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

Glucocorticoids are steroid hormones involved in the stress response, with a well-established role in promoting cardiovascular risk factors including obesity and diabetes. The focus of glucocorticoid research has shifted from understanding control of blood levels, to understanding the factors that control tissue steroid concentrations available for receptor activation; it is disruption of these tissue-specific factors that has emerged as underpinning pathophysiological mechanisms in cardiovascular risk, and revealed potential therapeutic targets. However, the field is hampered by the inability at present to measure concentrations of steroid within individual tissues and indeed within component cell types. This research project explores the potential for steroid measurements using mass spectrometry-based tissue imaging techniques combining matrix assisted laser desorption ionization with on-tissue derivatisation with Girard T and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (OTCD-MALDIFTICRMS). A mass spectrometry imaging (MSI) platform was developed and validated to quantify inert substrate and active product (11-dehydrocorticosterone (11DHC), corticosterone (CORT) respectively) of the glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in rodent tissues. A novel approach to derivatising keto-steroids in tissue sections using Girard T reagent was developed and validated. Signals were boosted (10⁴ fold) by formation of GirT hydrazones compared to non-derivatised neutral steroids. Active and inert glucocorticoids were detected in a variety of tissues, including adrenal gland and brain; in the latter, highest abundance was found in the cortex and hippocampus. The MSI platform was also applied to human biopsies and murine tissues for the analysis of other ketosterols such as androgens and oxysterols. Proof-of-principle validation that the MSI platform could be used to quantify differences in enzyme activity was carried out by following in vivo manipulation of 11β-HSD1. Regional steroid distribution of both substrate and product were imaged at 150-200μm resolution in mouse brain sections, and the identification confirmed by collision induced dissociation/liquid extraction surface analysis (CID-LESA). To validate the technique, the CORT/11DHC ratios (active/inert) were determined in 11β- HSD1 deficient mice and found to be reduced (KO vs WT; cortex (49 %*); hippocampus (46 %*); amygdala (57 %)). Following pharmacological inhibition by administration of UE2316, drug levels peaked at 1 h in tissue and at this time point, a reduction in CORT/11DHC ratios were also determined, although to a lesser degree than in KO mice, cortex (22%), hippocampus (25 %) and amygdala (33 %). The changes in ratios appeared driven by accumulation of DHC, the enzyme substrate. In brains of mice with 11β-HSD1 deficiency or inhibition, decreases in sub-regional CORT/11DHC ratio were quantified, as well as accumulation of an alternative 11β- HSD1 substrate, 7-ketocholesterol. MSI data correlated well with the standard liquid chromatography tandem mass spectrometry (LC-MS/MS) in whole brain homogenates. Subsequently, the MSI platform was also applied to measure the dynamic turnover of glucocorticoids by 11β-HSD1 in metabolic tissues using stable isotope tracers (Cortisol-D4 (9,11,12,12-D4) (D4F). D4F was detected in plasma, liver and brain after 6 h infusion and after 48 h in adipose. D3F generation was detected at 6 h in plasma and liver; at 24 h in brain specifically in cortex, hippocampus and amygdala; and at 48 h in adipose. The spatial distribution of d3F generation in brain by MSI closely matched enzyme localisation. In liver, an 11β-HSD1-riched tissue, substantial generation of d3F was detected, with a difference in d4F/d3F ratios compared with plasma (ᴧTTRᴧ 0.18± 0.03 (6 h), 0.27± 0.05 (24 h) and 0.38±0.04 (48 h)). A smaller difference in TTR was also detected between plasma and brain (ᴧTTR 0.09 ± 0.03 (24 h), 0.13±0.04 (48 h)), with no detectable regeneration in adipose. After genetic disruption of 11β-HSD1, d3F generation was not detected in plasma or any tissues, suggesting that 11β-HSD1 is the only enzyme carrying out this reaction. After pharmacological inhibition, a similar pattern was seen. The circulating concentration of drug peaked at 2 h and declined towards 4 h, with same pattern in liver and brain. The ᴧTTR ratios 2HPD between plasma and liver (0.27±0.08vs. 0.45± 0.04) and brain (0.11±0.2 vs. 0.19± 0.04) were smaller following drug administration than vehicle, indicating less d3F generation. Extent of enzyme inhibition in liver responded quickly to the declining drug, with ᴧTTR returning to normal by 4 h (0.38± 0.06). ᴧTTR had not normalised 4HPD in brain (0.12±0.02, suggesting buffering of this pool. In adipose, UE2316 was not detected and nor were rates of d3F altered by the drug. Two possible phase I CYP450 metabolites were identified in the brain differing in spatial distribution. In conclusion, MSI with on-tissue derivatisation is a powerful new tool to study the regional variation in abundance of steroids within tissues. We have demonstrated that keto-steroids can be studied by MALDI-MSI by using the chemical derivatisation method developed here and exemplified its utility for measuring pharmacodynamic effects of small molecule inhibitors of 11β-HSD1. This approach offers the prospect of many novel insights into tissue-specific steroid and sterol biology.

