Studies on Copepods from the EEZ of India-Bay of Bengal and Andaman Sea

The main objectives of the study are: To study the seasonal distribution of copepods with special reference to their qualitative and quantitative distribution, with notes on biodiversity in the Andaman Sea and the Bay of Bengal. To study the spatial and temporal distribution of copepods in the Andaman Sea and the Bay of Bengal.To understand the hydrography and the environmental characteristics of the Andaman Sea and the Bay of Bengal and their role in the distribution and biomass of copepods.To study the vertical migration/diurnal migration of the copepods. To study the difference between the coastal and oceanic composition of copepods in the study area and the factors responsible for it.

Feasibility study of biofuel production from freshwater fern, Azolla sp in Anzali Wetland

Production of bioethanol through acidic and enzymatic hydrolysis of aquatic Azolla sp., as a new source of bio-mass, has been performed, as a means to control increasing growth and reducing undesirable effects of this plant in Anzali lagoon. After sampling, drying and crushing, Azolla was hydrolyzed, using diluted acid and enzyme. Diluted acid hydrolysis was done using both autoclave and a high-pressure system (Batch Synth® Microwave synthesizer). The effects of temperature and time (in autoclave) and concentration of acid (in both) were compared. Cellubrix®, a ommercial cellulase source, was used for enzymatic hydrolysis process. The amounts of reducing sugars, glucose and furfural, released from hydrolyzate, were measured. To produce alcohol, Sacchromyces cerevisiae (to ferment sixcarbon sugars), Zygowilliopsis californica and Pichia stipitis (to ferment five-carbon and sixcarbon sugars) were used. Maximum amounts of glucose (4.83% w/w) and reducing sugars (14.15% w/w) were obtained using acid hydrolysis in autoclave. In the microwave oven, maximum glucose (5.04% w/w) and reducing sugars (13.27 w/w) were obtained at 180 and 200 °C, respectively. Under these conditions, maximum produced furfural was 1.54 g/L. The difference between amounts of furfural obtained from acid hydrolysis of Azolla in microwave oven compared to autoclave was statistically significant. Amounts of alcohol produced and its yields were 3.99 g/L and 33.13% for S. cerevisiae in 48 hours, 3.73 g/L and 30.45% for Pichia stipites in 48 hours, and 3.73 g/L and 30.45% for Z. californica in 24 hours after inoculation, respectively, with significant differences. Statistical comparison of results showed significant differences (P<0.05) in glucose production, at different conditions. Amounts of reducing sugars and glucose increased after optimization of levels of acid, time, and temperature. The overall optimum released sugar and glucose were obtained with 1.67% (w/v) acid using autoclave. Higher temperatures in microwave oven caused a significant increase (P<0.05) in furfural. Furfural severely inhibits fermentation. Hence, regarding the issues of energy consumption and time, amounts of inhibiting substances and sugar production, autoclave is found to be superior to the high temperature and pressure, generated in microwave oven, for hydrolyzing Azolla. Furthermore, given the amounts of Azolla in Anzali lagoon, it may be recommendable to use this plant as a biomass resource.

Short-term response of the Ca cycle of a montane forest in Ecuador to low experimental CaCl2 additions

The tropical montane forests of the E Andean cordillera in Ecuador receive episodic Sahara- dust inputs particularly increasing Ca deposition. We added CaCl2 to isolate the effect of Ca deposition by Sahara dust to tropical montane forest from the simultaneously occurring pH effect. We examined components of the Ca cycle at four control plots and four plots with added Ca (2 × 5 kg ha?1 Ca annually as CaCl2) in a random arrangement. Between August 2007 and December 2009 (four applications of Ca), we determined Ca concentrations and fluxes in litter leachate, mineral soil solution (0.15 and 0.30 m depths), throughfall, and fine litterfall and Al con- centrations and speciation in soil solutions. After 1 y of Ca addition, we assessed fine-root bio- mass, leaf area, and tree growth. Only < 3% of the applied Ca leached below the acid organic layer (pH 3.5?4.8). The added CaCl2 did not change electrical conductivity in the root zone after 2 y. In the second year of fertilization, Ca retention in the canopy of the Ca treatment tended to decrease relative to the control. After 2 y, 21% of the applied Ca was recycled to soil with throughfall and litterfall. One year after the first Ca addition, fine-root biomass had decreased significantly. Decreasing fine-root biomass might be attributed to a direct or an indirect beneficial effect of Ca on the soil decomposer community. Because of almost complete association of Al with dissolved organic matter and high free Ca2+ : Al3+ activity ratios in solution of all plots, Al toxicity was unlikely. We conclude that the added Ca was retained in the system and had benefi- cial effects on some plants.

