This thesis includes studies on N-, mucin type O-, and glycosaminoglycan (GAG)-linked glycosylation in human biospecimens. Glycosylation plays a central role in biological processes, including protein folding, immune surveillance, and regulation of cell growth. The structures of GAG are regulated in a tissue-specific manner. Heparan sulfate (HS) and chondroitin sulfate (CS) are the two types of GAGs targeted in this thesis. Human leukocytes express both CS and HS GAGs with CS being the more abundant type; however, little is known regarding the properties and structures of GAG chains, their ranges of variability among normal subjects, and changes in structure associated with disease conditions. We measured the relative and absolute disaccharides abundances of HS and CS for purified B, T, NK cells, monocytes, and polymorphonuclear leukocytes (PMNs) using size exclusion chromatography-mass spectrometry (SEC-MS). We found that all leukocytes express HS chains with levels of sulfation more similar to heparin than to organ-derived HS. In addition, CS abundances varied considerably in a leukocyte cell type specific manner. Therefore, our results established the ranges of GAG structures expressed on normal leukocytes as well as necessary for subsequent inquiry into disease conditions. Glioblastoma (GBM) accounts for 30% of human primary brain tumors. It is deadly and highly invasive. In past decades, most GBM research focused on pathophysiological changes in genome. There remains a dearth of knowledge regarding alterations in glycomics, glycoproteomics, and proteomics during GBM tumorigenesis. Therefore, we developed a comprehensive platform for high-throughput sample preparation with surface digestion for tissue microarrays, LC-MS/MS data dependent acquisition, and semi-automated data analysis to integrate glycomics, glycoproteomics, and proteomics for different grade of tumor and different subtypes of GBM. By analyzing GBM tissue microarrays, we found tumor grade and subtype specific changes to the expression of biomolecules. We also identified approximately 100 site-specific N- and mucin type O-glycosylations, the majority of which were previously unreported. Overall, our results improved the fundamental understandings about GBM pathogenesis. / 2018-11-02T00:00:00Z

Synthetic drugs such as piperazines are among the most commonly abused drugs and are typically consumed by younger populations. Because of their popularity, developing optimized analytical strategies designed to improve detection and interpretation of synthetic piperazines is of interest to the forensic community. To improve the likelihood that a substance of interest is detected, careful evaluation into the mass spectrometry signal is required. However, with all analytical pursuits, there is a limit at which the substance cannot be detected with certainty; thus a threshold is commonly referred to as the limit of detection (LOD). Formally, the LOD is the minimum amount of analyte (concentration, mass, number of molecules, etc.) that can be detected at a known confidence level. The purpose of this research was to use common analytical methods to calculate the LOD and verify the results with previous work at the Boston University forensic toxicology laboratory. Data from the Liquid Chromatography-tandem Mass Spectrometer (LC-MS/MS) was previously generated and consisted of signal intensity information in the form of peak height and peak area, from titrations of eight synthetic piperazines that included: Benzylpiperazine (BZP), 1-(3-chlorophenyl)-piperazine (mCPP), 3-trifluoromethylphenylpiperazine monohydrochloride (TFMPP), methylbenzylpiperazine (MBZP), 1-(4-fluorobenzyl)-piperazine (FBZP), 2,3-dichlorophenylpiperazine (DCPP), para-fluorophenylpiperazine (pFPP) and para-methoxyphenylpiperazine (MeOPP). Generally, the LOD is determined by first evaluating the signal in the absence of analyte and determining the probability that signal, , crosses the signal threshold, . The signal threshold is based upon the false detection rate the laboratory can withstand for a given interpretation scheme. In instances where very small levels of false detections can be tolerated, a large is chosen. In other circumstances, where noise detection can adequately be interpreted, a low is chosen. In chromatography and radiography the typical one sided =0.003. The number of molecules for each analyte at each concentration (20 ng/mL, 50 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL) was determined and used throughout this work. Peak area signals and ratios versus number of molecules for each analyte were used to, first, visually inspect the linearity of signal to analyte level. It was determined that using internal standards improved linearity, as expected; however, the data suggested that absolute signal intensity was sufficient to compute the LOD for these compounds. Generally accepted methods of calculating LOD were not used for this research as the signal from the blank was not detected most likely due to the sensitivity of the instrument used. This study used an extrapolation of the data and propagation of errors method to calculate the LOD as the signal from the blank was not needed. For all eight analytes, the LOD calculated was similar to the lowest concentration (20 ng/mL) used when validating this method. This research needs to be expanded on to include more concentration points and see the plateau effect at higher concentrations. This will provide information to analytical chemists when a blank signal is not available about how the LOD can be calculated with high confidence.