Hereditary endometrial cancer (EC) is a Lynch syndrome (LS) related cancer variant and 2-10% of all EC are hereditary. The aim of this study was to develop a method for analysis of microsatellite instability (MSI) as such analysis would assist in identifying potential LS patients with EC at an early state of their disease, before a possible second cancer is developed in another organ. Twenty-six patients with adenocarcinoma in the endometrium, diagnosed at Uppsala University Hospital in Sweden between 1993 and 2012, were included in the study. Seven of these patients were also diagnosed with LS and the rest were sporadic EC. DNA was extracted from the patients’ formalin-fixed and paraffin-embedded tissues. The extracted DNA was subjected to a multiplex PCR with fluorescently labelled primers and then analysed by using capillary electrophoresis. Of the sporadic EC, 26% was MSI-High, which correlates well with published data. Of the LS patients, 83% was MSI-High. The outcome of this project resulted in that MSI analysis is now a validated and established method used in the process of identifying potential LS among patients with EC.

PURPOSE: African Americans (AA) are at a higher risk of colorectal cancer (CRC) and some studies report a higher frequency of microsatellite instability (MSI) in this population while others report lower frequency compared to Caucasians. AIM: To determine and evaluate the association of race and clinical factors with MSI frequency through meta-analysis. METHODS: Twenty-two studies out of 15,105 (1997-2015) were evaluated after a search in different literature databases, using keywords "colorectal cancer, microsatellite instability, African Americans, Caucasians and Hispanics". We used random effect meta-analysis to calculate the MSI frequency in all studies as well as in African American and Caucasian samples. Meta-regression analysis was used to assess the univariate effect of race, gender, age, tumor location and stage on MSI frequency. RESULTS: The overall MSI frequency among CRCs was 17% (95% CI: 15%-19%, I-2 = 91%). In studies with available race data, The MSI rate among AAs, Hispanics and Caucasians were 12%, 12% and 14% respectively and was not significantly different. Sub-group analysis of studies with racial information indicates MSI OR of 0.78 for AAs compared to Caucasians. CONCLUSION: CRCs demonstrate an overall MSI frequency of 17%. MSI frequency differences between AAs and Caucasians were not pronounced, suggesting that other factors contribute to the racial disparity. The methodological approaches and biological sources of the variation seen in MSI frequency between different studies need to be further investigated.

No description available.

Made available in DSpace on 2018-08-02T00:01:45Z (GMT). No. of bitstreams: 1 tese_10281_TeseDoutoradoRichardTello2016.pdf: 12331551 bytes, checksum: 0dae4547527893319ca299b5e22f6234 (MD5) Previous issue date: 2016-09-01 / Durante os últimos dez anos, as Interfaces Cérebro Computador (ICC) baseadas em Potenciais Evocados Visuais de Regime Permanente (SSVEP) têm chamado a atenção de muitos pesquisadores devido aos resultados promissores e as altas taxas de precisão atingidas. Este tipo de ICC permite que pessoas com dificuldades motoras severas possam se comunicar com o mundo exterior através da modulação da atenção visual a luzes piscantes com frequência determinada. Esta Tese de Doutorado tem o intuito de desenvolver um novo enfoque dentro das chamadas ICC Independentes, nas quais os usuários não necessitam executar tarefas neuromusculares para seleção visual de objetivos específicos, característica que a distingue das tradicionais ICCs-SSVEP. Assim, pessoas com difculdades motoras severas, como pessoas com Esclerose Lateral Amiotrófca (ELA), contam com uma nova alternativa de se comunicar através de sinais cerebrais. Diversas contribuições foram realizadas neste trabalho, como, por exemplo, melhoria do algoritmo extrator de características, denominado Índice de Sincronização Multivariável (ou MSI, do Inglês), para a detecção de potenciais evocados; desenvolvimento de um novo método de detecção de potenciais evocados através da correlação entre modelos multidimensionais (tensores); o desenvolvimento do primeiro estudo sobre a influência de estímulos coloridos na detecção de SSVEPs usando LEDs; a aplicação do conceito de Compressão na detecção de SSVEPs; e, fnalmente, o desenvolvimento de uma nova ICC independente que utiliza o enfoque de Percepção Fundo-Figura (ou FGP, do Inglês).

