A química de produtos naturais da família Asteraceae tem sido foco de estudo do Núcleo de Pesquisa em Produtos Naturais e Sintéticos (NPPNS) da FCFRP-USP que relatou diversas novas moléculas, com destaque para a classe das lactonas sesquiterpênicas. Para essas substâncias já foram atribuídas diversas atividades farmacológicas tais como antioxidante, anti-inflamatória, antimicrobiana, analgésicas e tripanossomicida. A atividade antitumoral da lactona sesquiterpênica goyazensolido (GOYA) foi avaliada no NPPNS e esse estudo revelou uma atividade farmacológica interessante paras as linhagens de tumor cutâneo e cerebral. Com isso, viu-se nesta classe de substâncias uma oportunidade para explorar o potencial antitumoral assim como sua distribuição e metabolismo. Para compreender melhor a distribuição dessa substância na pele, o modelo in vitro de penetração utilizando células de Franz e pele de orelha de porco como membrana foi aplicado. Para esse estudo um método de geração de imagem MALDI-MSI para avaliação da distribuição do GOYA na pele, assim como, um método por UPLC-MS/MS foram desenvolvidos. Para o desenvolvimento e validação do método de MALDI-MS as substâncias doxorrubicina e minoxidil, com estudos de penetração já estabelecidos, foram utilizadas. Para avaliação da distribuição de GOYA em sistema nervoso central (SNC) um modelo em insetos, utilizando gafanhotos foi aplicado. Nesse experimento os gafanhotos receberam a dose de 500 ?M de GOYA, e foram coletadas amostras de hemolinfa, fezes e cérebro nos tempos de 15 e 30 minutos, assim como amostras de gafanhoto total. Para determinação de GOYA nas amostras de gafanhoto um método quantitativo por UPLC-MS/MS e um método de geração de imagem por DESI-MSI foram desenvolvidos. Com os estudos de penetração cutânea pudemos concluir que MALDI-MSI foi capaz de confirmar a distribuição de minoxidil nas amostras de pele, no entanto, não se mostrou uma técnica eficaz para determinação de doxorrubicina. A técnica de MALDI-MSI, em adição ao método de UPLC-MS/MS foi capaz de revelar que o GOYA não penetrou na pele estando acumulado na sua camada superior, provavelmente no estrato córneo. Nos ensaios de distribuição em SNC foi possível observar através do método de UPLC-MS/MS que o GOYA está presente no cérebro, hemolinfa e fezes do gafanhoto. Com isso podemos concluir que o modelo utilizado é um bom modelo de predição de permeação a barreira-hematoencefálica, bem como para estudos de metabolismo. Conclui-se também que o método desenvolvido para essa finalidade foi adequado. A técnica de DESI-MS apesar de não gerar resultados positivos para permeação cerebral revelou a presença de GOYA no intestino do animal no tempo de 30 minutos, o que caracteriza uma rápida eliminação de GOYA do organismo / The Natural Products\' Chemistry has been the focus of the \"Núcleo de Pesquisa em Produtos Naturais e Sintéticos\"(NPPNS) at School of Pharmaceuthical Science of Ribeirão Preto, University of São Paulo (FCFRP- USP). NPPNS reported a variety of unknown molecules from Asteraceae family, highlighting the sesquiterpene lactones (STL). STL showed important pharmacological activities, such as antiinflammatory, antimicrobial, analgesic and trypanossomicide. The STL goyazensolide (GOYA) exhibit antitumor activity for skin and central nervous system (CNS) cancer cell lines . With that the research group saw in this class of compounds a chance to explore the antitumor potential as well as unveil its distribution and metabolism. For a better understanding of the distribution of this compound in the skin, the Franz cell in vitro model using ear pig skin was applied. For that, a MALDI-MSI method was developed to assess the distribution of GOYA in the skin, along with a UPLC-MS/MS method, to confirm the results. In order to develop and validate a MALDI-MSI method, doxorubicin and minoxidil, known substances in the cutaneous penetration studies, were used. An insect model with locust was applied for the investigation of GOYA distribution in CNS. The locust received a 500 ?M GOYA dose and hemolymph, brain and feces samples were collected in 15 and 30 minutes, as well as the entire locust. In order to assess GOYA in the locust samples, an UPLC-MS/MS method was developed, and for distribution in the entire locust a DESI-MSI was developed. The MALDI-MSI method developed for cutaneous penetration study was able to confirm the results for minoxidil experiments and allowed us to see the distribution of this compound in the skin. Unfortunately for doxorubicin MALDI-MSI by the is source analythe dissociation. The MALDI-MSI and the UPLC-MS/MS was able to show that GOYA does not permeate the skin, but is in the skin, probably interacting with the stratum corneum barrier. In the CNS studies we could see through the UPLC-MS/MS method that GOYA is present in the brain, hemolymph and feces in the in vivo model. With that we can conclude that the in vivo insect model is a good alternative for the metabolism and blood-brain-barrier studies. Also we can conclude that, although the DESI-MSI technique was not suitable for CNS permeation studies, it can be applied for metabolism studies, as it revealed the presence of GOYA in the intestine with a 30 minutes experiment, what characterizes a fast distribution and elimination of GOYA in the living organism