Innate immune cells, such as macrophages, are trained by the unique microenvironment of the tissue they occupy. This tissue influence can include the extracellular matrix, the presence of inflammatory stimuli or signals and, in some tissues, the microbiota. Most studies, however, have examined such tissue specific training in health whereas little is known about the possibility of immune re-training in the lung following acute inflammation. The lung extracellular matrix is important for mechanical stability and structural support, as well as influencing inflammation via altering cell adhesion, migration, survival, proliferation and differentiation. Matrix alterations are a feature of a number of significant chronic respiratory diseases that carry high clinical unmet need. These include idiopathic pulmonary fibrosis, cystic fibrosis and chronic obstructive pulmonary disease (COPD). On the other hand, the impact on matrix after acute inflammation and whether it is returned to its pre-infection state is relatively unexplored. In murine models, macroscopic examination of the lung following acute inflammation implies a return to a reasonable homeostatic state. However, using more sensitive techniques, we now show that this is not the case. In this thesis we test the premise that a more thorough interrogation of lung extracellular matrix by mass spectrometry will reveal long term alterations that are not visible by histology. After influenza virus infection, we demonstrate that heightened extracellular matrix persists in the lung tissue, often forming structures that were not present in health. Furthermore, basement membrane components, for example collagen IV and laminin, are reduced. In vitro investigations show that individual extracellular matrix components affect lung macrophage activity. For example, hyaluronan and fibronectin alter macrophage expression of microRNA species known to influence toll-like receptor responsiveness and fibrosis. We also describe an alteration in microRNA species in response to influenza virus infection as well as a non-infectious model of pulmonary inflammation using carbon nanotubes. Collectively, this implies that altered matrix composition impacts on the inflammatory tone of the lung innate immune system. It is therefore feasible that such changes following severe lung inflammation could be overcome by targeting abnormal matrix production or degradation.

The contamination of 316L stainless steel surfaces within an electrospray ionisation source of a mass spectrometer is investigated. An accelerated method of contamination is used. Following initial test method development and investigation of the contamination resulting on the ion transfer components (sample cone, outer cone and extraction cone), flat samples are employed within the ionisation source. This enables characterisation of the contamination composition, morphology and build-up with time. Blood plasma is introduced into the mass spectrometer as it is a widely analysed substance that is known to result in contamination. The contamination from a mixture of human blood plasma, diluted in methanol, and a water/acetonitrile mobile phase is found to contain inorganic NaCl crystals embedded in a matrix of organic residues. The morphology shows self-organising features as the contamination builds. A model is proposed to explain the morphology, involving rapid evaporation of the droplets that impinge on the stainless steel surface. Two types of surface modification are considered for the stainless steel: electrochemically grown films and coatings deposited by vapour deposition. A method for electrochemical film growth is developed, enabling nanoporous films to be formed on the stainless steel in 5 M sulphuric acid at 60°C by square wave pulse polarisation between active or transpassive and passive potentials. The films are characterised using glow discharge optical emission spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, Rutherford backscattering spectroscopy and nuclear reaction analysis. The films are shown to be chromium- and molybdenum-rich relative to the substrate, and to consist mainly of sulphates, oxides and hydroxides. The morphology and composition of the films are discussed in relation to the polarisation conditions and mechanism of film formation. A range of vapour deposited coatings are considered: TiN, TiC, TiB2, Graphit-iC, and diamond-like carbon coatings with Si and N2 dopants and with varying sp2:sp3 ratios. In addition, a hydrophobic coating is deposited on the stainless steel by immersion, in order to provide a significant variation in surface energy. Surface analysis of the coatings is carried out, considering their sp2:sp3 ratios, their electrical conductivities, their water contact angle, and the various components of the surface energy. The contamination build-up on the surface of uncoated 316L stainless steel is compared with that on stainless steel with the various surface modifications. A method for quantification of the build-up of contamination on flat samples is developed using white light interferometry. The surface modifications which result in the slowest contamination build-up with time are then applied to the ion transfer components of the mass spectrometer. The robustness of the mass spectrometric response for the selected coated surfaces is compared with that of the uncoated stainless steel. The electrochemically grown films and two of the doped diamond-like carbon coatings are found to be successful in reducing the build-up of contamination.

