The PI3K/AKT/mTOR signaling pathway is commonly dysregulated in cancer. The goal of this thesis project was to assess the hypothesis of a strong correlation between PI3K/AKT/mTOR pathway activity and the response to targeted therapies, by using a protein quantitation technique called immuno-matrix assisted laser desorption/ionization (iMALDI). The use of iMALDI as a clinical tool was demonstrated by automating an established iMALDI assay for quantifying plasma renin activity. The results from the automated method gave high correlation coefficients of ≥0.98 with a clinical LC-MS/MS method and could be performed significantly faster than with manual sample preparation. The 7.5-fold faster analysis compared to LC-MS/MS, reduction in human error, and higher throughput, demonstrated the suitability of this assay for clinical use. The automated iMALDI platform was then adapted for use with cancer cell lines and tissue analysis, targeting the kinases AKT1 and AKT2 as surrogate proteins for signaling pathway activity. Using minute amounts (10 µg/capture), AKT1 and AKT2 expression and phosphorylation stoichiometry (PS) were successfully quantified via their C-terminal tryptic peptides, which encompassed key phosphorylation sites. After assay optimization, the assays were analytically validated for linear range, accuracy, and interferences. In addition, PS cut-off values based on measurement errors were established for confident PS quantitation. The functionality of the assay was demonstrated with cell lines, and flash-frozen and FFPE tissue lysates, with, on average, lower AKT1/AKT2 measurements obtained from FFPE samples. The developed assays were sensitive and precise enough to detect differences between matched normal and adjacent tumor tissues. To answer the hypothesis, patient-derived xenograft (PDX) mouse-model tumors treated with Herceptin, Everolimus, a combination of both (E+H), or with no treatment, were assessed for molecular patterns linked to tumor response. One mouse from the E+H group showed a partial response, with elevated total and phosphorylated AKT1/AKT2. Unfortunately, overlapping values between treatment groups were obtained in this study, and the large within-group spread and the low number of biological replicates made it difficult to confirm a definite correlation between PI3K/AKT/mTOR pathway activity and response to treatment. A follow-up study with additional protein targets, a larger number of samples, and serial biopsies will be required to determine if there is, in fact, a correlation between PI3K/AKT/mTOR pathway activity and response to treatment. / Graduate / 2019-10-05

Submitted by TARCISIO TORRE LOURENÇO null (tarcisiolourenco16@yahoo.com.br) on 2016-12-09T13:14:54Z No. of bitstreams: 1 Dissertação Tarcísio com a ficha.pdf: 10339060 bytes, checksum: 23f48d3539cc446a8efd713c0a0b082d (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-13T10:58:12Z (GMT) No. of bitstreams: 1 lourenco_tt_me_bot.pdf: 10339060 bytes, checksum: 23f48d3539cc446a8efd713c0a0b082d (MD5) / Made available in DSpace on 2016-12-13T10:58:12Z (GMT). No. of bitstreams: 1 lourenco_tt_me_bot.pdf: 10339060 bytes, checksum: 23f48d3539cc446a8efd713c0a0b082d (MD5) Previous issue date: 2016-03-30 / O ciclo estral da vaca é composto por 2-3 ondas de crescimento folicular, no qual vários folículos são recrutados e iniciam um novo crescimento. Durante o período denominado desvio folicular, um folículo se torna dominante e os outros entram em atresia. Este processo envolve um mecanismo ainda não completamente compreendido, incluindo proteínas específico, como já estabelecido pela expressão gênica. O objetivo do presente estudo foi caracterizar as proteínas do fluído folicular a fim de identificar macromoléculas relacionadas ao desenvolvimento dos folículos de vacas zebuínas nã-gestantes. Foram colhidos os ovários de 25 vacas mestiças não-gestantes em um abatedouro. A presença do corpo lúteo foi anotada para cada ovário. O líquido folicular foi colhido utilizando-se a imersão do ovário em meio líquido e ultrassonografia. De acordo com a mensuração do diâmetro folicular, foram formados 3grupos, folículos pequenos (≤6,5mm, n=25), médios (>6,5mm a ≤9mm, n=9) e grandes (>9,0mm, n=11). Após 2 centrifugações (600xg/10 minutos e 15.000xg/30 minutos, 4ºC) o sobrenadante foi separado e utilizado para determinação da concentração de proteína total (método de Bradford). A eletroforese foi conduzida sob condições desnaturantes