graphite but none more so than MALDI-TOF spectrometry. The biocarbons contained readily desorbed discrete ions with m/z values of 701, 685, 465, 453, 429, and 317. All of the above findings were used to develop a model for the structure of carbonized charcoal that is consistent with the biocarbon's oxygen content, microporosity and surface area, electrical conductivity, radical content, and its MALDI-TOF spectra. The removal of inorganic mineral constituents from various biomass feedstocks was achieved via simple washing/soaking techniques using two different aqueous media; deionized water and citric acid. The most effective and consistent demineralization treatment for removing minerals from biomass involved a hot 0.1 molL-1 citric acid percolation treatment, ca. 67% of inorganic mineral matter was removed. Mineral matter at the levels present in typical biomass derived charcoals and carbons had no significant influence upon the surface area or the electrical resistivity in carbonaceous materials after high heat treatment (950 C).

BACKGROUND: There is an increased need to replace materials derived from fossil sources by renewables. Sugar-cane derived carbohydrates are very abundant in Brazil and are the cheapest sugars available in the market, with more than 400 million tons of sugarcane processed in the year 2007. The objective of this work was to study the preparation of sugar acrylates from free sugars and free acrylic acid, thus avoiding the previous preparation of protected sugar derivatives, such as glycosides, or activated acrylates, such as vinyl acrylate. RESULTS: Lipase catalyzed esterification of three mono- and two disaccharides with acrylic acid, in the presence or absence of molecular sieves was investigated. The reactions were monitored by high-performance liquid chromatography (HPLC) and the products were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The main products are mono- and diacrylates, while higher esters are formed as minor products. The highest conversion to sugar acrylates was observed for the D-glucose and D-fructose, followed by D-xylose and D-maltose. Molecular sieves had no pronounced effect on the conversion CONCLUSIONS: A feasible method is described to produce and to characterize sugar acrylates, including those containing more than two acrylate groups. The process for production of these higher esters could potentially be optimized further to produce molecules for cross-linking in acrylate polymerization and other applications. The direct enzymatic esterification of free carbohydrates with acrylic acid is unprecedented.

Depuis le séquençage complet du génome humain, la génomique se focalise sur la détection des modifications de l'ADN des gènes potentiellement impliqués dans les maladies humaines. Les modifications de l'ADN sont au niveau de la séquence ou épigénétique. Ces polymorphismes, fréquents, rares, connus ou inconnus, peuvent être identifiés par le développement de nouvelles méthodes de séquençage de l'ADN pour chaque individu. Ce projet a pour but le développement de méthodes d'analyse de l'ADN par clivage d'une chimère ARN/ADN et par MALDI-TOF MS. Elle repose sur l'utilisation d'une nouvelle classe d'ADN polymérases qui peut incorporer également des ribonucléotides. La chimère ARN/ADN, simple ou double brin, contient trois bases désoxynucléotides et une quatrième base sous sa forme ribonucléotide. Elle est ensuite clivée après chaque ribonucléotide par l'hydroxyde de sodium. Les fragments de clivage se terminent par un ribonucléotide qui possède un groupement 3'- phosphate terminal. Ils sont dessalés par des billes échangeuses de cation et leurs masses sont analysées par MALDI-TOF MS. La comparaison des masses de l'individu avec celles de la séquence de référence est significative d'un changement dans la séquence d'ADN. Les fragmentations en phase gazeuse de la chimère d'ARN/ADN ont également été étudiées. Cette méthode est adaptée pour l'étude d'un grand nombre d'individus dans une région limitée du génome grâce à la capacité haut-débit du MALDI-TOF MS. Cette méthode, rapide et efficace, possède de nombreuses applications tel que: le génotypage, le génotypage allèle-spécifique en multiplex, le microhaplotypage en multiplex, l'analyse de la méthylation de l'ADN et le séquençage