Analysis of bio-derived platform chemicals in ionic liquid media by desorption/ionization mass spectrometry

In this work, desorption/ionization mass spectrometry was employed for the analysis of sugars and small platform chemicals that are common intermediates in biomass transformation reactions. Specifically, matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometric techniques were employed as alternatives to traditional chromatographic methods. Ionic liquid matrices (ILMs) were designed based on traditional solid MALDI matrices (2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA)) and 1,3-dialkylimidazolium ionic liquids ([BMIM]Cl, [EMIM]Cl, and [EMIM]OAc) that have been employed as reaction media for biomass transformation reactions such as the conversion of carbohydrates to valuable platform chemicals. Although two new ILMs were synthesized ([EMIM][DHB] and [EMIM][CHCA] from [EMIM]OAc), chloride-containing ILs did not react with matrices and resulted in mixtures of IL and matrix in solution. Compared to the parent solid matrices, much less matrix interference was observed in the low mass region of the mass spectrum (< 500 Da) using each of the IL-matrices. Furthermore, the formation of a true ILM (i.e. a new ion pair) does not appear to be necessary for analyte ionization. MALDI sample preparation techniques were optimized based on the compatibility with analyte, IL and matrix. ILMs and IL-matrix mixtures of DHB allowed for qualitative analysis of glucose, fructose, sucrose and N-acetyl-D-glucosamine. Analogous CHCA-containing ILMs did not result in appreciable analyte signals under similar conditions. Small platform compounds such as 5-hydroxymethylfurfural (HMF) and levulinic acid were not detected by direct analysis using MALDI-MS. Furthermore, sugar analyte signals were only detected at relatively high matrix:IL:analyte ratios (1:1:1) due to significant matrix and analyte suppression by the IL ions. Therefore, chemical modification of analytes with glycidyltrimethylammonium chloride (GTMA) was employed to extend this method to quantitative applications. Derivatization was accomplished in aqueous IL solutions with fair reaction efficiencies (36.9 – 48.4 % glucose conversion). Calibration curves of derivatized glucose-GTMA yielded good linearity in all solvent systems tested, with decreased % RSDs of analyte ion signals in IL solutions as compared to purely aqueous systems (1.2 – 7.2 % and 4.2 – 8.7 %, respectively). Derivatization resulted in a substantial increase in sensitivity for MALDI-MS analyses: glucose was reliably detected at IL:analyte ratios of 100:1 (as compared to 1:1 prior to derivatization). Screening of all test analytes resulted in appreciable analyte signals in MALDI-MS spectra, including both HMF and levulinic acid. Using appropriate internal standards, calibration curves were constructed and this method was employed for monitoring a model dehydration reaction of fructose to HMF in [BMIM]Cl. Calibration curves showed wide dynamic ranges (LOD – 100 ng fructose/μg [BMIM]Cl, LOD – 75 ng HMF/μg [BMIM]Cl) with correlation coefficients of 0.9973 (fructose) and 0.9931 (HMF). LODs were estimated from the calibration data to be 7.2 ng fructose/μg [BMIM]Cl and 7.5 ng HMF/μg [BMIM]Cl, however relatively high S/N ratios at these concentrations indicate that these values are likely overestimated. Application of this method allowed for the rapid acquisition of quantitative data without the need for prior separation of analyte and IL. Finally, small molecule platform chemicals HMF and levulinic acid were qualitatively analyzed by DESI-MS. Both HMF and levulinic acid were easily ionized and the corresponding molecular ions were easily detected in the presence of 10 – 100 times IL, without the need for chemical modification prior to analysis. DESI-MS analysis of ILs in positive and negative ion modes resulted in few ions in the low mass region, showing great potential for the analysis of small molecules in IL media.

SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

How effective are broad-scale nutrient mass balances for determining the sustainability of lot-feed manure application?