e redutoras, em gel de separação de poliacrilamidaà 12%. A concentração de progesterona e estradiol do líquido folicular foi determinada a fim de identificar os folículos saudáveis. As proteínas diferenciais identificadas pela eletroforese foram submetidas à espectrometria de massas e a ontologia gênica foi investigada nos bancos de dados disponíveis. Foram encontradas 45 bandas proteicas em 45 amostras de líquido folicular. A média da concentração ± desvio padrão da progesterona foi de 129,91 ± 186,43, considerando todos os folículos. Os resultados mostram que houve uma expressão diferenciada de proteínas nas diferentes categorias de folículos. / The estrous cycle of the cow consists of 2-3 follicular waves, in which several follicles are recruited and initiate growth. During the period called follicular deviation, one follicle becomes dominant and the other come into atresia.This process involves mechanisms not yet fully understood, including specific proteins as already determined by gene expression. As a result, the objective of this study was to characterize the proteins of the follicular fluid to identify macromolecules related to the development of follicles from Zebu cow. The ovaries of nonpregnant cows 25 were harvested. The presence of luteum body was noted for each ovary. Follicular fluid was collected using ultrasound. According to the measurement of follicular diameter was 3 separate groups, small follicles (≤6,5mm, n = 28), medium (> 6.5mm to ≤9mm, n = 7) and large (> 9,0mm, n = 11). After 2 centrifugations (600xg / 10 minutes, 15.000xg / 30 minutes, 4 ° C) the supernatant was separated and used for determination of total protein concentration (Bradford method). Electrophoresis was conducted under denaturing and reducing conditions on polyacrylamide separating gel at 12%. The concentration of progesterone and follicular fluid estradiol was determined to identify healthy follicles. The differential proteins identified by electrophoresis will be submitted for MALDI TOF MS / MS approach and gene ontology will be investigated in the databases available. They found 45 protein bands in electrophoresis in 45 follicular fluid samples. The mean ± standard deviation of progesterone concentration was 129.91 ± 186.43 considering all follicles. The results show that there was a differential expression of proteins in different categories of follicles.

Orientador: Marcos Nogueira Eberlin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T11:19:58Z (GMT). No. of bitstreams: 1 Marques_LygiadeAzevedo_M.pdf: 2432568 bytes, checksum: 7bf6003b0fa0df13e1ba0f91d7ebf00b (MD5) Previous issue date: 2006 / Resumo: Neste trabalho aplicamos técnicas avançadas de espectrometria de massas, (MALDI-TOF e ESI-MS) na análise de micotoxinas em alimentos e na tipificação e verificação de fraudes em perfumes. Aplicamos a técnica MALDI-TOF em análises de micotoxinas, e esta mostrou excelente desempenho nas análises de aflatoxinas e ocratoxina e vantagem sobre a técnica de escolha atual, o método ELISA. Esta vantagem é principalmente maior especificidade através de maior exatidão em medidas de massas e, portanto, maior confiabilidade. O Planejamento de experimento foi uma ferramenta valiosa para obtenção das melhores condições e estudo dos parâmetros de interferência. O limite de detecção encontrado para a técnica foi da ordem de 25 pg para aflatoxinas e de 1 ng para ocratoxina, com perspectiva de melhoria através de aumento da massa amostral em estudos futuros para adaptação da metodologia de extração na matriz de interesse à técnica MALDI-TOF. A técnica ESI-MS foi utilizada para a tipificação e detecção de perfumes proporcionando, através da análise de componentes principais (PCA), a diferenciação com segurança entre perfumes originais, falsos e inspirados, utilizando como indicadores componentes polares não majoritários característicos de cada categoria avaliada. Este estudo abre caminho para que esta técnica seja utilizada na avaliação de perfumes que estão sob suspeita de falsificação com auxilio de uma biblioteca de "fingerprint" de perfumes por ESI-MS. O emprego da técnica de MALDI-TOF também é uma opção vantajosa para o monitoramento da qualidade de grãos quanto a presença de toxinas indesejáveis, bem como ameaças de bioterrorismo. / Abstract: In this work we applied advanced mass spectrometry techniques (MALDI-TOF and ESI-MS) to micotoxin analysis in food and for the typification and detection of counterfeit