Background Customer satisfaction is important for clinical microbiology laboratories and the most important service aspect is the reliability of responses. One important indicator of the quality of care is turnaround time for a sample referred to a laboratory. Aim This study describes and evaluates an improvement of the blood culture process and evaluates the staff’s experiences of the changes brought by the improvement project. Methods The blood culture process during evenings and nights was re-designed in a cooperation project between the laboratories of clinical microbiology and clinical chemistry in a mid-size Swedish county council. Typing with matrix-assisted laser desorption/ionization time of flight (MALDI ToF) and rapid antibiotic susceptibility testing were also introduced. To describe staff experi-ences semi-structured interviews were performed with twelve of the staff involved. Results The time from sampling to susceptibility testing and typing, for patients with cefotaxime resistant enterobacteriaceae, was before the improvement project on average 55 hours compared to 43 hours after closure of the project. In the qualitative content analysis four categories were found which represented the experience of the staff: patient focus, changed knowledge, cooperation and driving forces. Discussion The study of the implementation of the improvement showed that laboratory staff could handle the change well. The change from traditional biochemical typing, used for over 50 years, to MALDI ToF is indeed a paradigm shift. Nevertheless, nobody was disappointed over the fact that some of the fundamental previous microbiological laboratory work routines were laid to rest.

Due to the technology development, the diet habit has completely changed. It accompanied by the metabolite diseases relevant to blood glucose and lipids, which are dependent with the atherosclerosis and cardiovascular disease. In this study, we using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) to characterize triglycerides in human plasma. In the other, the glycosylated hemoglobin in human whole blood was detected by liquid electrospray laser desorption ionization (Liquid ELDI/MS). Triglycerides are energy source (9 kcal/g) in human body, derived from glycerol and three fatty acids. It is a main constituent of vegetable oil and animal fats. In clinical diagnosis, human plasma was mixed with triglyceride Kit to react to the final 520 nm UV-absorbing substance, then the concentration was quantified consistent with the calibration line by UV/Visible spectrometry. By the way, it needed Kit chemicals for one trial. MALDI-TOF/MS is a simple and easy method to operate to detect complex compounds in human plasma, only need to optimize the parameters (solvent collection, sample dilution, matrix selection, sample pretreatment ) to form a homogeneous crystals. The developed ¡§seed layer¡¨ method can reduce the sweet spot effect and cause a lower with-in spot variation (RSD < 20%) compared to ¡§premix¡¨ method (RSD >30%). Combined with statistic software 2D peak distribution, a semi-quantification can be observe of 24 different triglyceride concentration human plasmas. The level of glycosylated hemoglobin (HbA1c) in whole blood is currently the most important measurement of long-term control of the glycemic state of diabetes. As a result of the interferences of high concentrations of metabolites, proteins and salts in whole blood, tedious sample cleanup procedures must be performed prior to subjecting the sample solutions to conventional LC/MS and MALDI analyses for the detection of HbA1c. Electrospray laser desorption ionization mass spectrometry (ELDI/MS), a two-step ambient ionization technique, has been developed to characterize analytes directly from the liquid sample surface. One drop of the diluted hole blood (1/10, v/v in water) was placed on the stainless steel plate. The sample droplet was irradiated with a pulse laser, the desorbed analytes were post-ionized in an electrospray (ESI) plume (ESI solution: 70% methanol in water, 0.1% acetic acid), and the analyte ions were detected by a ion trap mass analyzer. Through this study, the protocol for efficiently characterizing HbA1c present in a drop of diluted whole blood with ELDI/MS was established. We successfully detected the ion signal of HbA1c with ELDI/MS. Quantification of the level of HbA1c in the whole blood of diabetic patients was achieved by calculating the ratio of the ion peak area of the glycosylated and non-glycosylated hemoglobin ions. A linear relationship exists for the quantitative results of HbA1c in whole blood of 20 diabetic patients obtained between ELDI/MS and that through conventional spectroscopic measurement.

