Ce travail a pour objectif de mettre au point un procédé d’oligomérisation de rutine et d’esculine par la laccase de Trametes versicolor. Un procédé de synthèse en parallèle et d’analyse en ligne par SEC-UV et par MALDI-TOF a été mis au point. L’analyse par MALDI-TOF a révélé la formation d’un simple pontage, allant jusqu’au degré d’oligomérisation 6 pour la rutine et 9 pour l’esculine. Un pontage par liaison éther a été observé par FTIR dans le cas des oligorutines. L’analyse par RMN a démontré la mise en place de liaisons tant C-C que C-O localisées sur la partie phénolique et la partie sucre des monomères. De faibles pH et températures favorisent l’allongement de la chaîne, alors que l’augmentation de la constante diélectrique du solvant ou de la température augmente la production des oligomères de rutine. La limitation de la masse de ces oligomères serait due à une inhibition de l’enzyme, provoquée par les capacités chélatantes des oligomères. Une diminution du pouvoir antioxydant et une augmentation du pouvoir inhibiteur de la xanthine oxydase ont pu être observées lors de l’accroissement de la masse des oligomères de rutine. Ces deux activités sont améliorées lors de l’accroissement de la masse des oligomères d’esculine. Pour ces deux types d’oligomères, la solubilité dans l’eau est fortement accrue. Dans le cas des oligorutines, cette forte augmentation a été corrélée à la mise en place d’un réseau dense de liaisons hydrogène observé par modélisation moléculaire. Globalement, l’approche par modélisation moléculaire dans le vide et dans le solvant a permis de dégager des relations structure-activité, reliant notamment le nombre de liaisons hydrogène à la solubilité / The aim of this work is the elaboration of rutin and esculin oligomerization process by the laccase from Trametes versicolor. A parallel synthesis process and on-line analysis of reaction media by SEC-UV and MALDI-TOF have been elaborated. The MALDI-TOF analysis has revealed the formation of simple bridges between rutin and esculin units, up to degree of oligomerization of 6 and 9 respectively. An ether bond has been observed by FTIR spectrometry for the rutin oligomers. Finally, the NMR analysis has revealed the formation of C-C and C-O bridges both on phenolic and the sugar parts of the flavonoids. At low pH and temperature, the elongation of the chain is favored, whereas increasing the dielectric constant of the solvent or the temperature favors the production of rutin oligomers. The limitation of oligomers mass is explained by the inhibition of the enzyme, probably due to the highest chelation properties of oligomers. In the case of oligorutin, a decrease of antiradical activity and an increase of xanthine oxidase inhibitory activity have been observed when the oligomers molecular mass increases. In the case of esculin oligomers, these two activities increase with the increase of the oligomers mass. For these two types of oligomers, the water solubility is considerably increased. For the oligorutins, this augmentation has been correlated to a dense network of H-bonds, which has been demonstrated by molecular modeling. Globally, the molecular modeling approach in vacuum and in solvent has allowed to establish structure-activity relationship

No controle da qualidade da água, parâmetros microbiológicos são avaliados e devem estar de acordo com os limites estipulados em portarias e resoluções. Todas as metodologias de monitoramento microbiano demandam o cultivo dos organismos-alvo. Em muitos casos, as cepas isoladas devem ser analisadas por teses fenotípicos para determinação da espécie. Por meio de inóculos de E. coli, demonstrou-se que a variação na técnica de plaqueamento se deve especialmente à falta de consistência entre as diluições. Além disso, para a análise de réplicas, comarando-se as médias de dois métodos de diluição, foi demonstrado que ser utilizado apenas uma série de diluição derivada de um único inóculo. Utilizando-se culturas puras de E. coli, E. faecalis e P. aeruginosa, a recuperação foi similar entre os meios seletivos respectivos (m-Endo, m-EI, m-PA-C) e meio não seletivo (Tripticase Soy Agar). A utilização da técnica de membrana filtrante resultou em contagens significativamente maiores para E.coli e E. faecalis em comparação ao método de spread plate. A microbiota natural presente na água potável (torneira) não influenciou significativamente as contagens de