The biophysical properties, biological activity and function of macromolecular systems are highly dependent on their structure. Structure-activity relationships of proteins and their binding partners are critical for drug discovery, biochemical and medical research. While the gas-phase environment might present as an unusual venue from which to explore protein structure, for over the past two decades, nano-electrospray ionization (nESI) coupled to mass spectrometry (MS) has been recognized as having great potential for analysis of protein structure and protein non-covalent complexes. In conjunction with related technique of ion mobility (IM), mass spectrometry (IM-MS) provides insights into protein native-like conformations and any structural changes in may undergo upon ligand binding or alternations induced via physical parameters such as temperature, pressure or solution conditions. As most proteins tend to exist as multiple domains; from the distribution of oligomeric states in the Protein Data Base (PDB) 86% of proteins exist as oligomers; the work presented in this thesis focuses on application of MS techniques to probe the tertiary and quaternary structure of various large and multimeric protein complexes, their dynamics and/or conformational changes. Wherever relevant, the gas-phase studies reported here are complemented by other techniques, such as hydrogen deuterium exchange MS (HDX), molecular modelling (MD) and analytical ultracentrifugation (AUC). Firstly, the dynamics of intact monoclonal antibodies (mAbs) and their fragments are explored with IM-MS. Variations observed in conformational landscapes occupied by two mAb isotypes are rationalized by differences in disulfide linkages and non-covalent interactions between the antibody peptide chains. Moreover, mAb intrinsic flexibility is compared to other multimeric protein complexes in terms of collision cross section distribution span. Secondly, variable temperature MS (VT-MS) and variable temperature IM-MS (IM-MS) are used to probe unfolding and dissociation of four standard multimeric protein complexes (TTR, avidin, conA and SAP) as a function of the of analysis environment temperature. VT-MS is found to allow for decoupling of their melting temperature (Tm) from the protein complex dissociation temperature (TGPD). Whereas, VT-IM-MS is used to investigate structural changes of these protein complexes at elevated temperatures and provide insights into the thermally induced dissociation (TID) mechanism, as well as strength of the non-covalent interactions between subunits. Thirdly, VT-(IM)-MS methodology is applied to study behaviour of three mAbs: IgG1, IgG4 and an engineered IgG4 of increased thermal stability. Such analysis shows to be promising for comparative thermal stability studies for proteins of therapeutic interest. Lastly, the structure of ATP-phosphoribosyltransferase (MtATPPRT), an enzyme catalysing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, is explored. Conformational changes occurring upon feedback allosteric inhibition by L-histidine are probed with MS, IM-MS, HDX-MS and AUC. Reported results serve as the basis for IM-MS/HDX-MS based screening method to be used for screening of a library of novel and promising anti-tuberculosis agents.