Chapter 1. Deciphering the toxicity of Helicobacter pylori clinical isolates from gastric diseases patients using MALDI-TOF/MS and multivariate analysis. Helicobacter pylori (H. pyloyi) infection is associated with gastric diseases such as gastric polyp, chronic gastritis, gastric ulcer, gastric cancer, etc. In fact, most of the people infected not have the symptoms of gastric diseases due to the high degree of variability of gene with H. pyloyi and the specific immune responses of the hosts. In order to investigate the relationship between H.pylori and gastric diseases, the clinical strains of H. pylori isolated from patients from nine gastric diseases were extracted from the optimized extraction and analysis by MALDI-TOF/MS, then the high reproducible spectra were combined with multivariate statistical analysis including Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), Discriminant Analysis (DA) . In the result of PCA, there is no specific potential marker to discriminate the clinical strains to nine gastric diseases. In the result of HCA, the strains from different gastric diseases were clustered together means they have the similarity of the protein and metabolite. In the result of DA, the strains from gastric and non-gastric cancer were discriminanted by the discriminant function composed of thirty-eight discriminant variables in the spectra. This discriminant function would be confirmed by other clinical strains isolated from gastric diseases patients in the future and then would help to predict the the similarity of the protein and metabolite of the strains isolated from the gastric diseases patients whether gastric cancer or not. Chapter 2. Biomarker discovery in the stools of gastric cancer patients using MALDI-TOF/MS. According to the statistics of Republic 100 years from the Department of Health, cancer was the first of the ten lesding to death. With the modern change of eatiog habbits, gastrointestinal cancer has increased steadily. Gastrointestinal cancer accompanied occult gastrointestinal bleeding, and it is commonly detected by the fecal occult blood test (FOB). FOB including Guaiac-based fecal occult-blood test and immunochemical tests. Guaiac-based fecal occult-blood tests make use of the pseudoperoxidase activity of heme, and the reagent turns blue after oxidation by oxidants or peroxidases in the presence of an oxygen donor such as hydrogen peroxide, so it would have the potential of false-positive result. Immunochemical tests, which use antibodies detect against human hemoglobin with great sensitivity, but the tests are limited by loss of hemoglobin antigenicity at room temperature and require processing in a laboratory. In order to decrease the false-positive of detecting heme and decreasing the cost of the detection against hemoglobin in stools, in the study, we ues the distill water to extract the heme (m/z 616) and hemoglobin in stools and analysis with the reflectron and linear mode of MALDI-TOF/MS. In this study, at first, we used the stimulated stomach acid decomposing the hemoglobin to release the heme, to stimulate the gastrointestinal bleeding. Second, we used the distill water to extract the hemoglobin in stools, and detected by the linear mode of MALDI-TOF/MS, and the detection limit of MALDI-TOF/MS against hemoglobin in stool was better than the immunochemical tests. Third, the same strategy was applied to fifty-nine patients (including nineteen esophageal cancer patients, twenty gastric cancer patients and colorectal cancer patients) stools to detect heme and hemoglobin by MALDI-TOF/MS and the results were compared with the fecal occult blood test. In the detection of heme, MALDI-TOF/MS had not detect heme, but the Guaiac-based fecal occult-blood test had detected, it would be that the stools had the oxidants (not heme) to react the reagent. In addition, MALDI-TOF/MS had detected heme, but the Guaiac-based fecal occult-blood test had no results, those cases would be catched up in the future. In the detection of hemoglobin, using immunochemical tests to be the reference index, MALDI-TOF/MS had the false-negative result might come from the complicated matrix effect of stools, so that the hemoglobin could not form the good crystalline with matrix CHCA. The false-positive results of MALDI-TOF/MS might come from the criteria of hemoglobin signal.

We gathered normal and colorectal cancer tissues from 18 patients after tumor surgery. The tumor tissues represent caner stages from T1 to T4 (TNM system).Two-dimensional electrophoresis and MALDI-TOF techniques were utilized to identify the differentially expressed proteins. Our studies showed that there are about 18 differentially expressed proteins in normal and tumor tissues (p<0.05) which 13 proteins increased in tumor were Keratin 8, Protein disuflde isomerase A3 precursor, Keratin 18, Fractalkine precursor, LDH-B, Tropomyosin alpha 4 chain, Tropomyosin alpha 3 chain, chloride intracellular channel protein 1, PTTG1(Pituitary tumor-transforming protein 1),