No / No Abstract

L’agence européenne du médicament (EMA) stipule que les entreprises pharmaceutiques se doivent d’améliorer continuellement le contrôle de leur production afin de garantir la qualité des médicaments et préserver la santé des patients. Les outils analytiques classiques sont, par exemple, capables d’examiner l’intégralité d’un comprimé pharmaceutique pour contrôler la qualité et la quantité des substances actives utilisées et estimer leurs profils de libération dans l’organisme. Ils ne permettent cependant pas de cartographier la distribution des composés chimiques pourtant considérée comme un critère important pour la qualité du médicament. Des techniques d’imagerie chimique comme la MSI MALDI sont donc utilisées afin de déterminer par une analyse unique la répartition spatiale et la structure des composés constituant les médicaments. Toutefois, la MSI MALDI nécessite une préparation des échantillons relativement complexe et génère des données de grande taille difficilement exploitables. Ces caractéristiques, ainsi que l’absence de spectromètre de masse adapté à l’analyse de formes pharmaceutiques solides, complexifient la mise en place de la MSI MALDI au sein de laboratoires industriels. Les travaux réalisés durant cette thèse ont eu pour objectifs d’améliorer le protocole de préparation des échantillons, d’optimiser le système d’acquisition et de mettre en place les outils chimiométriques et informatiques nécessaires à l’analyse des images MALDI au sein de l’entreprise Technologie Servier. Les développements réalisés et les résultats obtenus ont finalement permis la résolution de problématiques inexplicables jusqu’alors par les techniques analytiques classiques. / European Medicines Agency (EMA) recommendations stipulate that pharmaceutical companies have to continually improve manufacturing efficiency to ensure drug product quality. The commonly used analytical tools provide information about drug substance quality and dosage or the drug release profile by dissolving the whole tablet. However these analytical tools are not able to highlight the distribution of chemical compounds contained in the tablet. This is why chemical imaging such as MALDI MSI are used to extract the spatial and spectral information from pharmaceutical solid dosage forms. This hyperspectral imaging technique needs complex sample preparation and generates huge dataset. These two features, as well as the lack of optimized mass spectrometers to study tablets, make difficult the implementation of the MALDI MSI in industrial laboratories. During this thesis, the sample preparation protocol has been improved, the mass spectrometer has been optimized to analyze tablets and chemometrics tools has been developed in order to implement MALDI MSI within Technologie Servier company.

Yes / Failure of systemic cancer treatment can be, at least in part, due to the drug not being delivered to the tumour at sufficiently high concentration and/or sufficiently homogeneous distribution; this is termed as “pharmacokinetic drug resistance”. To understand whether a drug is being adequately delivered to the tumour, “precision pharmacology” techniques are needed. Mass spectrometry imaging (MSI) is a relatively new and complex technique that allows imaging of drug distribution within tissues. In this review we address the applicability of MSI to the study of cancer drug distribution from the bench to the bedside. We address: (i) the role of MSI in pre-clinical studies to characterize anti-cancer drug distribution within the body and the tumour, (ii) the application of MSI in pre-clinical studies to define optimal drug dose or schedule, combinations or new drug delivery systems, and finally (iii) the emerging role of MSI in clinical research. / Spanish Medical Oncology Society (SEOM) for contribution with a grant for research abroad of Dr. Jove, “Instituto Carlos III” for contribution with a “Río Hortega” Grant (nº CM17/00008) for Dr. Jove