O metabolismo celular é substancialmente alterado durante a oncogênese, progressão tumoral e outros processos patológicos. Tem sido frequentemente analisado para compreensão dos processos que permitem o crescimento dos tecidos, reprodução, manutenção da homeostase e resposta a sinais extracelulares. Dentre os vários métodos para caracterização de alterações metabólicas, a espectrometria de massas tem contribuído significativamente para a identificação e quantificação de proteínas envolvidas no metabolismo, e também para a caracterização do metaboloma. A análise proteômica baseada em espectrometria de massas permite estudos qualitativos em grande escala, adequados para a busca e descoberta de analitos relevantes. A análise proteômica dirigida complementa esse caráter com qualidade quantitativa para proteínas alvo pré-selecionadas. Neste trabalho foram desenvolvidos métodos de proteômica dirigida para o monitoramento de alterações quantitativas nos níveis de proteínas envolvidas na via glicolítica do metabolismo da glicólise. Para tal peptídeos proteotípicos para cada proteína foram identificados e padronizados utilizando a estratégia de monitoramento de reações múltiplas. O painel foi aplicado para obter resultados das alterações que ocorrem em um modelo de progressão tumoral. Com esta estratégia empregada, foi possível selecionar e utilizar vários peptídeos representativos das enzimas da via glicolítica e também de algumas proteínas relevantes ao câncer. A utilização de peptídeos sintéticos facilitou substancialmente o processo de desenvolvimento do método. Por fim, com a metodologia desenvolvida, foi demonstrado para células MCF7, que o fator EGF alterou a expressão das proteínas da via glicolítica, aumento no fluxo para via das pentoses e capacidade aumentada da respiração celular. Este estudo, portanto, sugere uma nova disposição do metabolismo celular dado o conhecimento estabelecido em relação aos efeitos na respiração, como o efeito Warburg. / Cellular metabolism is altered during ontogenesis, cancer progression and several other pathological events. Because of that, the metabolism is constantly analyzed in order to provide further comprehension of processes that allow tissue growth, reproduction, homeostasis maintenance, and response to extracellular signals. Among the methods great diversity of methods used to characterize metabolic alterations, mass spectrometry has been contributing significantly to identify and quantify proteins involved in a diversity of metabolic pathways, but also to monitor the changes in metabolome. Proteomics based on mass spectrometry allows highthroughput in-depth qualitative resources for discovery phases stages, providing the new relevant candidates for further biochemical characterization. In a complementary way, targeted proteomics allows precise quantitative analyses of such selected protein targets. Here, it was developed a targeted proteomics method for multiplex monitoring glycolytic pathway enzymes and relevant cancer-related proteins. For that, proteotypic peptides representing proteins of interest were selected and studied in detail to be incorporated in a multiple reaction monitoring assay. The developed method was applied to monitor the alterations in the glycolytic pathway in a cancer progression model. Using targeted proteomics strategies, we selected and applied for quantification several proteotypic peptides representing glycolitic enzymes and cancerrelated proteins. The use of synthetic peptides allowed faster method development and more sensitive methods. The application of such methods, demonstrated alterations in glycolytic pathways and cancer-related proteins promoted in MCF7 cells treated with EGF. Also, an activation of pentose-phosphate pathway was suggested and increase in cellular oxygen consumption. This study, therefore, suggests changes in the cellular metabolism that differs from the classic Warburg effect observed during cancer development.