perfumes. MALDI-TOF was applied to micotoxin analysis, which showed excellent performance for the analysis of aflatoxins and ochratoxin with advantage over the current technique of choice, the ELISA method. This advantage is mainly its greater specifity due the exactness of the measurements, therefore with higher reliability. The surface analysis was a valuable tool to attain the best conditions and study the interference of several parameters. The detection limit found for the technique was 25 pg for aflatoxins and 1 ng for ochratoxins, with perspective of improvement through increase of the sample mass in future studies for adaptation of the methodology of extration in the matrix of interest for the MALDI-TOF technique. The ESI-MS technique was used for typification and detection of counterfeit perfumes, providing, through principal component analysis (PCA), the characterization of original, counterfeit and inspired perfumes, using as minoritarian polar compounds as diagnostic ions of each perfume category evaluated. We envisage that the method can be used to establish a ESI-MS fingerprinting library of perfumes for comparison with those from samples under investigation, and that such a library could be updated constantly by the addition of ESI-MS of new perfumes even before they are commercially released. MALDI-TOF technique is also an advantageous option for the monitoring of crop quality relating to the presence of undesirable toxins, as well as bioterrorism threats by micotoxin poisoning. / Mestrado / Quimica Analitica / Mestre em Química

La miniaturisation et la complexification croissante des composants microélectroniques induit une sensibilisation et une fragilisation accrue des composants vis-à-vis des contaminations présentes dans les zones de productions appelées “salles blanches”. Dans ces espaces, le contrôle actuel de la contamination organique n’est pas suffisant puisqu’il ne permet pas d’éviter la contamination de surface des plaquettes de silicium et des optiques des robots de production utilisés pour la photolithographie. Un contrôle accru des concentrations des contaminants organiques dans les atmosphères des salles blanches devient donc nécessaire et de nouvelles méthodes analytiques doivent être développées et validées. Dans le cadre de ce travail, des méthodes d’analyse ont été développées et validées afin de disposer d’une gamme d’outils permettant un suivi rigoureux des contaminations. Ces outils permettent d’identifier et de quantifier les contaminations surfaciques des plaquettes de silicium par des composés organiques semi-volatils (phtalates et organophosphorés) mais aussi de déterminer les concentrations de composés organiques volatils présents dans l’atmosphère des salles blanches. Ces méthodes font appel aux technologies du WOS/ATD-GC-MS « Wafer Outgassing System/Automated Thermal Desorber–Gas Chromatography–Mass Spectrometry » et de la DART-ToF-MS « Direct Analysis in Real Time-Time of Flight–Mass Spectrometry » pour les analyses de surfaces et au PTR-ToF-MS « Proton Transfer Reaction – Time of Flight - Mass Spectrometry » pour l’analyse de l’atmosphère. / The recent advances in the miniaturization and complexification of microelectronic components induce an increase in the sensitivity of these components regarding the organic contamination present in the production zone called “clean room”. Although, the control of organic contamination in the clean room is very rigorous it does not avoid the contamination of silicon wafer surfaces and robot lenses used in the photolithography process. The later implies that new analytical methodologies should be developed and validated. In this work, analytical methods were developed and validated in order to have a panel of tools which allows careful monitoring of organic contaminants. These tools allow the identification and quantitation of the contamination of silicon wafer surface by semi-volatiles organic compounds (phthalates and organophosphates) and the determination of volatile organic compounds concentrations in the clean room atmosphere. These methods uses the WOS/ATD-GC-MS « Wafer Outgassing System/Automated Thermal Desorber–Gas Chromatography–Mass Spectrometry » technology and the DART-ToF-MS « Direct Analysis in Real Time-Time of Flight–Mass Spectrometry » technology for wafer surface analysis and the PTR-ToF-MS « Proton Transfer Reaction – Time of Flight - Mass Spectrometry » technology for gas-phase analysis.