E. coli, E. faecalis e P. aeruginosa em meios seletivos. Entretanto, na água mineral engarrafada inoculada artificialmente com P. aeruginosa, a contagem foi significativamente maior na réplica estéril. Na análise de água marinha, e na réplica não estéril inoculada com E. faecalis, a contagem foi signifivativamente menor e as células de P. aeruginosa produziram colônias atípicas. Analisando-se amostras do meio ambiente (biofilmes de estação de tratamento de água, lodo de esgoto e água de córrego contaminado), 45 colônias típicas isoladas em meios de cultura seletivos foram identificadas por meio do sequenciamento do gene 16S rRNA, testes bioquímicos e MALDI-TOF. Os resultados de sequenciamento do gene 16SrRNA tiveram baixa correlação com a identificação dos organismos por testes bioquímicos (46,6% em nível de gênero e 20% em nível de espécie) e MALDI-TOF (60% em nível de gênero e 48,8% em nível de espécie). Para as cepas identificadas como E. coli e Enterococcus spp., a correlação entre o sequenciamento e MALDI-TOF foi de 75% e 62%, respectivamente. Uma vez que a quantificação de micro-organismo por membrana filtrante depende do micro-organismo em análise e do tipo de água, é necessário que os laboratórios realizem testes de padronização antes de implementar essa metodologia. Os resultados demonstram que os métodos convencionais utilizados não são adequados e suficientes para a análise da qualidade da água e, portanto, novas metodologias devem ser empregadas. Idealmente devem ser utilizadas técnicas baseadas em características fenotípicas, genômica e proteômica cujos resultados são complementares e fornecem uma identificação mais acurada. / All over the world regulatory agencies specify microbiological water quality parameters to guarantee water safety. Conventional microbiological water quality analysis is based on the cultivation of the target organisms. In many analysis protocols, phenotypic assays of isolated strains are mandated for species determination. Reference samples are required for quality assessment and quality control of analytical protocols. Using E. coli as a model organism, it was demonstrated here that the variability of plate counts of reference cultures is caused mainly by the spread of counts in individual serial dilutions. Besides, comparing the means obtained by two dilution approaches, it was demonstrated that in the analysis of replicates it is possible to use only a set of dilutions derived for a unique inoculum. Recovery of pure cultures of E. coli, E. faecalis and P. aeruginosa was similar on selective culture media (m-Endo, m-EI, m-PA-C) and with the non-selective medium (Tripticase Soy Agar). The membrane filter technique yielded significantly higher counts for E.coli and E. faecalis in comparison to spread plate method. The autochthonous microbiota of potable water (tap water) did not influence the counts of E. coli, E. faecalis and P. aeruginosa on selective culture media. However, the sterile replica of mineral water spiked with P. aeruginosa showed higher counts for these bacteria. For marine water analysis, the non-sterile replica spiked with E. faecalis yielded low counts and P. aeruginosa produced an atypical colony. Both, sample-induced variation in target strain recovery and colony appearance on culture plates indicates the requirement for method evaluation tests on particular sample matrices before implementing routine microbiological analysis by culture-based methods in the laboratory. 45 typical colonies obtained from environmental samples in selective culture media (biofilms from activated sludge, drinking water treatment plants and water from creek contaminated with raw sewage) were analyzed by 16S rRNA gene sequencing, biochemical phenotypic assays and MALDI-TOF. Sequencing showed low correlation with phenotypic assays (46,6% at the genus level and 20% at the species level) and MALDI-TOF (60% at the genus level and 48,8% at the species level). On the other hand, the strains identified as E. coli an Enterococcus spp. by 16S rDNA gene sequencing demonstrated a high correlation with MALDI-TOF (75% and 62%, respectively). Since the results showed that conventional methods aren´t suitable and sufficient to assess water quality, new technologies should be employed. Ideally, techniques should be based on phenotypic, genomic and proteomic features since the results are complementary and provide a more accurate identification.

