The feasibility of Fourier transform infrared (FTIR)-imaging spectroscopy as a tool to discriminate samples from patients suffering from benign prostatic hyperplasia (BPH) and prostate cancer (CaP) samples in blood serum was investigated. Prostate cancer is known to be an age related disease, with the risk of developing the disease dramatically increasing in men past forty years old. Currently the PSA blood test is notoriously unreliable and is non specific for CaP thus leading to overtreatment of the disease. It is important therefore to develop diagnostic method that is non-invasive, reliable, and specific for CaP.In order to achieve the objective of establishing a robust protocol, which could be applied to a clinical study, obtaining optimal sample preparation for the FTIR analysis of serum smears, had to be achieved. A protocol was developed to prepare the serum samples prior to their FTIR analysis. First, the samples were centrifuged with ultrafiltration devices of different sizes to obtain several fractions which were then smeared to obtain thin films of serum. The spectra from the larger (>100 kDa components) and medium (containing the 10–100 kDa components) fractions were utilised for both a pilot and a clinical study, while the spectra from the smaller fractions (containing the 3–10 and <3 kDa components) were affected by fringing and could therefore not be used. A major novelty of this project involved the application of FTIR-imaging to the analysis of serum smears. The use of the Focal Plane Array detector system enabled the collection of a spectral image containing 16,384 spectra, on which a Quality Testing and pre-processing techniques were applied to select the “good spectra” and reject the spectra that failed the Quality Test. Several types of substrates were assessed to determine the most appropriate for the analysis of the smears and it was established that the spectra obtained from the serum smeared on CaF2 windows gave the most reproducible results. 5 BPH and 5 CaP samples were analysed for the pilot study following the developed protocol. While no clear separation was observed in the Principal Component Analysis (PCA) plots between the BPH and the cancerous samples, a trend emerged throughout the results, with the CaP samples clustering together and the BPH samples scattered around them. A larger clinical study was conducted with 60 BPH samples and 60 CaP samples. PCA was applied on the “good spectra” and while the over 100 kDa fraction did not show a clear separation between the two types of samples, the 10–100 kDa fraction showed a distinct classification between the BPH and CaP samples. An artificial neural network was then applied to create a model using patients from the database used for the PCA analysis to determine whether the discrimination between the two types of samples could be increased or highlight different classification trends. For the >100 kDa fraction, the sensitivity value was calculated to be 97.8% and the specificity value was calculated to be 44.3% while the sensitivity and the specificity value for the 10 to 100 kDa fraction were calculated to be 78.9% and 60% respectively. A complementary study using mass spectrometry was carried out on healthy and diseased samples to identify the components contained within the different fractions and determine whether they could be correlated with the components identified from the spectral features of the FTIR data. While no quantitative information was obtained from this study, the components found in the different fractions were identified, confirming the results of the FTIR studies.

Le taux de mortalité associé aux infections par le S. aureus résistant à la méticilline (SARM) est le plus élevé parmi les infections staphylococciques. Le SARM est aussi la plus fréquente des bactéries multirésistantes, plaçant souvent le médecin en situation d'impasse thérapeutique. Une meilleure compréhension des phénomènes de résistance aux antibiotiques permettrait de mieux détecter ces bactéries et concevoir de nouveaux antibiotiques. Dans ce contexte, ces travaux de thèse visaient à étudier le phénotype de résistance à la méticilline de S. aureus par une approche métabolomique.La première partie de ce travail a été dédiée au développement des outils nécessaires aux analyses métabolomiques globales de S. aureus. Ces développements ont impliqué la mise place d'un protocole de préparation d'échantillon spécifique et multi-étapes, de méthodes de chromatographie liquide couplée à la spectrométrie de masse