Made available in DSpace on 2015-08-20T17:10:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-17. Added 1 bitstream(s) on 2015-08-20T17:25:51Z : No. of bitstreams: 1 000841833_20170417.pdf: 281313 bytes, checksum: 8f2aa9fdfa5ffa94e65eb440978fe805 (MD5) Bitstreams deleted on 2017-04-17T13:37:16Z: 000841833_20170417.pdf,. Added 1 bitstream(s) on 2017-04-17T13:38:04Z : No. of bitstreams: 1 000841833.pdf: 2174199 bytes, checksum: 085bf6e5fb3ed50c4ce7758cc704e700 (MD5) / Terminalia catappa Linn. pertence à Família Combretaceae e é comumente chamada amendoeira-da-praia ou chapéu de sol. A família Combretaceae reúne numerosas espécies que têm sido estudadas por seu uso pela população para fins medicinais. É originária da Malásia, mas, muitas vezes, cultivada e muito característica da costa brasileira. A partir do extrato hidroalcoólico das folhas, foram detectados como constituintes principais a substância majoritária punicalagina (anômeros α e β), o composto punicalina, ácido galágico e ácido elágico. Identificouse, também, as substâncias 1,2,3-Tri-O-galoil-β-D-glicose e possíveis isômeros, 1,6- Di-O-galoil-β-D-glicose, HHDP-acetilglicosídeo, HHDP-hexosídeo, ácido elágico hexosídeo, ácido galágico e apigenina 8-C-(2'-galoil)-β-D-glicopiranosil. Estes metabolitos foram confirmados por experimentos FIA-ESI-IT-MSn (Direct Flow Analysis-Electrospray ionization-Íon Trap-Tandem Mass Spectrometry) and High Performance Liquid Chromatography (HPLC) acoplada a arranjo de fotodiodos (PDA). Ácido elágico, ácido gálico e galato de metila foram confirmados por experimentos de co-injecção de padrão. Punicalagina foi detectada e isolada da fração hidrometanólica como uma mistura de anômeros e confirmada por experimentos de RMN 1H e 13C, mono e bidimensionais. O fracionamento da fração acetato proveniente do extrato hidroalcoólico por MPLC (Medium Pressure Liquid Chromatography) permitiu o isolamento dos metabólitos ácido galágico e apigenina 8-C-(2'-galoil)-β-D-glicopiranosil. A configuração absoluta dos anômeros da punicalagina e do composto apigenina 8-C-(2'-galoil)-β-D-glicopiranosideo foi estabelecida por experimentos de dicroísmo circular (DC). A determinação quantitativa dos principais metabolitos secundários foi realizada por HPLC-PDA, possibilitando, assim, a determinação da concentração e teor (%) de... / Terminalia catappa Linn. belongs to Combretaceae family and is commonly called almond-the-beach or sun hat.The Combretaceae family gathers numerous species that has been studied for its use by the population for medicinal purposes. It is from Malaysia, but often cultivated and very characteristic of the Brazilian coast. From the alcoholic extract of the leaves were detected as main constituents the major substance punicalagin (α and β anomers), the punicalin compound, gallagic acid and ellagic acid. It was identified as well, the 1,2,3-Tri-O-galloyl-β-D-glucose substances, and possible isomers, 1,6-Di-O-galloyl-β-D-glucose, HHDP acetilglicoside, HHDPhesoside, ellagic acid hexoside, gallagic acid and apigenin 8-C (2 '-galloyl)-β-Dglucopyranosyl. These metabolites were confirmed by FIA-ESI-IT-MSn experiments (Direct Flow Analysis Electrospray ionization-Ion-Trap Tandem Mass Spectrometry) and High Performance Liquid Chromatography (HPLC) coupled with diode array (PDA). Ellagic acid, gallic acid and methyl gallate were confirmed by standard coinjection experiments. Punicalagin was detected and isolated from the hydromethanol fraction as a mixture of anomers experiments and confirmed by 1H and 13C NMR, mono- and bi-dimensional. The ethyl acetate fraction from fractionation of the hydroalcoholic extract by MPLC (Medium Pressure Liquid Chromatography) allowed the isolation of galágico acid metabolites and 8 apigenin-C (2'- galloyl) -β-Dglucopyranosyl. The absolute configuration of anomers of the compound of punicalagin and apigenin 8-C (2'-galloyl) -β-D- glucopyranosyl was established by experiments circular dichroism (CD). Quantitative determination of the main secondary metabolites was performed by HPLC-PDA, thus allowing the determination of concentration and content (%) of metabolites present in the alcoholic extract. They were first quantified in hydroalcoholic extract of this species, the...