Sowohl in der Klinischen Labormedizin, der Klinischen Mikrobiologie als auch in der Pathologie ist die Massenspektrometrie (MS) ein bedeutender Bestandteil der Diagnostik geworden. Der Fortschritt in der Gerätetechnik ermöglicht in kurzer Zeit viele, hochaufgelöste Spektren zu generieren. Diese Informationsvielfalt macht die manuelle Auswertung durch den Anwender sehr kompliziert bis unmöglich. Aus diesem Grund ist die Unterstützung durch bioinformatische Programme notwendig. Für die Reproduzierbarkeit der Ergebnisse und die Qualitätskontrolle ist es essentiell, dass die verwendeten Algorithmen transparent und die Programme als Open Source Software (OSS) frei verfügbar sind (Aebersold and Mann, 2003). Das Ziel dieser Arbeit war die Entwicklung von MALDIquant, einer unter der GNU General Public License (GPL) stehenden, flexiblen OSS, die für die o.g. Anwendungsbereiche modernste Algorithmen für die komplette Analyse bietet und in der freien Programmiersprache R (R Core Team, 2014) geschrieben ist. Im Zusammenspiel mit dem dazugehörigen Paket MALDIquantForeign ist MALDIquant in der Lage die üblichen Dateiformate der verschiedenen MS-Geräte zu verarbeiten. Dadurch ist MALDIquant hersteller- und geräteunabhängig und eignet sich nicht nur für MALDI/TOF, sondern für alle zweidimensionalen MS-Daten. Angefangen vom Datenimport über die Prozessierung bis hin zur Analyse der Spektren bietet MALDIquant eine komplette Analyse-Pipeline und implementiert state-of-the-art Methoden. Neben weit verbreiteten Verfahren zur Baseline Correction und Peak Detection zeichnet sich MALDIquant besonders durch ein hervorragendes Peak Alignment aus. Dieses ist sehr genau und aufgrund des Fokus auf die Peaks schneller als die meisten anderen Verfahren und weitestgehend unabhängig von der Qualität der Intensitätenkalibrierung. Eine weitere Stärke von MALDIquant ist die Möglichkeit, eigene Algorithmen zu integrieren, sowie den Ablauf der Analyse den individuellen Bedürfnissen anzupassen. In der beispielhaften Analyse der Daten von Fiedler et al. (2009) konnten durch MALDIquant Peaks gefunden werden, die Patienten mit Pankreaskarzinom von nicht erkrankten Probanden unterscheiden. Einige dieser Peaks wurden bereits in anderen Publikationen beschrieben. Neben diesem Beispiel hat MALDIquant seine Nützlichkeit bereits in verschiedenen Anwendungsbereichen und Publikationen bewiesen, wie etwa in Ouedraogo et al. (2013) oder Jung et al. (2014).:Bibliographische Beschreibung (III) Abbildungsverzeichnis (V) Tabellenverzeichnis (VII) Abkürzungsverzeichnis (IX) 1 Einleitung (1) 1.1 Intention (1) 1.2 Eigene Beiträge (2) 1.3 Übersicht (3) 2 Hintergrund (5) 2.1 Proteomik (5) 2.2 Massenspektrometrie (6) 2.3 Bioinformatik (7) 3 Methoden (9) 3.1 Überblick (9) 3.2 Import der Rohdaten (9) 3.3 Transformation der Intensitäten (11) 3.4 Korrektur der Grundlinie (11) 3.5 Kalibrierung der Intensitäten (13) 3.6 Identifizierung von Merkmalen (15) 3.7 Kalibrierung der m/z-Werte (17) 3.8 Nachbearbeitung (19) 4 Ergebnisse (23) 4.1 Implementierung (23) 4.2 Anwendungsbeispiel Fiedler et al. 2009 (23) 4.3 Vorbehandlung der Daten aus Fiedler et al. 2009 mit MALDIquant (24) 4.4 Multivariate Analyse (24) 4.5 Mögliche Biomarker (26) 5 Diskussion (29) 6 Zusammenfassung (31) 7 Literaturverzeichnis (35) A Publikation (45) B Übersicht Codeumfang (49) C Analyse Fiedler et al. 2009 (51) D Erklärung über die eigenständige Abfassung der Arbeit (69) E Lebenslauf (71) F Danksagung (75)

Introduktion Antibiotikaresistens är ett globalt växande problem. Till gruppen β-laktamantibiotika hör piperacillin-tazobaktam och cefotaxim som båda verkar genom att försvaga cellväggen med kovalenta bindningar till peptidoglykanlagret som lyserar cellen. E. coli och K. pneumoniae tillhör gruppen Enterobacteriaceae, som är en del av den humana tarmfloran och ofta förekommande vid urinvägsinfektion och sepsis. Utvidgat Spektrum β-Laktamas (ESBL) är ett enzym som finns hos Enterobacteriaceae och som hydrolyserar β-laktamantibiotika. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) är en kvalitativ analysteknik för detektion av kemiska föreningar i avseende på massa och laddning. Kännedom om antibiotikametaboliters molekylvikt vid hydrolys möjliggör detektion. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) är en högsensitiv kvantifieringsmetod som separerar molekyler i avseende på polaritet för vidare detektion i avseende på massa och laddning. Syfte Syftet med denna studie var att vidareutveckla en snabb och effektiv metod för att påvisa nedbrytning av piperacillin-tazobaktam och cefotaxim i blodplasma med LC-MS/MS. Material och Metod Tiofaldigt sjunkande koncentrationer av piperacillin-tazobaktam från 2000 till 2 µg/ml, och cefotaxim med koncentrationerna 500 till 0,5 µg/ml analyserades med MALDI-TOF MS, dels intakt men även med bakterierna E. coli och K. pneumoniae med uttryck av olika resistensmekanismer. Vid optimerade koncentrationer spikades plasmaprover med nedbrutet antibiotika som sedan kvantifierades med LC-MS/MS. Resultat Lägsta detektionsgräns med MALDI-TOF MS för intakt och hydrolyserat piperacillin-tazobaktam var 20/2,5 µg/ml. För cefotaxim var lägsta gränsen 5 µg/ml. Med kliniskt relevanta blodkoncentrationer gick hydrolys inte att detektera för. Med tre bakteriekolonier/50 µl kunde dock hydrolys detekteras och kvantifieras med LC-MS/MS. Slutsats Detektion av β-laktamantibiotika är möjligt med både MALDI-TOF MS och LC-MS/MS. För att påvisa hydrolys krävdes större mängder bakterier än förväntat med LC-MS/MS. / Introduction Antibiotic resistance is a global growing problem. Piperacillin-tazobactam and cefotaxime are parts of the group β-lactam antibiotics. The common feature is to inhibit the cell wall synthesis by covalent bindings to the peptidoglycan layer and thereby causing lysis of the bacterial cell. E. coli and K. pneumoniae are members of the Enterobacteriaceae which is a part of the human normal flora but also are commonly associated with urinary tract infections which sometimes develops into to sepsis. Extended Spectrum β-Lactamases (ESBLs) are enzymes with hydrolytic abilities acting on β-lactam antibiotics, expressed by Enterobacteriaceae. The qualitative, Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to detect chemical compounds in the ratio of mass to charge in accordance to their molecular weight. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) is a highly sensitive two-step method of quantification which first separate molecules by their polarity attraction force and then by the ratio of mass to charge. Aim The aim of this study was to develop a fast and efficient method to determine degradation of piperacillin-tazobactam and cefotaxime in blood plasma by LC-MS/MS. Method Tenfold dilution of piperacillin-tazobactam in concentrations of 2000 to 2 µg/ml, and cefotaxime in concentrations of 500 to 0,5 µg/ml where analyzed by MALDI-TOF MS, intact and also with the bacteria E. coli and K. pneumoniae with different expression of antibiotic resistance. Optimized concentrations where fixed in blood plasma and then quantified by LC-MS/MS. Result The detection limit by using MALDI TOF MS of hydrolyzed as well as non-hydrolyzed piperacillin-tazobactam was 20/2,5 µg/ml. The detection limit in cefotaxime was 5 µg/ml. Hydrolysis could not be detected in clinically fixed blood concentrations. Detection and quantification of hydrolysis by LC-MS/MS was possible in a concentration of three bacteria colonies/50 µl. Conclusion It is possible to detect hydrolysis in both MALDI TOF MS and LC-MS/MS. A larger amount of bacteria than expected was needed to demonstrate hydrolysis In LC-MS/MS.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Candida albicans is an opportunistic fungal pathogen that may cause mucutaneous infection and/or disseminated candidasis if the host defense system is impaired (such as those in HIV patients). Cell surface of C. albicans is the frontier where initial interplay between host-pathogen takes place and therefore is of great importance in understanding the mechanism of hostpathogen interaction. MALDI-TOF-MS analysis of intact fungal cells yielded mass signatures for rapid species differentiation, strain grouping and yeast morphogenesis monitoring. Cell surface biotinylations at low temperature (4°C), enzymatic digestion of the intact fungal cell surface proteins ("whole cell shaving"), biotin-avidin