Nutrient mass balances have been used to assess a variety of land resource scenarios, at various scales. They are widely used as a simple basis for policy, planning, and regulatory decisions but it is not clear how accurately they reflect reality. This study provides a critique of broad-scale nutrient mass balances, with particular application to the fertiliser use of beef lot-feeding manure in Queensland. Mass balances completed at the district and farm scale were found to misrepresent actual manure management behaviour and potentially the risk of nutrient contamination of water resources. The difficulties of handling stockpile manure and concerns about soil compaction mean that manure is spread thickly over a few paddocks at a time and not evenly across a whole farm. Consequently, higher nutrient loads were applied to a single paddock less frequently than annually. This resulted in years with excess nitrogen, phosphorus, and potassium remaining in the soil profile. This conclusion was supported by evidence of significant nutrient movement in several of the soil profiles studied. Spreading manure is profitable, but maximum returns can be associated with increased risk of nutrient leaching relative to conventional inorganic fertiliser practices. Bio-economic simulations found this increased risk where manure was applied to supply crop nitrogen requirements (the practice of the case study farms, 200-5000 head lot-feeders). Thus, the use of broad-scale mass balances can be misleading because paddock management is spatially heterogeneous and this leads to increased local potential for nutrient loss. In response to the effect of spatial heterogeneity policy makers who intend to use mass balance techniques to estimate potential for nutrient contamination should apply these techniques conservatively.

Methods and tools for mass spectrometric lipidome analysis

The analysis of lipid compositions from biological samples has become increasingly important. Lipids have a role in cardiovascular disease, metabolic syndrome and diabetes. They also participate in cellular processes such as signalling, inflammatory response, aging and apoptosis. Also, the mechanisms of regulation of cell membrane lipid compositions are poorly understood, partially because a lack of good analytical methods. Mass spectrometry has opened up new possibilities for lipid analysis due to its high resolving power, sensitivity and the possibility to do structural identification by fragment analysis. The introduction of Electrospray ionization (ESI) and the advances in instrumentation revolutionized the analysis of lipid compositions. ESI is a soft ionization method, i.e. it avoids unwanted fragmentation the lipids. Mass spectrometric analysis of lipid compositions is complicated by incomplete separation of the signals, the differences in the instrument response of different lipids and the large amount of data generated by the measurements. These factors necessitate the use of computer software for the analysis of the data. The topic of the thesis is the development of methods for mass spectrometric analysis of lipids. The work includes both computational and experimental aspects of lipid analysis. The first article explores the practical aspects of quantitative mass spectrometric analysis of complex lipid samples and describes how the properties of phospholipids and their concentration affect the response of the mass spectrometer. The second article describes a new algorithm for computing the theoretical mass spectrometric peak distribution, given the elemental isotope composition and the molecular formula of a compound. The third article introduces programs aimed specifically for the analysis of complex lipid samples and discusses different computational methods for separating the overlapping mass spectrometric peaks of closely related lipids. The fourth article applies the methods developed by simultaneously measuring the progress curve of enzymatic hydrolysis for a large number of phospholipids, which are used to determine the substrate specificity of various A-type phospholipases. The data provides evidence that the substrate efflux from bilayer is the key determining factor for the rate of hydrolysis.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Avaliação da acidez de bio-óleos obtidos pela pirólise de óleos vegetais pós-consumo

O descarte irregular do óleo vegetal pós-consumo diretamente na rede de esgoto vem causando grandes problemas para o meio ambiente. Atualmente, essa problemática tem se intensificado devido ao aumento de produção e consumo destes óleos, o que por conseqüência aumenta o despejo desordenado. No presente estudo foi realizada a obtenção do bio-óleo a partir da pirólise térmica em atmosfera de nitrogênio, a 400C, por 20 minutos, do óleo vegetal pós-consumo, provenientes das seguintes oleoginosas: soja, milho, girassol e canola. As pirólises não-catalíticas apresentaram uma geração média entre 40 e 50% de um bio-óleo, de elevado índice de acidez, 81,8 mg NaOH/g. Na pirólise catalítica, a argila ácida K10 foi o catalisador que apresentou melhor eficácia para geração de um bio-óleo de menor índice de acidez. A concentração ótima do catalisador foi de 5%(m/m), gerando 482 % de um bio-óleo com índice de acidez de 43,8 mg NaOH/g. A caracterização dos líquidos pirolíticos obtidos foi realizada através da técnica de espectrofotometria na região do infravermelho (FTIR) e cromatografia em fase gasosa acoplada a espectrômetro de massas (CG/EM) que monstraram que a trioleína, o triglicerídeo do ácido oléico, foi craqueado, gerando o hexadecanoato de octadecila e o oleato de eicosila, ésteres do respectivo ácido graxo