Made available in DSpace on 2014-11-10T11:09:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-20Bitstream added on 2014-11-10T11:57:25Z : No. of bitstreams: 1 000781145.pdf: 6824903 bytes, checksum: c08fff186aec6d4637f211665ab36871 (MD5) / O objetivo central desse trabalho foi sintetizar novas arquiteturas supramoleculares homo- e heterometálicas direcionadas para o reconhecimento molecular de hóspedes a partir da estratégia da auto-montagem envolvendo íons de metais de transição e ligantes exodentados. A primeira parte deste trabalho consistiu na síntese e caracterização espectroscópica de complexos polinucleares homometálicos inéditos a partir da auto-montagem entre as subunidades angulares [PdCl2(N N)] {N N = N,N,N´,N´-tetrametiletilenodiamina (tmen), 2,2´-bipiridina (bpy), 1,10-fenantrolina (fen)} e o ligante tridentado 1,3,5-tris(4- piridil)imidazol (tpyHImz). Os sólidos [{Pd(bpy)}6(tpyHImz)4]Cl12·6H2O (1B), [{Pd(tmen)}6(tpyHImz)4]Cl12·30·H2O (2B) e [{Pd(phen)}6(tpyHImz)4]Cl12·6H2O (3B) são estáveis às condições ambientes e solúveis em dmso. Já a espécie [Pd6(μ-Pz)6(μ-bpym)3] (4B) foi formada a partir da reação de auto-montagem entre o bloco de construção linear [Pd2(μ- bpym)Cl4] (bpym = 2,2’-bipirimidina) e o ânion pirazolato (Pz). Estas espécies foram caracterizadas pelas técnicas de análise elementar, espectroscopia vibracional no IV, RMN, espectrometria de massas (ESI-MS). A partir da análise dos dados espectroscópicos e dos resultados provenientes dos cálculos quânticos ab initio baseados na teoria do funcional de densidade (DFT), foi possível propor as estruturas das espécies 1B-4B. Para os compostos 1B-3B, sugere-se a formação de espécies supramoleculares cíclicas discreta catiônicas A2 6A3 4 em que seis subunidades angulares ditópicas [Pd(N N)]+2 (N N = tmen, bpy, phen) atuam como vértices e quatro subunidades angulares tritópicas tpyHImz (A3 4) formariam as arestas da nanoestrutura. No caso da espécie 4B, espera-se uma supramolécula hexanuclear de topologia triangular neutra (A2 6L4 3) em que três subunidades lineares bpym (L4) atuam como arestas enquanto que os grupos “Pd(μ-Pz)2Pd” (A2) formariam os... / The main goal of this work was to synthesize new homo- and heterometallic supramolecular structures with molecular recognition properties self-assembled from metal ions and exodentate ligands. The first part involved the preparation and spectroscopic characterization of polynuclear homometallic complexes obtained from self-assembly reactions between angular subunits [PdCl2(N N)] {N N = N,N,N´,N´-tetramethylethylenediamine (tmen), 2,2´-bipyridine (bpy), 1,10-phenanthroline (fen)} and the tridentate ligand 1,3,5-tris(4- pyridyl)imidazole (tpyImz). The solids [{Pd(bpy)}6(tpyHImz)4]Cl12·6H2O (1B), [{Pd(tmen)}6(tpyHImz)4]Cl12·30·H2O (2B) e [{Pd(phen)}6(tpyHImz)4]Cl12·6H2O (3B) are air stable and sluble in dmso. The compound [Pd6(μ-Pz)6(μ-bpym)3] (4B) was self-assembled from the linear building block [Pd2(μ-bpym)Cl4] (bpym = 2,2’-bipyrimidine) and pyrazolate anion (Pz). These polynuclear species were characterized bi elemental analyses, IR and NMR spectroscopies and mass spectrometry (ESI-MS). From the inspection of spectroscopic data and DFT calculations, structural models for compounds 1B-4B were proposed. For complexes 1B-3B, it was suggested the formation of cyclic metallosupramolecules A2 6A3 4 in which six angular ditopic subunits [Pd(N N)]+2 (N N = tmen, bpy, phen) act as vertices whereas four tritopic subunits tpyHImz (A3 4) give rise to the sides of the nanostructure. In the case of 4B, it is expected a hexanuclear triangular-shaped metallomacrocycle (A2 6L4 3) in which the core “Pd(μ-Pz)2Pd” acts as vertices of the triangle whereas the sides are composed by three bischelating 2,2’-bipyrimidine ligands. The second part of this work was carried out in collaboration with Universidade de Barcelona. A series of heterometallic metallamacrocycles have been constructed from self-assembly reactions between the Au(I) organometallic compounds [(AuPy)2(μ2-diphosphane)] {diphosphane = 1,2-bis(diphenylphosphanyl)ethane (dppe), ...