à haute résolution (LC/HRMS) et l'implémentation d'une base de données spectrales dédiée.Dans ces conditions, nous avons pu extraire et identifier jusqu'à 210 métabolites intracellulaires, ce qui représente la plus large couverture métabolique de S. aureus obtenue à ce jour. Dans un second temps, nous avons appliqué l'approche développée à l'étude de souches SARM et de souches sensibles à la méticilline (SASM). Après optimisation rigoureuse des conditions de culture, l'étude approfondie de 24 souches cliniques nous a permis de dégager une signature métabolique du phénotype de la résistance à la méticilline, signant notamment l'implication des voies de biosynthèse de la paroi et de la capsule bactérienne.Le séquençage complet du génome de ces 24 souches combiné à l'étude complémentaire de 16 souches de SARM de fonds génétiques différents nous a ensuite permis de souligner la pertinence de cette signature métabolique vis-à-vis du phénotype étudié. Enfin, l'étude de souches isogéniques en présence d'antibiotique a également confirmé l'implication de la synthèse de la paroi dans la distinction SARM/SASM. / The mortality rate associated with Methicillin resistant S. aureus (MRSA) is the highest among the staphylococcal infections. A better understanding of antibiotic resistance phenomena would lead to better detect these bacteria and design new antibiotics. In this context, this PhD work aimed to study the MRSA phenotype by using a metabolomics approach. The first part of this work has been dedicated to developing a global metabolomic analysis of S. aureus: i.e., the setting up of a reliable sample preparation protocol, the development of liquid chromatography-high resolution mass spectrometry methods, and the implementation of a dedicated spectral database. Under these conditions, 210 metabolites have been extracted and identified in bacterial cell extracts, which is the widest metabolic coverage of S. aureus obtained to date.Secondly, we have performed a metabolomic study of MRSA and methicillin susceptible S. Aureus (MSSA) strains. The analysis of 24 clinical strains has allowed us to highlight a metabolic signature of MRSA phenotype of which metabolites involved in bacterial wall and capsule biosynthesis pathways were part. The complete genome sequencing of these 24 strains combined with the complementary study of 16 MRSA strains of different genetic backgrounds have reinforced the relevance of this metabolic signature. Finally, the study of isogenic strains in the presence of an antibiotic has confirmed the involvement of metabolites of the wall and capsule biosynthesis pathways in the distinction of MRSA/MSSA phenotypes.

Předložená dizertační práce s názvem “Rastrovací elektronová mikroskopie a její aplikace pro senzitivní vzorky” pojednává o problematice rastrovací elektronové mikroskopie v kontextu instrumentálního a metodologického vývoje vedoucího k inovativnímu řešení, které je dobře aplikovatelné zejména v mikrobiologickém výzkumu. Součástí práce je rozprava o historii a současném stavu elektronové mikroskopie (EM) jakožto vědecké zobrazovací a analytické techniky, tato část se nachází v úvodních kapitolách. Nepopiratelný přínos EM v biologických a lékařských oborech je dokazován mnoha citovanými vědeckými publikacemi. Předložená dizertační práce přináší novinky z oblasti přípravy preparátů a kryogenní rastrovací elektronové mikroskopie (cryo-SEM) vyvinuté na pracovišti Ústavu přístrojové techniky AV ČR, v.v.i. v Brně. Jedná se především o návrhy a výrobu speciálních držáků vzorků a vývoj nových metodik v oblasti přípravy mikrobiologických preparátů vedoucích k nalezení optimálních parametrů jednotlivých procesů. V experimentální části se nachází ověření metodologických postupů při studiu hydratovaných a na elektronový svazek senzitivních preparátů. Následné srovnání různých přístupů na definovaném biologickém systému z oblasti mikrobiologie přispívá k rozšíření interpretace doposud známých výsledků. Mezi zkoumanými mikrobiologickými kmeny byly biofilm-pozitivní bakterie Staphylococcus epidermidis a kvasinky jako Candida albicans a Candida parapsilosis, jež jsou považovány za klinicky významné, protože se podílejí na vzniku závažných infekcí zejména u imunokompromitovaných pacientů. Dále byl studován vliv růstu biofilmu bakterie Bacillus subtilis na biodeteriorizaci a biodegradaci poly--kaprolaktonových fólií. Vývoj v oblasti cryo-SEM byl aplikován ve výzkumu mikrobů s biotechnologickým potenciálem, jako jsou např. Cupriavidus necator a Sporobolomyces shibatanus.