affinity enrichment of biotinylated peptides, liquid chromatography mass spectrometry (LC-MS) based proteomic approach were employed for unambiguous identification of cell wall/cell wall associated proteins and the exposed peptide segments of these proteins. SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) based CWP quantification analyses were performed to monitor CWP accumulation level change in response to hyphae induction. Information on surface exposed peptide segments and regulation of cell wall/surface protein during morphogenesis provided new candidates to the pool of potential peptide targets for protective vaccine development. A New type of "fluorous" (fluorinated alkane) affinity gained popularity due to its low level nonspecific protein/peptide binding. Fluorous labeling reagents that target primary amine groups in proteins/peptides were synthesized and characterized. The acid labile linker in the labeling reagents allows cleavage of the bulky fluorous tag moiety and the long oligo ethylene glycol (OEG) spacer after fluorous affinity purification. Upon collision induced decomposition, the labeled peptide ion yielded a characteristic fragment that could be retrieved from the residual portion of fluorous affinity tag, and serve as a marker to indicate that the relevant peptide had been successfully labeled. Results showed that both the protein/peptide labeling and affinity enrichment/separation process were highly efficient.

O diagnóstico rápido e eficiente da mastite subclínica é importante para reduzir a persistência da doença e os prejuízos decorrentes. O objetivo do estudo foi avaliar a técnica de espectrometria de massas (MS) por ionização e dessorção a laser assistida por matriz - tempo-de-vôo (MALDI-TOF) para identificação de bactérias causadoras de mastite subclínica bovina por dois métodos: 1) a partir de bactérias isoladas por cultivo icrobiológico; 2) a partir da recuperação de bactérias diretamente do leite, visando eliminar completamente a necessidade de cultivo microbiológico para identificação dos patógenos. O estudo foi composto por dois experimentos (1 e 2), no experimento 1 foram utilizadas 33 amostras de leite provenientes de animais das raças Gir e Holandesa de quatro fazendas leiteiras para a identificação icrobiológica e MALDI-TOF MS. As amostras com resultados conflitantes foram confirmadas por sequenciamento do gene 16S rRNA. Os resultados de cultura microbiológica foram Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) e Estafilococos coagulase negativo (ECN) (n = 10). Para todas as amostras de Streptococcus agalactiae, resultados similares foram observados para a identificação microbiológica e por MALDI-TOF MS. De 13 isolados de Staphylococcus aureus, 11 foram igualmente identificados por MALDITOF, 1 isolado foi identificado como Staphylococcus haemolyticus por sequenciamento do gene 16S rRNA, e o outro isolado isolado com resultado conflitante foi caracterizado como cultura mista de Staphylococcus aureus e Enterococcus faecalis. Em relação às amostras de ECN, todas as amostras do grupo foram identificadas por MALDI-TOF em nível de gênero (S. simulans, S. epidermidis, S. Haemolyticus, S. Chromogens e S. aureus coagulase negativa). No experimento 2 foi avaliado o método de recuperação de bactérias presentes em leite e sua identificação por MALDI-TOF MS utilizando o método de contaminação experimental de leite com Escherichia coli, Enterococcus faecalis e Staphylococcus aureus. A identificação de patógenos recuperados diretamente do leite foi possível quando a concentração de E. faecalis e S. aureus foi de 106 ufc/mL, e de 107 ufc/mL para E.coli. Concluímos que o uso de MALDI-TOF MS pode acelerar a identificação de patógenos causadores de mastite subclínica bovina podendo contribuir para a adoção de medidas de controle e tratamento mais adequado. / Rapid and efficient diagnosis of subclinical mastitis is important to reduce the persistence of the disease and its losses. This study aimed to evaluate the technique of mass spectrometry (MS) and desorption ionization by matrix assisted laser time-offlight (MALDI-TOF) for identification of bacteria causing bovine subclinical