Submitted by MARCEL PRATAVIEIRA null (marcelprata93@hotmail.com) on 2016-03-07T15:52:49Z No. of bitstreams: 1 Dissertação - Marcel Pratavieira - 2016.pdf: 8130455 bytes, checksum: a459e73ed4ef0757431f12802bc7f15b (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-07T19:51:13Z (GMT) No. of bitstreams: 1 pratavieira_m_me_rcla.pdf: 8130455 bytes, checksum: a459e73ed4ef0757431f12802bc7f15b (MD5) / Made available in DSpace on 2016-03-07T19:51:13Z (GMT). No. of bitstreams: 1 pratavieira_m_me_rcla.pdf: 8130455 bytes, checksum: a459e73ed4ef0757431f12802bc7f15b (MD5) Previous issue date: 2016-02-23 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As abelhas têm sido utilizadas como modelos robustos e influentes para o estudo de memória e aprendizagem, contribuindo para o melhor entendimento das bases da cognição. Nesse contexto, diferentes metabólitos foram caracterizados por desempenharem funções distintas no processo de aprendizagem e formação de memória em insetos. Considerando que pouco se sabe sobre os metabólitos em relação ao desenvolvimento das habilidades cognitivas em A. mellifera, ou mesmo em relação aos comportamentos reflexos (condicionados e/ou não condicionados), o presente estudo teve como objetivo a análise metabolômica do cérebro de abelhas submetidas ao ensaio comportamental de reflexo de extensão de probóscide (REP). Para isto, foi padronizada a técnica de análise metabolômica com o uso do sistema LC-ESI- MS e MSn, construindo-se inicialmente uma biblioteca de compostos característicos de cérebro de abelhas (neurotransmissores, aminoácidos livres, poliaminas, nucleotídeos, nucleosídeos, ácidos orgânicos, etc). Nesta primeira abordagem, dentre os 112 compostos da biblioteca, 48 foram identificados e quantificados; alguns destes compostos foram únicos para o grupo controle (cadaverina, espermina, glicose, uracila e n-acetil-L-glutamato 5-semialdeido), enquanto que outros foram únicos para o grupo REP (fenilalanina, betaína, espermidina, serina e creatina). Dentre os compostos identificados em ambos os grupos, apenas 5 compostos apresentaram diferenças quantitativas estatisticamente significantes (arginina, asparagina, guanosina monofosfato, putrescina e 4-guanitinobutanoato). Visando o estudo dos perfis metabólicos regionais (em cada região do cérebro), foi também padronizado o protocolo experimental utilizando-se a estratégia MALDI Spectral Imaging e o desenvolvimento de um método semi-quantitativo de metabólitos. Esta estratégia permitiu o mapeamento e o estudo da distribuição espacial dos metabólitos identificados em cortes de cérebro de abelhas, bem como uma melhor compreensão da distribuição dessas moléculas nas diferentes estruturas do cérebro e sua correlação com o comportamento ensaiado. As duas estratégias aplicadas mostraram-se complementares e fundamentais para a compreensão da cognição em abelhas. De modo geral, o ensaio de REP parece estimular intensa atividade cerebral, alto gasto energético, intensa sinalização química e a ativação de algumas cascatas metabólicas específicas, tais como a rota metabólica da prolina e arginina. Nos indivíduos do grupo REP a arginina provavelmente foi catabolizada nas sínteses de creatina, 4-guanidinobutanoato, putrescina e espermidina. Esses processos bioquímicos provavelmente foram importantes para coordenar o reconhecimento da molécula de sacarose, e associar esse odor com um comportamento reflexo para a extensão da probóscide (estímulo não condicionado). / The honeybee Apis mellifera has long served as an invertebrate model organism for learning and memory research, contributing to a better understanding of cognition bases. In this context, different metabolites (especially neurotransmitters) were characterized by play distinct roles in learning process and in memory formation in insects. Whereas little is known about the metabolites in relation to the development of cognitive skills in A. mellifera, the present study aims to perform a metabolomic analysis of the honeybee brains submitted to the behavioral test of proboscis extension reflex (PER). For this, has been standardized a metabolomic analysis technique through the use of LC-ESI-MS and MSn system. Initially a low molecular weight compounds library was created, containing characteristic compounds of bee brain (neurotransmitters, free amino acids, polyamines, nucleotides, nucleosides, organic acids, etc.). In this first approach, from the library of 112 compounds, 48 compounds were identified and quantified; some of these compounds were only identified in the control group (cadaverine, spermine, glucose, uracil and N-acetyl-L-glutamate 5- semialdehyde), while others were only identified in PER group (phenylalanine, betaine, spermidine, serine and creatine). Among the compounds identified in both groups, only five compounds showed statistically significant differences in quantitative results (arginine, asparagine, guanosine monophosphate, putrescine and 4-guanidinebutanoate). In order to study the metabolic profiles by regions (within each brain region) it was also standardized an experimental protocol using a novel semi-quantitative method of MALDI Spectral Imaging strategy. This strategy allowed the study and the mapping of the spatial distribution of metabolites identified in honeybee brain sections, as well as a better understanding of the distribution of molecules in the different brain structures and their correlation with the behavior tested. Both strategies applied proved to be complementary and essential to the understanding of cognition in bees. Overall, in this study the PER test seems to stimulate intense brain activity, high energy expenditure, intense chemical signaling and activation of some specific metabolic pathways, such the arginine and proline metabolic pathway. In individuals of PER group arginine was probably catabolized in the synthesis of creatine, 4-guanidinobutanoato, putrescine and spermidine. This biochemical process were probably important to coordinate the recognition of sucrose molecule and associate this odor with a reflex behavior for the proboscis extension (unconditioned stimulus). / FAPESP: 2014/05376-1