"Benzo Fury" compounds and derivatives are enactogens similar to 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in various aspects. These compounds are similar in structure to MDMA and MDA, as well as, elicit similar effects such as elevated mood, euphoria and hallucinations. This similarity in effect increases the potential for abuse as MDMA has become less prevalent in some regions as the use of these new psychoactive substances (NPSs) has increased. The benzofury compounds are used as legal alternatives to MDMA because of their marketing as “not for human consumption”. With the relative ease in obtaining NPSs via the Internet, it is possible that these drugs may soon be prevalent in the United States. The project’s goal was to separate, detect, and quantitate the benzofury compounds and derivatives as well as MDA and MDMA in one method of analysis using ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). The project also examined which method of sample preparation is more effective for these compounds. Six benzofury compounds were researched: 5-(2-aminopropyl)benzofuran) (5-APB), 6-(2-aminopropyl)benzofuran) (6-APB), 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB), 6-(2-aminopropyl)-2,3-dihydrobenzofuran (6-APDB), 1-(benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) and 1-(benzofuran-6-yl)-N-methylpropan-2-amine (6-MAPB) as well as MDMA and MDA. These drugs were analyzed in blood. A liquid-liquid extraction (LLE) method and solid phase extraction (SPE) method were examined to determine which would be better for the separation, detection and quantitation of the benzofury compounds. For the development of the overall method, accuracy, precision, calibration curve, carryover, limit of detection, limit of quantitation, analyte stability, and recovery were examined. The accuracy of the methods examined was greater than +/- 20%. For most analytes, the precision within-run and between-run did not exceed 20%, regardless of the sample preparation method used. A weighting of 1/x was applied to the calibration curve regardless of sample preparation method utilized. The carryover was less than 2% with the SPE method having less carryover (0.02% to 0.50%) than the LLE method (0.05% to 1.56%). The limit of quantitation was determined to be greater than 10 ng/mL. While this was unexpected, the limit of detection calculations determined that this was correct. Using the LLE method in combination with the UFLC-MS/MS method developed, the limit of detection was determined to be at least 9.98 ng/mL. Compared to the LLE method, the SPE limit of detection was lower and calculated to be 3.75 ng/mL. The percent recovery was examined for each of the analytes. It was determined that the SPE was capable of recovering 80% or more of the benzofury compounds and derivatives regardless of the concentration level. The LLE was not as successful in the recovering the benzofury compounds, the best recovery occurred at the 200 ng/mL level with only 65% or less recovered. Analyte stability exhibited a general decrease with variation prior to day 7 and then remains relatively stable until day 14. It was anticipated that the quantitation of the drugs might be complicated due to the similarity in structure between the isomers as well as the similarity of structure between all of the compounds. While this may still be the case, the difficult separation resulted in a re-evaluation and alterations to the UFLC-MS/MS method to correct for these issues. With the change in the UFLC-MS/MS method, further method optimization is required to achieve the appropriate accuracy and limit of quantitation. It was found that the best combination of sample preparation and detection of the benzofury compounds and derivatives is to use SPE followed by an UFLC-MS/MS method.

No description available.

No description available.

Atualmente as ag&ecirc;ncias reguladoras est&atilde;o focadas no controle de res&iacute;duos de antimicrobianos, pesticidas entre outros que s&atilde;o prejudiciais no meio ambiente, sa&uacute;de humana e animal e que podem ser encontrados em alimentos, &aacute;gua de rio entre outros, como por exemplo, res&iacute;duos de sulfonamidas (SAs) no leite bovino. A quantifica&ccedil;&atilde;o desses compostos em amostras complexas requer o preparo da amostra, extra&ccedil;&atilde;o, pr&eacute;concentra&ccedil;&atilde;o dos analitos e elimina&ccedil;&atilde;o dos interferentes que s&atilde;o necess&aacute;rios para o desenvolvimento de m&eacute;todos cromatogr&aacute;ficos. Por isso, &eacute; vantajoso que se utilize m&eacute;todos simples, r&aacute;pidos, baixo consumo de reagente, alta sensibilidade, maior precis&atilde;o anal&iacute;tica e automa&ccedil;&atilde;o das an&aacute;lises. Neste contexto, este estudo explora uma das t&eacute;cnicas de preparo de amostra baseadas em sorvente mais empregadas a SPE na sua vertente automatizada, SPE on-line. Recentemente, o uso de novos sorventes mais seletivos t&ecirc;m