mastitis by two methods: 1) from bacteria isolated by microbiological culture, 2) from bacteria recovered directly from milk, to eliminate completely the need for microbiological culture for identification of pathogens. The study consisted of two experiments (1 and 2).In experiment 1, 33 milk samples from Gir and Holstein animals collected in four dairy farms were used for microbiological identification and MALDI-TOF MS. The samples with different results were confirmed by sequencing the 16S rRNA. These samples were identified by microbiological culture as Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) and Staphylococcus coagulase negative (SCN) (n = 10). For all strains of Streptococcus agalactiae, similar results were observed for microbiological identification and MALDI-TOF MS. From 13 isolates of Staphylococcus aureus, 11 were also identified by MALDI-TOF, one isolate was identified as Staphylococcus haemolyticus by 16S rRNA sequencing , and the other discrepant sample was charactezided a mixed culture of Staphylococcus aureus and Enterococcus faecalis. Regarding the SCN samples, all samples of this group were identified by MALDI-TOF at gender level (S. simulans, S. epidermidis, S. haemolyticus, S. Chromogens and S. aureus coagulase negative). In experiment 2, we evaluated the method of recovery of bacteria present in milk and their identification by MALDI-TOF MS using experimental method of contamination of milk with Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. The identification of pathogens recovered directly from milk was possible when the concentration of E. faecalis and S. aureus was 106 ufc/mL and 107 ufc/mL for E.coli. We conclude that the use of MALDI-TOF MS can accelerate the identification of pathogens causing bovine subclinical mastitis may contribute to the adoption of control measures and appropriate treatment.

Matrix Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) mass spectroscopy is used in the characterization of synthetic polymers. MALDI allows for determination of: modal, most probable peak (M P), molecular number average (MN), molecular weight average (MW), polydispersity (PD), and polymer spread (PSP). We evaluate a new sample preparation method using Induction Based Fluidics (IBF) to kinetically launch and direct nanoliter volumes to a target without contact. IBF offers signal improvement via field enhanced crystallization. This is the first study to discuss filed enhanced crystallization in MALDI sample preparation. IBF can increase signal/noise (S/N) and signal intensity for polystyrene (PS), poly(methyl methacrylate) (PMMA), and poly(ethylene glycol) (PEG) across a mass range of 2,500 to 92,000 Da showing more accurate PSP. Increases in S/N range up to: 279% for PS, 140% for PMMA, and 660% for PEG. Signal intensities increased up to: 438% for PS, 115% for PMMA, and 166% for PEG. Cross-polarization microscopy indicates dramatic morphology differences between IBF and micropipette. Finally, we speculate as to why IBF nanoliter depositions afford higher S/N values in experiments conducted in different instrumental configurations even without optimization. Next we sought to investigate whether nanoliter volumes of concentrated polar liquids and organic monomers launched to targets using IBF can be verified through the real time charge measurements. We show that using a nanoliter IBF dispensing device and nanocoulomb meter, charge measurements made on nanoliter drops in real time are correlated with the droplets surface area following Gauss’s Law. We infer the "induction only" formation of the double layer showing the ability to determine nanoliter volumes, nearly instantaneously, in real time. Implications are presented from these IBF measurement observations on improving/monitoring MALDI quantitation and its quality control. Polymer-dye interactions were further investigated using PMMA composites made from a polar metalloporphyrin [5-(4',4',5',5'-tetramethyl[1',3',2']dioxaborolan-2'-yl)-10,20-diphenylporphyrinato]zinc(II) (Zn(II)Bpin-DPP) in select weight %s (wt%s). Fluorescence spectroscopy has revealed that the porphyrin was well dispersed within the composite. Differential Scanning