Dans le cadre de la qualité environnementale des matériaux, il est essentiel d'apporter la preuve que les matériaux à base de ciments adjuvantés ne relarguent pas ou peu de matières organiques lors de leur contact avec l'eau constituant une solution lixiviante. Les additifs organiques, tels que les agents de mouture et les superplastifiants, constituent deux classes d'adjuvants organiques utilisés de manière systématique dans la fabrication ou la formulation des matériaux cimentaires, notamment quand ils sont en contact avec l'eau potable (conduites et châteaux d'eau). Pour évaluer le piégeage de ces composés organiques par une pâte de ciment CEM I, cinq montages de lixiviation dynamique CTG-LEACHCRETE ont été mis en place et adaptés pour l'étude de pâtes de ciment formulées avec des adjuvants organiques. La seconde partie de ce travail a pour objectif de mettre au point des techniques analytiques sensibles pour la détection de traces des constituants du superplastifiant et de l'agent de mouture directement dans les produits de la lixiviation de pâtes de ciment (les lixiviats) grâce aux techniques de spectrométrie de masse MALDI-TOF et Py-THM-MS. Enfin, l'application du protocole global de "lixiviation dynamique couplée à la spectrométrie de masse" nous permet d'apprécier la présence des composés organiques suite à des essais de lixiviation de pâtes de ciment formulées avec de l'agent de mouture et du superplastifiant. Cette démarche nous a permis d'obtenir de nombreux résultats donnant des informations sur les mécanismes de piégeage des différents additifs organiques par une pâte de ciment / Evidence that materials used by the industry are not damageable for the environment has become a major issue. In cement industry, organic admixtures such as grinding aids or superplasticizers ar widely used. In particular, they constitute cementitious materials in concrete contacting water like in water pipes and water tower. It is therefore essential to test whether these organic coumpounds are enventually dissolved into water by leaching. In this aim, five different dynamic leaching tests were developed and applied to a CEM I cement paste formulated with organic admixtures. In paralell, highly sensitive analytical methods based on MALDI-TOF and Py-THM-MS mass spectrometry techniques were designed in order to detect traces of leached superplasticizers or grinding aids. The dynamic leaching tests coupled to mass spectrometry allowed us to detect the presence of organic compounds in the leachate, and to better understand the mechanisms involved in the trapping of additives into a cement paste

Introduction: Hypoxia is found in solid cancerous tumours. The presence of hypoxia within tumours inhibits anti-cancer treatment strategies such as chemotherapy from being completely effective and it is suspected that multiple mechanisms contribute to the resistance. Methods: In this project a systems biology approach was applied to determine how the toxicity of doxorubicin is affected by hypoxia at the metabolome level. A multitude of analytical techniques were applied to analyse the intracellular metabolism of a monolayer of cancer cells (MDA-MB-231). Metabolic profiling was used to determine metabolite markers related to hypoxia-induced chemoresistance. For this gas chromatography mass spectrometry (GC-MS) and ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) were used. Furthermore, network-based correlation analysis was developed as a novel tool to bridge the gap between metabolomics dataset and systems biology modelling. This methodology was applied to elucidate novel metabolic pathways as potential therapeutic targets to overcome hypoxia-induced chemoresistance. This algorithm determines significant correlation differences between different physiological states, and through applying graph-theory on large genome scale models; it is possible to construct a metabolic network of the pathways connecting the pair-wise correlation. Finally, imaging mass spectrometry using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was developed as a tool for in situ metabolite analysis to investigate the metabolic response to chemotherapy in multi-tumour spheroids (MTSs). Results: Metabolic fingerprinting analysis characterised a snapshot of