recebido grande interesse como os l&iacute;quidos i&ocirc;nicos (ILs) imobilizados na s&iacute;lica. Diferentes abordagens para a s&iacute;ntese da s&iacute;lica modificada por ILs foram estudadas. O sorvente sintetizado pela rea&ccedil;&atilde;o sol-gel atrav&eacute;s via cat&aacute;lise b&aacute;sica apresentou maior efici&ecirc;ncia de extra&ccedil;&atilde;o para as SAs. Os resultados obtidos por espectroscopia vibracional na regi&atilde;o do infravermelho e microscopia eletr&ocirc;nica de varredura sugerem que o IL foi ancorado &agrave; s&iacute;lica. O sorvente baseado em s&iacute;lica modificada por IL ([C4MIM] [PF6]) foi selecionada como fase extratora para o isolamento e pr&eacute;-concentra&ccedil;&atilde;o de SAs em leite bovino utilizando um sistema SPE on-line -LC-ESIQToF / MS. O m&eacute;todo desenvolvido mostrou um LOQ de 5-7,5 &micro;g mL-1, enquanto que a faixa de recupera&ccedil;&atilde;o relativa da extra&ccedil;&atilde;o foi de 74-93%. A faixa de precis&atilde;o e exatid&atilde;o intra e inter-dia obtida foi de 1,5-12,5 e 2,3-13,1, respectivamente. Outra vertente explorada neste trabalho foi o uso de colunas tubulares abertas (OT) em cromatografia l&iacute;quida. At&eacute; agora, as colunas OT foram usadas principalmente para an&aacute;lise de pept&iacute;deos e prote&iacute;nas, e seu potencial para a separa&ccedil;&atilde;o de pequenas mol&eacute;culas ainda n&atilde;o foi totalmente explorado. Preparou-se uma coluna de SPE de 5 cm em um tubo capilar aberto composto por 84 canais (10 &#956;m de di&acirc;metro interno (id) cada) de um capilar de fibra de cristal e a parede interna de cada canal foi revestida com um pol&iacute;mero org&acirc;nico -OD &ndash;DVB &ndash; mPLOT SPE). A mPLOT SPE foi acoplada com uma coluna tubular aberta PS-DVB de 10 &#956;m x 2 m com detec&ccedil;&atilde;o em espectrometria de massa por nanospray (OTLC-MS) no modo positivo. Num estudo comparativo com colunas de SPE monol&iacute;ticas e empacotadas a mPLOT SPE-OTLC melhorou significativamente a pr&eacute;concentra&ccedil;&atilde;o e o formato do pico das SAs e seus metabolitos (~ 300 Da). A baixa backpressure e a geometria da mPLOT SPE combinada com a filtra&ccedil;&atilde;o autom&aacute;tica (AFFL). / Nowadays health regulatory agencies are focusing on the presence of residues of antimicrobials, pesticides among other that are harmful to the environment, human and animal health that are found on food, river water among others, as example, the residues of sulfonamides (SAs) in bovine milk. The quantification of these compounds in complex matrices requires the sample preparation, extraction and preconcentration of the analytes, besides the elimination of the interferents for the development of chromatographic methods high sensitivity and analytical selectivity. Therefore, it is advantageous to use simple, fast methods that exhibit high selectivity, low reagent consumption, high sensitivity, higher analytical precision and automation of analyzes. In this context, this study explores one of the most employed sample preparation techniques, solid phase extraction (SPE) on automatization approach, the on-line SPE. Recently, the use of new sorbents more selective have received great interest as the ionic liquids (ILs) immobilized on the silica surface. Different approaches to the synthesis of silica modified by ILs were studied. The sorbent synthesized by sol-gel methodology through basic catalysis showed higher extraction efficiency for SAs. The results obtained by infrared spectroscopy and scanning electron microscopy suggested that IL was anchored to the silica surface. The silica modified by IL ([C4MIM] [PF6]) was selected as sorbent for the isolation and preconcentration of SAs in bovine milk using an on-line SPE system -LC-ESI-QToF / MS. The method showed a LOQ of 5-7.5 &#956;g mL-1, while the relative extraction recovery range was 74-93%. The accuracy and precision intra- and inter-day range was 1.5-12.5 and 2.3-13.1, respectively. Another aspect explored was the use of open tubular column (OT) for separation of small molecules. OT columns have been used primarily for peptide and protein analysis, and their potential for small molecules separation has not yet been fully explored. A 5 cm SPE column (mPLOT) was prepared in an open capillary tube composed by 84 channels (10 &#956;m inner diameter (id) each) of a crystal fiber capillary and the inner wall of each channel was coated by organic polymer (PS-OD&ndash;DVB). The mPLOT was coupled with a 10 &#956;m x 2 m PS-OD-DVB open tubular column with positive mode nanospray mass spectrometry (OTLC-MS) detection. Compared to the use of monolithic / particulate SPE columns, SPE-OTLC mPLOT significantly improved the preconcentration and peak forms of SAs and their metabolites (~ 300 Da). Low backpressure, mPLOT geometry and the combination with automatic filtration (AFFL) was essential to sample preparation and separation of small molecules on complex matrices.