Made available in DSpace on 2015-03-03T11:52:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-28Bitstream added on 2015-03-03T12:07:18Z : No. of bitstreams: 1 000806902_20150828.pdf: 529126 bytes, checksum: 0ba8a9d77911f516559558294b02f40f (MD5) Bitstreams deleted on 2015-08-28T16:08:57Z: 000806902_20150828.pdf,. Added 1 bitstream(s) on 2015-08-28T16:10:00Z : No. of bitstreams: 1 000806902.pdf: 2067465 bytes, checksum: 40c8b4a1cf5b96c8d6428c73cac91c66 (MD5) / A abordagem reducionista na busca por substâncias bioativas em produtos naturais nem sempre é capaz de compreender em sua totalidade a dinâmica envolvida nas respostas farmacológicas de matrizes complexas como extratos vegetais. Inserido neste paradigma, este trabalho visou ao desenvolvimento de métodos e abordagens racionais que permitissem o entendimento das relações moleculares em produtos naturais bioativos. Para tanto, espécies vegetais da família Chrysobalanaceae com potencial citotóxico, depositadas na extratoteca do NuBBE, foram os objetos de estudo na aplicação de metodologias para a determinação de sua composição metabólica e para a avaliação do potencial citotóxico. Os extratos hidroalcoólicos de Couepia grandiflora, Hirtella hebeclada, Licania hoehnei, Licania kunthiana, Licania huminis e Parinari excelsa foram analisados por CG-EM, CLAE-DAD-EM/EM e CLAE-autoEM/EM para a identificação in situ de sua composição metabólica, através de comparação dos dados espectrais a uma base de dados contendo valores de massas de alta resolução e espectros de UV de todos os metabólitos secundários já relatados para a família Chrysobalanaceae. O uso de ferramentas de análise multivariada, como a deconvolução empírica proposta pelo software AMDIS e a extração espectral desenvolvidas pelos algoritmos RAMSY e MCR-ALS, assistiram à desreplicação ao extrair os espectros dos compostos, mesmo em baixa resolução cromatográfica. A desreplicação das espécies de Chrysobalanaceae permitiu a detecção in situ de metabólitos primários (aminoácidos, ácidos e açúcares) e secundários (flavonoides-O-glicosilados e polímeros de proantocianidinas). Adicionalmente ao estudo de determinação metabólica das matrizes vegetais, realizaram-se ensaios de atividade citotóxica. Resultados preliminares da bioatividade ante diferentes linhagens de Saccharomyces cereviceae, obtidos... / The reductionist approach for the search of bioactive compounds in natural products is not always being able to understand the complete dynamics involved in the pharmacological responses of complex matrices such as plant extracts. Based on this paradigm, this study aimed to develop rational methods approaches to runderstand the molecular relationships present in bioactive natural products. Chrysobalanaceae plant species with cytotoxic potential, deposited NuBBE`s bank of extracts, were the objects of study in the application of methodologies for defining the metabolic composition and evaluate their cytotoxic potential. Couepia grandiflora, Hirtella hebeclada, Licania hoehnei, Licania kunthiana, Licania huminis and Parinari excelsa ethanol extracts were analyzed by GC-MS, HPLC-DAD-MS/MS and HPLC-autoMS/MS for in situ metabolic identification based on spectral comparison with a high resolution mass spectra and UV spectral database developed using previously reported data for Chrysobalanaceae. The use of multivariate analysis tools, the empirical deconvolution software AMDIS and the spectral extraction algorithms RAMSY and MCR-ALS assisted the dereplication by extracting spectral data for pure compounds even at low chromatographic resolution. The dereplication of Chrysobalanaceae species allowed the in situ detection of primary metabolites (amino acids, and sugars) and secondary (O-glycosylated-flavonoids and proanthocyanidins polymers). In addition to the metabolic determination of Chrysobalanaceae extracts, were performed cytotoxicity bioassays. Preliminary results of cytotoxicity against Saccharomyces cereviceae, carried out during the construction of the bank of extracts, indicated cytotoxic potential, especially for Hirtella hebeclada and Parinari excelsa. However, the cytotoxic assay against different tumor cell lines showed that the extracts were not active, which may be related to which may be...