Upgrading bio-oil over different solid catalysts

Solid acid 40SiO(2)/TiO2-SO42- and solid base 30K(2)CO(3)/Al2O3-NaOH were prepared and compared with catalytic esterification activity according to the model reaction. Upgrading bio-oil by solid acid and solid base catalysts in the conditioned experiment was investigated, in which dynamic viscosities of bio-oil was lowered markedly, although 8 months of aging did not show much viscosity to improve its fluidity and enhance its stability positively. Even the dehydration by 3A molecular sieve still kept the fluidity well. The density of upgraded bio-oil was reduced from 1.24 to 0.96 kg/m(3), and the gross calorific value increased by 50.7 and 51.8%, respectively. The acidity of upgraded bio-oil was alleviated by the solid base catalyst but intensified by the solid acid catalyst for its strong acidification. The results of gas chromatography-mass spectrometry analysis showed that the ester reaction in the bio-oil was promoted by both solid acid and solid base catalysts and that the solid acid catalyst converted volatile and nonvolatile organic acids into esters and raised their amount by 20-fold. Besides the catalytic esterification, the solid acid catalyst carried out the carbonyl addition of alcohol to acetals. Some components of bio-oil undertook the isomerization over the solid base catalyst.

Dilute phosphoric acid-catalysed hydrolysis of municipal bio-waste wood shavings using autoclave parr reactor system

The visibility of using municipal bio-waste, wood shavings, as a potential feedstock for ethanol production was investigated. Dilute acid hydrolysis of wood shavings with H3PO4 was undertaken in autoclave parr reactor. A combined severity factor (CSF) was used to integrate the effects of hydrolysis times, temperature and acid concentration into a single variable. Xylose concentration reached a maximum value of 17 g/100 g dry mass corresponding to a yield of 100% at the best identified conditions of 2.5 wt.% H3PO4, 175 degrees C and 10 min reaction time corresponding to a CSF of 1.9. However, for glucose, an average yield of 30% was obtained at 5 wt.% H3PO4, 200 degrees C and 10 min. Xylose production increased with increasing temperature and acid concentration, but its transformation to the degradation product furfural was also catalysed by those factors. The maximum furfural formed was 3 g/100 g dry mass, corresponding to the 24% yield. (C) 2011 Elsevier Ltd. All rights reserved.

Determination of geographical origin of distillers dried grains and solubles using isotope ratio mass spectrometry

In recent years distillers dried grains and solubles (DDGS), co-products of the bio-ethanol and beverages industries, have become globally traded commodity for the animal feed sector. As such it is becoming increasingly important to be able to trace the geographical origin of commodities in case of a contamination incident or authenticity issue arise. In this study, 137 DDGS samples from a range of different geographical origins (China, USA, Canada and European Union) were collected and analyzed. Isotope ratio mass spectrometry (IRMS) was used to analyze the DDGS for ²H/¹H, ¹³C/¹²C, ¹⁵N/¹⁴N, ¹⁸O/¹⁶O and ³⁴S/³²S isotope ratios which can vary depending on geographical origin and processing. Univariate and multivariate statistical techniques were employed to investigate the feasibility of using the IRMS data to determine botanical and geographical origin of the DDGS. The results indicated that this commodity could be differentiated according to their place of origin by the analysis of stable isotopes of hydrogen, carbon, nitrogen and oxygen but not with sulfur. By adding data to the models produced in this study, potentially an isotope databank could be set up for traceability procedures for DDGS, similar to the one established already for wine which will help in feed and food security issues arising worldwide.

Analytical strategies for the comprehensive profiling of histone post translational modifications by mass spectrometry and implications for functional analyses

Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi.

Identification de nouveaux substrats des kinases Erk1/2 par une approche bio-informatique, pharmacologique et phosphoprotéomique