Bordetella pertussis is a gram-negative, human pathogen and etiologic agent of whooping cough (pertussis), a highly contagious, acute respiratory illness. In this study, the analysis of whole immunproteome and subproteomes of this microorganism was performed. The soluble cytoplasmic proteomes of B. pertussis Tohama I strain and a local isolate Saadet were separated by 2DE. By Western blot analysis, we identified 25 immunogenic proteins of three categories. In the first group, there were well-known proteins of the pathogen The second group comprised proteins which were already shown antigenic in certain pathogenic bacteria, but not in B. pertussis before. The third group of proteins were those which have not been shown to be immunogenic in any pathogen till the present study such as putative chromosome partition protein, preprotein translocase SecA subunit, carbamoyl-phosphate synthase large chain, PRP synthase, putative substrate-CoA ligase, lysyl-tRNA synthetase, fumaryl acetoacetase, putative peptidyl-prolyl cis-trans isomerase, aspartate-semialdehyde dehydrogenase, putative DNA-binding protein and a putative outer membrane protein. In our surfaceome study, surface proteins of two strains were identified by 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these techniques 45 proteins were identified by 2DE and 226 proteins by geLC-MS/MS. The immunogenicity of surface proteins on 2DE gels were analyzed by Western blotting and among 11 identified immunogenic proteins glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase were found to be immunogenic for the first time in Bordetella. It was also found that 16 proteins were differentially expressed in B. pertussis Saadet and Tohama I. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I. In the secretome study, we identified 40 proteins by 2DE and 357 proteins by geLC-MS/MS. It was found that 12 proteins were immunogenic by Western blot analysis and the immunogenicity of putative secreted protein (BP1047) was shown for the first time in this study. In our study, PT subunit 2 and putative outer protein D (BopD) were more abundant in Saadet while one protein, glutamate synthase subunit beta was expressed at a higher level in Tohama I. Four proteins were expressed only in Saadet (two capsular polysaccharide biosynthesis protein, protein FimX and putative outer membrane permeability protein). The present study comprehensively covered almost the entire proteome of a crucial pathogen, demonstrated many novel antigens and identified hundreds of membrane-bound proteins, cell surface-associated and extracellular proteins. Thus, it is anticipated to greatly aid in a better understanding of pathogen-host relations, rational design of novel drugs and developing new generation vaccines against B. pertussis.

The surface-induced dissociation (SID) method is introduced into a Bruker matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI TOF MS) as an efficient ion fragmentation method. Ion trajectory calculations using the SIMION 7.0 ion optics program are performed and results are combined with simple unimolecular decay calculations in order to study the kinetics of the SID processes. In this instrument, the observation time frame for SID fragments lies in the submicrosecond region, allowing the specific detection of submicrosecond fragmentation channels. MALDI-produced protonated peptides in the mass range of 700 - 1500 Da and radical ions produced by laser irradiation of fullerenes C60 and C70 are fragmented at a gold surface coated with a self-assembled monolayer of alkanethiol to obtain TOF SID TOF mass spectra. For the SID of peptides in the hyperthermal energy regime, a fragmentation time frame of tens to a few hundreds of nanoseconds was calculated for the observed fast fragmentation channels (Chapters 3 and 4). Theoretical and experimental peak shape comparisons assuming unimolecular decay kinetics indicated a log rate constant in the range 6 - 7 (Chapter 4). Energy and mass resolved kinetic studies are also carried out. The contribution of special structural features to peptide fragmentation and the possibility of different fragmentation mechanisms such as sequential and parallel pathways are investigated. The results indicate a unimolecular decay process for observed fast peptide fragments ruling out a surface-shattering mechanism. Fullerene ions, especially C60+., showed a fragmentation behavior producing C2n+. fragments with an even number of C units at collision energies in the range of 100 - 400 eV (Chapter 5). At around 400 eV, additional small fragments appeared that are apart by only a single C unit. According to the calculated fragmentation times and the theoretical and experimental peak shape comparisons assuming unimolecular decay kinetics, both these processes may be approximated by parallel fast unimolecular decay processes with fragmentation time frames of tens to hundreds of nanoseconds although the poor theoretical and experimental peak shape matching for example in the decay of C60+. to C19+. may suggest deviations from a one-step unimolecular decay process.

Peptidoglycan, the major component of the bacterial cell wall, is essential for cell viability. Several important antibiotics disrupt peptidoglycan metabolism, including the β-lactams and vancomycin. There are several bacterial enzymes involved in peptidoglycan metabolism that are not yet the target of antibiotics, such as the lytic transglycosylases (LTs). Relatively little experimental characterization has been done on LTs, due largely to the difficulties of working with insoluble, heterogeneous, and highly variable peptidoglycan. This research develops a method for the generation of a soluble, homogeneous oligosaccharide substrate that can be used to study LTs. The approach taken was based on the enzymatic degradation of peptidoglycan into fragments of a specific nature, and their separation by HPLC. This work identifies the challenges associated with this approach, and discusses the potential flaws in the 'top-down' generation of a soluble substrate. / This thesis was typeset with LaTeX using Minion Pro and Myriad Pro typefaces.