Des exporteurs « ATP-Binding Cassette » (ABC) sont connus pour être responsables de la résistance contre un large spectre d'agents antibiotiques ou chimiothérapeutiques chez les bactéries et les cellules de mammifères. L'échange hydrogène / deutérium associé à la spectrométrie de masse (HDX-MS) a été utilisé pour caractériser les changements conformationnels de deux exporteurs ABC bactériens, BmrA et BmrC/BmrD, en présence et en absence de nucléotide. Les cinétiques locales d'HDX ont montré la nature hautement dynamique des domaines intra-cellulaires (ICDs) dans la forme apo, ce qui n'était pas attendu d'après les structures cristallographiques aux rayons X des protéines homologues. Dans la conformation ouverte vers l'extérieur (« outward facing »), domaines fixant les nucléotides (NBDs) interagissant, le mouvement des ICDs sont largement réduits pour les deux transporteurs. Les cinétiques d'HDX MS dans la conformation « outward facing » ont été déterminées en appliquant cette technique sur des mutants incapables d'hydrolyser les nucléotides et sur la forme sauvage inhibée au vanadate. La dynamique des NBDs, en particulier pour les régions qui interagissent au cours de l'hydrolyse de l'ATP, a été aussi diminuée dans la conformation « outward facing » comparativement à celle ouverte vers l'intérieur. L'ajout de différents agents connus pour être transportés par des transporteurs ABC n'a pas affecté la dynamique des NBDs. Par ailleurs, nous avons aussi appliqué l'HDX MS à la protéine GLIC, un homologue procaryote d'un « ligand-gated ion channel » pentamérique (pLGIC). Les cinétiques locales d'HDX sont en plein accord avec la structure cristallographique disponible et le changement de pH révèle des différences de deutération dans les régions d'interaction des sous-unités. / Some ATP-Binding Cassette exporters are known to be responsible for resistance against a broad spectrum of antibiotics and chemotherapeutic drugs in bacteria and mammalian cells. Amide hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) was applied to investigate the conformational changes in two different bacterial ABC exporters, BmrA and BmrC/BmrD, in the presence and absence of nucleotide. Local H/D exchange kinetics showed highly dynamic nature of ICDs in apo form which was not anticipated from X-ray structure of the homologue proteins. In outward facing (closed form) conformation the movement of ICDs were largely reduced for both transporters. The H/D exchange kinetics of closed form were determined by applying H/D exchange on mutants unable to hydrolyze nucleotides or on wild-type inhibited by vanadate. The dynamics of NBDs particularly for those regions which interact during ATP hydrolysis were also reduced in closed form as compared to open one. The addition of different drugs which are known to be transported by ABC transporters did not affect dynamics of NBDs. We further applied H/D exchange kinetics on a prokaryotic homologue of pentameric ligandgated ion channel (pLGIC) GLIC. Local H/D exchange kinetics were in full agreement with the available structure and change in pH showed differences in deuterium level for interacting regions of the subunits.