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.

Syftet med studien var att etablera Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry(MALDI-TOF MS) och onlinedatabasen Mass Spectrometry identification platform(MSI) som metod för identifiering av dermatofyter och mögelsvamp. Svamparna inkuberades 3 eller 6 dagar aerobt i 30°C på Sabouraud dextros agar. Två olika extraktionsmetoder användes innan analys med MALDI-TOF MS. Spektra analyserades med Flexcontrol version 3.4 och resulterade i scorevärde. De spektra som framkom skickades även till MSI för ytterligare analys där resultat erhölls som sannolikhet i procent. Av de ursprungliga 23 st prov identifierades totalt 22st(95,6%) efter 3 eller 6 dagars inkubation och med extraktionsmetod 1 eller 2. MSI databasen var mer omfattande än den interna databasen, men använde sig inte av de aktuella namnen. Behovet av artidentifiering kan ifrågasättas, då många dermatofyter och mögel inom samma släkte behandlas på samma sätt. Artbestämning för immunsupprimerade patienter kan däremot vara viktigt. Studien var lyckad och metoderna kommer tillämpas inom en snar framtid på Klinisk Mikrobiologi i Halmstad. / The purpose of this study was to establish Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry(MALDI-TOF MS) and the online database Mass Spectrometry identification platform(MSI) as a method for identification of dermatophytes and molds. The fungi were incubated 3 or 6 days aerobic in 30°C on Sabouraud dextrose agar. Two different extraction methods were used before analysis with MALDI-TOF MS. Spectra analyzed with Flexcontrol version 3.4 resulted in score values. The same spectra was later sent for analysis with MSI and the result obtained was seen as probability in percent. Of the original 23 samples a total of 22(95,6%) samples were identified after 3 or 6 days of incubation and by extraction method 1 or 2.  The MSI database was more comprehensive than the internal database, but did not use the current fungi names. The necessity of species identification can be questioned, since dermatophytes and molds within the same genera is treated the same way. Species identification for immunosuppressed patients could however be significant. The study was successful and the methods will be applied within a near future in the Clinical Microbiology laboratory in Halmstad.

La vectorisation de médicaments, qui permet de les acheminer sur les tissus cibles pour accroitre leur activité pharmacologique tout en limitant leur toxicité et effets indésirables, est un axe de recherche en forte expansion dans lequel les nanotechnologies sont un des facteurs clés. L’un des enjeux dans cette thèse a donc été de développer une méthode d’imagerie combinée entre la MSI et l’imagerie β pour l’étude de la biodistribution de nanoparticules de type graphène. Malgré l’étude de nanoparticules hétérogènes, les analyses ont permis de déterminer une signature carbonée répétable de l’oxyde de graphène en analyse LDI - MS et ainsi que des analyses reproductibles de MSI avec de CV inférieur à 30 %. De plus, la combinaison des deux techniques a permis d’obtenir la quantification absolue du GO en radioimagerie après exposition de souris à trois doses d’injection ainsi que la biodistribution à 25 µm de résolution spatiale de ces nanoparticules au sein des tissus grâce à l’apport de la MSI. Lors d’un second projet concernant l’étude de vecteurs micellaires polymériques encapsulant un médicament, une méthode MALDI - TOF a également été développée afin de détecter ces deux molécules simultanément. Cependant, les expérimentations réalisées ont montré le besoin de développer des protocoles de traitements tissulaire compatibles avec la MSI et permettant d’améliorer le seuil de sensibilité de cette technique analytique. / The vectorization of drugs, which allows them to be transported to target tissues to increase their pharmacological activity while limiting their toxicity and adverse effects, is a rapidly expanding research area in which nanotechnologies are one of the key factors. One of the challenges in this thesis was therefore to develop a combined imaging method between MSI and imaging β for the study of the biodistribution of graphene-type nanoparticles. Despite the study of heterogeneous nanoparticles, the analyses determined a repeatable carbon signature of graphene oxide in LDI - MS analysis and reproducible MSI analyses with CVs below 30%. In addition, the combination of the two techniques made it possible to obtain the absolute quantification of GO in radioimaging after exposure of mice to three injection doses as well as the biodistribution at 25 µm of spatial resolution of these nanoparticles within tissues thanks to the contribution of MSI. In a second project to study polymeric micellar vectors encapsulating a drug, a MALDI - TOF method was also developed to detect these two molecules simultaneously. However, the experiments carried out have shown the need to develop tissue treatment protocols compatible with MSI and making it possible to improve the sensitivity threshold of this analytical technique.