Calorimetry (DSC) showed that porphyrin acted as an antiplasticizer raising the glass transition (Tg) from 105 °C to 123 °C. Dielectric Analysis (DEA) was performed in the frequency range of 0.3 Hz to 100 kHz between -150 to 270 ⁰C. Permittivity (ε’), loss factor (ε’’) and dielectric response of beta (β), alpha beta (αβ), and conductivity relaxations were studied. Previous DEA data was limited to 190 ⁰C. This study brings analysis to 270 ⁰C which is start point for the first part of PMMA degradation. Thus forwarding DEA can be used to evaluate PMMA degradation. The electric modulus formalism is used to reveal the β and conductivity relaxations. The apparent activation energies (Ea) for the molecular relaxations are presented. AC (ζAC) and DC (ζDC) conductivity are also evaluated. Tan delta (δ), dissipation factor, evaluated between 1 Hz to 100 kHz was shown to increase with porphyrin loading although locally affected by free volume restriction. Havriliak-Negami (H-N) equation was fit using the complex electric modulus (M*) modified form and was performed on the conductivity region 160 to 190 ⁰C and degradation region 190 to 270 °C. Relaxations above the Tg were proven to be conductivity relaxations using four proofs. This is the first study to investigate PMMA degradation DEA with the complex electric modulus, M*, revealing a unique occurrence of increasing central relaxation times (s-1) and reducing electric loss modulus (M") frequency maxima (Hz) after the degradation temperature of 220 ⁰C was reached supporting current literature of the first of a two part degradation process that proceeds via end chain scission.

In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The calmodulin is a Ca+2-binding protein, important in a wide variety of cellular functions. The complex Ca+2/calmodulin interacts and regulates several enzymes and target-proteins, known as calmodulina-binding proteins (CaMBPs). This study identified comparatively the composition of CaMBPs in the brain of the workers honeybees Apis mellifera, aiming to relate the their behavior in the colony. For that, the CaMBPS from the foragers workers and nurses brain were purified by affinity chromatography, separated in gel 1D, digested and analysed to peptide mass fingerprinting (PMF) for identification. In PMF, 21 proteins were identified, being 2 just in foragers workers and 13 in nurses, considered specific-behavior protein. All proteins were classified according to their function and cellular location, it was observed a bigger intensity of CaMBPs related to metabolism, for both workers. Besides, the sequences were analyzed as for that the presence of IQ motif. The results here presented indicate that behavior change in the colony changes the CaMBPs composition and, possibly, in the protein function in the Apis melilifera workers brain. / A calmodulina é uma proteína ligante de Ca+2, importante em uma variedade de funções celulares. O complexo Ca+2/calmodulina interage e regula várias enzimas e proteínas-alvo, conhecidas como proteínas ligantes de calmodulina (CaMBPs). Neste estudo, identificou-se de forma comparativa a composição de CaMBPs no cérebro de abelhas operárias Apis mellifera, visando relacioná-la com o comportamento destas abelhas na colônia. Para isto, as CaMBPs do cérebro de operárias campeira e nutridora foram purificadas através de cromatografia de afinidade, separadas em gel 1D, digeridas e submetidas à análise por peptide mass fingerprinting (PMF) para identificação. Em análise PMF, 21 proteínas diferentes foram identificadas, sendo duas somente em operária campeira e 13 em nutridora, consideradas proteínas comportamento-específicas. Todas as proteínas foram classificadas quanto a sua função e localização celular, em que se observou maior expressão de CaMBPs relacionadas ao metabolismo, para ambas operárias. Além disso, as seqüências foram analisadas quanto a presença do sítio ligante de calmodulina. Os resultados apresentados aqui indicam que a mudança de comportamento na colônia leva a uma alteração na composição de CaMBPs e, possivelmente, na função destas proteínas no cérebro das abelhas operárias Apis mellifera. / Mestre em Genética e Bioquímica