cells exposed to various environmental perturbations. Metabolite markers associated with hypoxia-induced chemoresistance were related to metabolic pathways including gluconeogenesis, DNA synthesis and fatty acid synthesis. Furthermore, network-based correlation analysis revealed specific metabolites in the fatty acid synthesis pathways were contributing to drug resistance, which included malonyl-CoA, 3-oxoeicosanoyl-CoA, stearoyl-CoA and octadecanoic acid. To facilitate the detection of metabolites in ToF SIMS datasets, a series of metabolites standard spectra were acquired. Hypoxic metabolite markers detected in ToF-SIMS data of cell lysates included glycine, lactic acid and succinic acid, which were also shown to be metabolite markers in GC-MS metabolic data. Furthermore, MTS sections were imaged using ToF-SIMS to profile the chemical response to chemotherapy treatment within the oxygen gradient. Loadings from image PCA were explored to determine the metabolic response in the highly oxygenated outer region and hypoxic inner region of the MTS. Conclusion: A multitude of analytical techniques were able to contribute to elucidating the metabolic mechanisms associated with hypoxia-induced chemoresistance. Metabolic profiling combined with a systems biology approach was further able to identify potential underlying metabolic regulation of resistance. Finally ToF-SIMS was developed as a tool for metabolite analysis in complex biological systems in situ.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23410号 / 医博第4755号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 長尾 美紀, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.

SNAP and TOF are two non-contrast enhanced intracranial magnetic resonance angiography (MRA) techniques. Both techniques rely on incoming blood to produce a signal, although they have different sensitivity to blood flow velocity. In human beings, SNAP-MRA and TOF-MRA were frequently observed to show discrepancy in visibility of distal intracranial arteries. Whether this signal feature difference is associated with the blood flow of intracranial arteries has not been validated in patients. White matter hyperintensity is frequently observed in elderly and neurodegenerative patients, and may be contributed by the impairment of intracranial arteries. Whether the discrepancy between TOF-MRA and SNAP-MRA can serve as an imaging biomarker of the health of intracranial arteries and is associated with white matter hyperintensity remains unclear. In this study, we aim to characterize the disagreement of vessel visibility in SNAP and TOF, and explore its associations with blood flow and white matter lesions in patients with carotid artery atherosclerosis. In this study, we found this disagreement to be due to velocity sensitivity. The disagreement in the distal branches of the middle cerebellar and anterior cerebellar arteries were shown to be associated with white matter lesion volume. / 2022-06-04T00:00:00Z

Hepcidin is cysteine-rich cationic peptide produced by hepatocytes, secreted into blood plasma, and excreted in urine. Hepcidin is proposed to be the key regulator of iron metabolism and an evaluation of changes in the hepcidin level is important for diagnosis of several diseases. However, methods used for the hepcidin detection and determination in urine and serum have certain limitations. At present time MALDI-TOF MS based approaches have been applied for final analysis of urinary and/or serum hepcidin levels. Before MS analysis, separation of hepcidin from analyzed samples is an important and necessary step. The aim of this study was to compare the ability of several magnetic sorbents with different coating matrix and/or different terminal functionalized groups to adsorb hepcidin prior MS analysis. Either commercial magnetic sorbents containing -COOH groups or magnetic hydrophilic IDA-modified polymethacrylate microparticles P(HEMA-co-GMA)-IDA with immobilized metal ions were use for this purpose. Hepcidin was adsorbed to magnetic sorbents containing linked carboxyl groups (i.e. to weak cation exchange magnetic particles) at pH 6.8 independently on a nature of magnetic particle coating layer. Magnetic particles P(HEMA-co- GMA)-IDA with immobilized Cu(II) ions were found to adsorb hepcidin in a...