La phosphorylation est une modification post-traductionnelle omniprésente des protéines Cette modification est ajoutée et enlevée par l’activité enzymatique respective des protéines kinases et phosphatases. Les kinases Erk1/2 sont au cœur d’une voie de signalisation importante qui régule l’activité de protéines impliquées dans la traduction, le cycle cellulaire, le réarrangement du cytosquelette et la transcription. Ces kinases sont aussi impliquées dans le développement de l’organisme, le métabolisme du glucose, la réponse immunitaire et la mémoire. Différentes pathologies humaines comme le diabète, les maladies cardiovasculaires et principalement le cancer, sont associées à une perturbation de la phosphorylation sur les différents acteurs de cette voie. Considérant l’importance biologique et clinique de ces deux kinases, connaître l’étendue de leur activité enzymatique pourrait mener au développement de nouvelles thérapies pharmacologiques. Dans ce contexte, l’objectif principal de cette thèse était de mesurer l’influence de cette voie sur le phosphoprotéome et de découvrir de nouveaux substrats des kinases Erk1/2. Une étude phosphoprotéomique de cinétique d’inhibition pharmacologique de la voie de signalisation Erk1/2 a alors été entreprise. Le succès de cette étude était basé sur trois technologies clés, soit l’enrichissement des phosphopeptides avec le dioxyde de titane, la spectrométrie de masse haut débit et haute résolution, et le développement d’une plateforme bio-informatique nommée ProteoConnections. Cette plateforme permet d’organiser les données de protéomique, évaluer leur qualité, indiquer les changements d’abondance et accélérer l’interprétation des données. Une fonctionnalité distinctive de ProteoConnections est l’annotation des sites phosphorylés identifiés (kinases, domaines, structures, conservation, interactions protéiques phospho-dépendantes). Ces informations ont été essentielles à l’analyse des 9615 sites phosphorylés sur les 2108 protéines identifiées dans cette étude, soit le plus large ensemble rapporté chez le rat jusqu’à ce jour. L’analyse des domaines protéiques a révélé que les domaines impliqués dans les interactions avec les protéines, les acides nucléiques et les autres molécules sont les plus fréquemment phosphorylés et que les sites sont stratégiquement localisés pour affecter les interactions. Un algorithme a été implémenté pour trouver les substrats potentiels des kinases Erk1/2 à partir des sites identifiés selon leur motif de phosphorylation, leur cinétique de stimulation au sérum et l’inhibition pharmacologique de Mek1/2. Une liste de 157 substrats potentiels des kinases Erk1/2 a ainsi été obtenue. Parmi les substrats identifiés, douze ont déjà été rapportés et plusieurs autres ont des fonctions associées aux substrats déjà connus. Six substrats (Ddx47, Hmg20a, Junb, Map2k2, Numa1, Rras2) ont été confirmés par un essai kinase in vitro avec Erk1. Nos expériences d’immunofluorescence ont démontré que la phosphorylation de Hmg20a sur la sérine 105 par Erk1/2 affecte la localisation nucléocytoplasmique de cette protéine. Finalement, les phosphopeptides isomériques positionnels, soit des peptides avec la même séquence d’acides aminés mais phosphorylés à différentes positions, ont été étudiés avec deux nouveaux algorithmes. Cette étude a permis de déterminer leur fréquence dans un extrait enrichi en phosphopeptides et d’évaluer leur séparation par chromatographie liquide en phase inverse. Une stratégie analytique employant un des algorithmes a été développée pour réaliser une analyse de spectrométrie de masse ciblée afin de découvrir les isomères ayant été manqués par la méthode d’analyse conventionnelle.

Pirólise termoquímica de pós da fibra de coco seco em um reator de cilindro rotativo para produção de bio-óleo

This master thesis aims at developing a new methodology for thermochemical degradation of dry coconut fiber (dp = 0.25mm) using laboratory rotating cylinder reactor with the goal of producing bio-oil. The biomass was characterized by infrared spectroscopy with Fourier transform FTIR, thermogravimetric analysis TG, with evaluation of activation energy the in non-isothermal regime with heating rates of 5 and 10 °C/min, differential themogravimetric analysis DTG, sweeping electron microscopy SEM, higher heating value - HHV, immediate analysis such as evaluated all the amounts of its main constituents, i.e., lignin, cellulose and hemicelluloses. In the process, it was evaluated: reaction temperature (450, 500 and 550oC), carrier gas flow rate (50 and 100 cm³/min) and spin speed (20 and 25 Hz) to condensate the bio-oil. The feed rate of biomass (540 g/h), the rotation of the rotating cylinder (33.7 rpm) and reaction time (30 33 min) were constant. The phases obtained from the process of pyrolysis of dry coconut fiber were bio-oil, char and the gas phase non-condensed. A macroscopic mass balance was applied based on the weight of each phase to evaluate their yield. The highest yield of 20% was obtained from the following conditions: temperature of 500oC, inert gas flow of 100 cm³/min and spin speed of 20 Hz. In that condition, the yield in char was 24.3%, non-condensable gas phase was 37.6% and losses of approximately 22.6%. The following physicochemical properties: density, viscosity, pH, higher heating value, char content, FTIR and CHN analysis were evaluated. The sample obtained in the best operational condition was subjected to a qualitative chromatographic analysis aiming to know the constituents of the produced bio-oil, which were: phenol followed by sirigol, acetovanilona and vinyl guaiacol. The solid phase (char) was characterized through an immediate analysis (evaluation of moisture, volatiles, ashes and fixed carbon), higher heating value and FTIR. The non-condensing gas phase presented as main constituents CO2, CO and H2. The results were compared to the ones mentioned by the literature.