O objetivo deste trabalho foi o desenvolvimento de métodos visando às determinações de As, Cd, Cr(III), Cr(VI), Hg, Ni, Pb e Se em fertilizantes minerais por espectrometria de absorção atômica com atomização em forno de grafite (GF AAS) e espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES). Foi empregado Material de Referência Certificado de fertilizante inorgânico (SRM® 695, NIST) e amostra de candidato a Material de Referência Rastreado (MRR), mix-micro, cedida pelo LANAGRO de Goiânia. A análise da amostra MRR, obtida por fluorescência de Raios-X (XRF), mostrou a complexidade dessa matriz devido, principalmente à presença de elevados teores de Al, Si, Fe, Ti, Mn e Ca. Foram feitas otimizações do ICP OES visando condições robustas para determinações de As, Cd, Cr, Ni e Pb em soluções com nebulização direta e As, Hg e Se com nebulização combinada com geração de vapor. Foi verificado que os métodos de preparo de amostras, 3051 e 3052 recomendados pela USEPA (Agência de Proteção Ambiental dos Estados Unidos), podem ser aplicados apenas para determinação de As, Cd e Pb. Elementos fortemente ligados a matriz, como Cr e Ni, não foram quantitativamente extraídos. A digestão pelo método 3052 na presença de H3BO3 alterou o erro relativo de -36% para -15% para o Cr e a concentração de Ni também foi maior nesse caso. Para a determinação simultânea de As, Hg e Se no ICP OES por geração de vapor (VG-ICP OES), foi utilizado um procedimento de pré-redução para Se(VI), envolvendo aquecimento a 90°C das soluções em meio de HCl 6 mol L-1. A etapa adicional para redução de Se não prejudicou as determinações de As e Hg. A geração de vapor para a determinação simultânea dos elementos ocorreu com a separação gás-líquido dentro da câmara ciclônica. A extração usando banho e sonda ultrassônica, na presença de ácidos diluídos (HCl, HNO3, H3PO4), não influenciou consideravelmente as determinações de As, Cd, Cr, Ni e Pb por ICP OES e Se por GF AAS. O borbulhamento de ar atmosférico foi suficiente para uma eficiência de extração comparável aos dispositivos ultrassônicos. A utilização de pequenos volumes de ácidos concentrados mostrou ser adequada para melhorar a eficiência de extração, principalmente para o Ni. O HCl concentrado foi a condição mais adequada para a extração. Com relação à determinação de Cr(VI), foram estudadas diferentes soluções extratoras alcalinas e, além do uso do banho termostático, foi avaliada a extração utilizando ultrassom. Devido à complexidade das amostras de fertilizantes, estratégias foram adotadas para adequar o método da difenilcarbazida a essa matriz. Além da espectrofotometria de absorção molecular na região do visível, a determinação do teor de Cr(VI) nos extratos foi feita por GF AAS e as concentrações de Cr(VI) estavam abaixo do limite de detecção do método. / The aim of this work was the development of methods for the determination of As, Cd, Cr(III) Cr(VI), Hg, Ni, Pb and Se in mineral fertilizers by graphite furnace atomic absorption spectrometry (GF AAS) and inductively coupled plasma optical emission spectrometry (ICP OES). It was employed a Certified Reference Material (SRM® 695 - Trace elements in multi-nutrient fertilizer) from the National Institute of Standards and Technology (NIST) and an in-lab candidate of Reference Material (MR), mix-micro sample (raw material used in formulation of mineral fertilizers to improve micronutrient content) provided by LANAGRO (Laboratório Nacional Agropecuário, Goiania, Brazil). The analysis of MR sample by X-ray fluorescence (XRF) showed the matrix complexity, mainly due to the presence of high levels of Al, Si, Fe, Ti, Mn and Ca. The ICP OES optimization was carried out aiming rubust conditions for As, Cd, Cr, Ni and Pb determination by direct nebulization solutions, and As, Hg and Se with nebulization combined with vapor generation. It was found that the methods of sample preparation, USEPA 3051 and 3052 (US Environmental Protection Agency), can be applied only for the determination of As, Cd and Pb. Elements that are strongly bound to the matrix, such as Cr and Ni were not quantitatively extracted. Digestion by method USEPA 3052 in the presence of H3BO3 alter the relative error from -36% to -15% for Cr and Ni concentration was also higher in this case. For the simultaneous determination of As, Hg and Se by ICP OES vapor generation (VG-ICP OES), a pre-reduction procedure for Se (VI) was necessary, involving heating at 90 °C of the solutions in HCl 6 mol L-1. The additional