No description available.

Despite the significant progress in medicine, infectious diseases are life-threatening thanks to an increasing number of multiresistant strains of microorganisms and late detection of pathological agents. An opportunistic fungus Aspergillus fumigatus cause respiratory system diseases called aspergillosis. The invasive pulmonary aspergillosis affects immunocompromised patients after inhalation of ubiquitous conidia of A. fumigatus and results in 450,000 deaths per year. The biofilm formation in the infected tissue protects A. fumigatus against antimicrobial drugs. Late therapy may not be effective. Infection of immunocompromised patients and biofilm formation is characteristic also for gram negative bacteria Pseudomonas aeruginosa, which is due to the production of many factors of virulence and multiresistance a dreaded opportunistic pathogen. Scanning electron microscopy (SEM) provides detail information about morphology of microorganisms with the resolution in range of tens of nanometers that allows to observe microorganisms in the infected tissue and its pathological changes. Mass spectrometry allows to detect infection and its course based on identification of characteristic microbial molecules. The aim of this study was to optimize sample preparation of tissues infected with A. fumigatus or P....

No / AQ4N (banoxatrone) (1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione) is an example of a bioreductive prodrug in clinical development. In hypoxic cells AQ4N is reduced to the topoisomerase II inhibitor AQ4 (1,4-bis- {[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione). By inhibition of topoisomerase II within these hypoxic areas, AQ4N has been shown to sensitise tumours to existing chemo- and radiotherapy treatments. In this study the distribution of AQ4N and AQ4 in treated H460 human tumour xenografts has been examined by imaging matrix-assisted laser desorption/ionisation mass spectrometry. Images of the distribution of AQ4N and AQ4 have been produced that show little overlap. The distribution of ATP in the tumour xenografts was also studied as an endogenous marker of regions of hypoxia since concentrations of ATP are known to be decreased in these regions. The distribution of ATP was similar to that of AQ4N, i.e. in regions of abundant ATP there was no evidence of conversion of AQ4N into AQ4. This indicates that the cytotoxic metabolite AQ4 is confined to hypoxic regions of the tumour as intended.

The work presented herein is a method optimization for biomolecule detection and identification using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI). MALDI-MSI is a unique form of mass spectrometry that is highly multiplexed; it can simultaneously retain location information of the mass of multiple ions, allowing for correlation of morphology or pathology to reconstructed ion heat maps. There were three main objectives for the research - 1) A method optimization of sample preparation techniques for bottom-up proteomic MALDI-MSI was performed. This included the optimization of tissue wash steps, trypsin digestion incubation times, and matrix deposition techniques. The results included identifying the appropriate pH for the wash steps to optimize trypsin digestion, an overnight trypsin incubation to allow for complete digestion, and the inclusion of MCAEF – Matrix Coating Assisted by an Electric Field – during matrix coating for enhanced spectra. 2) An unbiased statistical data processing workflow for simultaneous processing of multiple datasets was performed. This was done using a thyroid hormone treated tadpole dataset to gain insight into the metabolism of anuran metamorphosis. Results found included a finalized data processing workflow that detected 5000 metabolite features from five organs were detected in pre-metamorphic tadpoles. Of these detected metabolites, 136 were significantly affected upon exposure to thyroid hormone and 64 metabolites were putatively identified. 3) A sample preparation technique for metabolomic analysis of formalin-fixed paraffin embedded (FFPE) colorectal liver metastasis samples was performed. Results included the importance of using a high mass resolution mass spectrometer while emphasizing more appropriate use of fresh-frozen tissue sections for metabolomic analysis. / Graduate