step for Se reduction did not impair Hg and As determination. The vapor generation for simultaneous elements determination with the gas-liquid separation occurring within the cyclonic spray chamber. The extraction using bath and ultrasonic probe in the presence of dilute acids (HCl, HNO3, H3PO4) did not influence considerably the determination of As, Cd, Cr, Ni and Pb by ICP OES and Se by GF AAS. Only atmospheric air bubbling was enough for an efficient extraction providing results comparable to ultrasonic devices. The use of small volumes of concentrated acids proved to be suitable for improving extraction efficiency, particularly for Ni. Concentrated HCl was the most suitable condition for extraction. For Cr(VI) determination, various solutions and alkaline extractant was studied, and the use of the thermostatic bath and ultrasound bath was evaluated. Due to the complexity of fertilizer samples, strategies were adopted to adapt diphenylcarbazide method to this matrix. Besides Cr(VI) determination in extracts by molecular absorption spectrophotometry in the visible region, it was also performed by GF AAS and Cr(VI) concentration was below limit of detection of the method.

O objetivo deste trabalho foi o desenvolvimento de métodos visando às determinações de As, Cd, Cr(III), Cr(VI), Hg, Ni, Pb e Se em fertilizantes minerais por espectrometria de absorção atômica com atomização em forno de grafite (GF AAS) e espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES). Foi empregado Material de Referência Certificado de fertilizante inorgânico (SRM® 695, NIST) e amostra de candidato a Material de Referência Rastreado (MRR), mix-micro, cedida pelo LANAGRO de Goiânia. A análise da amostra MRR, obtida por fluorescência de Raios-X (XRF), mostrou a complexidade dessa matriz devido, principalmente à presença de elevados teores de Al, Si, Fe, Ti, Mn e Ca. Foram feitas otimizações do ICP OES visando condições robustas para determinações de As, Cd, Cr, Ni e Pb em soluções com nebulização direta e As, Hg e Se com nebulização combinada com geração de vapor. Foi verificado que os métodos de preparo de amostras, 3051 e 3052 recomendados pela USEPA (Agência de Proteção Ambiental dos Estados Unidos), podem ser aplicados apenas para determinação de As, Cd e Pb. Elementos fortemente ligados a matriz, como Cr e Ni, não foram quantitativamente extraídos. A digestão pelo método 3052 na presença de H3BO3 alterou o erro relativo de -36% para -15% para o Cr e a concentração de Ni também foi maior nesse caso. Para a determinação simultânea de As, Hg e Se no ICP OES por geração de vapor (VG-ICP OES), foi utilizado um procedimento de pré-redução para Se(VI), envolvendo aquecimento a 90°C das soluções em meio de HCl 6 mol L-1. A etapa adicional para redução de Se não prejudicou as determinações de As e Hg. A geração de vapor para a determinação simultânea dos elementos ocorreu com a separação gás-líquido dentro da câmara ciclônica. A extração usando banho e sonda ultrassônica, na presença de ácidos diluídos (HCl, HNO3, H3PO4), não influenciou consideravelmente as determinações de As, Cd, Cr, Ni e Pb por ICP OES e Se por GF AAS. O borbulhamento de ar atmosférico foi suficiente para uma eficiência de extração comparável aos dispositivos ultrassônicos. A utilização de pequenos volumes de ácidos concentrados mostrou ser adequada para melhorar a eficiência de extração, principalmente para o Ni. O HCl concentrado foi a condição mais adequada para a extração. Com relação à determinação de Cr(VI), foram estudadas diferentes soluções extratoras alcalinas e, além do uso do banho termostático, foi avaliada a extração utilizando ultrassom. Devido à complexidade das amostras de fertilizantes, estratégias foram adotadas para adequar o método da difenilcarbazida a essa matriz. Além da espectrofotometria de absorção molecular na região do visível, a determinação do teor de Cr(VI) nos extratos foi feita por GF AAS e as concentrações de Cr(VI) estavam abaixo do limite de detecção do método. / The aim of this work was the development of methods for the determination of As, Cd, Cr(III) Cr(VI), Hg, Ni, Pb and Se in mineral fertilizers by graphite furnace atomic absorption spectrometry (GF AAS) and inductively coupled plasma optical emission spectrometry (ICP OES). It was employed a Certified Reference Material (SRM® 695 - Trace elements in multi-nutrient fertilizer) from the National Institute of Standards and Technology (NIST) and an in-lab candidate of Reference Material (MR), mix-micro sample (raw material used in formulation of mineral fertilizers to improve micronutrient content) provided by LANAGRO (Laboratório Nacional Agropecuário, Goiania, Brazil). The analysis of MR sample by X-ray fluorescence (XRF) showed the matrix complexity, mainly due to the presence of high levels of Al, Si, Fe, Ti, Mn and Ca. The ICP OES optimization was carried out aiming rubust conditions for As, Cd, Cr, Ni and Pb determination by direct nebulization solutions, and As, Hg and Se with nebulization combined with vapor generation. It was found that the methods of sample preparation, USEPA 3051 and 3052 (US Environmental Protection Agency), can be applied only for the determination of As, Cd and Pb. Elements that are strongly bound to the matrix, such as Cr and Ni were not quantitatively extracted. Digestion by method USEPA 3052 in the presence of H3BO3 alter the relative error from -36% to -15% for Cr and Ni concentration was also higher in this case. For the simultaneous determination of As, Hg and Se by ICP OES vapor generation (VG-ICP OES), a pre-reduction procedure for Se (VI) was necessary, involving heating at 90 °C of the solutions in HCl 6 mol L-1. The additional step for Se reduction did not impair Hg and As determination. The vapor generation for simultaneous elements determination with the gas-liquid separation occurring within the cyclonic spray chamber. The extraction using bath and ultrasonic probe in the presence of dilute acids (HCl, HNO3, H3PO4) did not influence considerably the determination of As, Cd, Cr, Ni and Pb by ICP OES and Se by GF AAS. Only atmospheric air bubbling was enough for an efficient extraction providing results comparable to ultrasonic devices. The use of small volumes of concentrated acids proved to be suitable for improving extraction efficiency, particularly for Ni. Concentrated HCl was the most suitable condition for extraction. For Cr(VI) determination, various solutions and alkaline extractant was studied, and the use of the thermostatic bath and ultrasound bath was evaluated. Due to the complexity of fertilizer samples, strategies were adopted to adapt diphenylcarbazide method to this matrix. Besides Cr(VI) determination in extracts by molecular absorption spectrophotometry in the visible region, it was also performed by GF AAS and Cr(VI) concentration was below limit of detection of the method.

Les stratégies de protéomique quantitative sans marquage sont très attractives dans le domaine de la recherche de biomarqueurs de pathologies. Cependant, elles requièrent une pleine maîtrise du schéma analytique et de sa répétabilité. Plus particulièrement, la préparation d’échantillons nécessite d’être suffisamment répétable pour ne pas impacter la qualité et la fiabilité des résultats. Les objectifs de cette thèse étaient de développer et d’optimiser des méthodes analytiques pour la protéomique quantitative, en particulier pour l’étape de préparation d’échantillons. Ainsi, un protocole innovant, simple, rapide et permettant l’analyse quantitative sans marquage d’un grand nombre d’échantillons avec une haute répétabilité a été développé et optimisé : le « Tube-Gel ». Par ailleurs, des préparations d’échantillons adaptées à différentes matrices biologiques pour la recherche de biomarqueurs ont été élaborées. Les méthodes mises au point et leur application ont permis de proposer des candidats biomarqueurs pour plusieurs pathologies : le glioblastome, les lymphomes B diffus à grandes cellules, et les complications survenant sur les greffons rénaux. / Label-free quantitative proteomics strategies are very attractive for diseases biomarkers researches. These approaches require the full control and the repeatability of the analytical workflow. In particular, the sample preparation has to be repeatable enough to ensure the quality and reliability of the results. Objectives of this work were to optimize and develop analytical methods for quantitative proteomics, with a special focus on the sample preparation step. Thus, an innovative, easy and fast protocol allowing the analysis of high sample numbers with high repeatability was developed and further optimized: the “Tube-Gel” protocol. Besides,sample preparations adapted to a variety of biological matrices were developed for the search of biomarkers. The developed methods and their application allowed the identification of potential biomarkers for a variety of diseases: glioblastoma, diffuse large B-cell lymphomas and renal transplants failures.