Optimization of immunofluorescence protocols for detection of biomarkers in colorectal and breast cancer tissues

Cancer remains an undetermined question for modern medicine. Every year millions of people ranging from children to adult die since the modern treatment is unable to meet the challenge. Research must continue in the area of new biomarkers for tumors. Molecular biology has evolved during last years; however, this knowledge has not been applied into the medicine. Biological findings should be used to improve diagnostics and treatment modalities. In this thesis, human formalin-fixed paraffin embedded colorectal and breast cancer samples were used to optimize the double immunofluorescence staining protocol. Also, immunohistochemistry was performed in order to visualize expression patterns of each biomarker. Concerning double immunofluorescence, feasibility of primary antibodies raised in different and same host species was also tested. Finally, established methods for simultaneous multicolor immunofluorescence imaging of formalin-fixed paraffin embedded specimens were applied for the detection of pairs of potential biomarkers of colorectal cancer (EGFR, pmTOR, pAKT, Vimentin, Cytokeratin Pan, Ezrin, E-cadherin) and breast cancer (Securin, PTTG1IP, Cleaved caspase 3, ki67).

Procedimiento de obtención de abono líquido orgánico rico en sustancias húmicas a partir de compost de origen vegetal

Obtención de un producto líquido rico en sustancias húmicas mediante un procedimiento sencillo, rápido y extrapolable a nivel industrial y cuya aplicación a nivel agrícola sea satisfactoria. La obtención de dicho abono se ha llevado a cabo gracias a la optimización del proceso de extracción de las sustancias húmicas presentes en muestras de compost de origen vegetal. Las condiciones óptimas en las que se llevó a cabo dicho proceso implicaron altos valores de pH (> 10) y temperaturas superiores a 100°C. La aplicación del producto obtenido bajo tales condiciones, a nivel agrícola, ha mostrado aspectos de enorme interés tanto desde el punto de vista del desarrollo vegetal como en relación a las características del suelo o de la microbiota asociada a dicho sustrato.

Un modelo de solución tecnológica a la sanidad vegetal

En este trabajo muestra cómo aplicar soluciones tecnológicas concretas a las principales tareas relativas a la sanidad vegetal. Proponemos un modelo de las tareas en términos de flujos de trabajo. Cada una de estas tareas se mapea con una aplicación software concreta y en el caso de la selección de un tratamiento fitosanitario en una situación específica se describe un método estructurado para la toma de decisiones que puede ser aplicable el proceso analítico jerárquico (Analytic Hierarchy Process, AHP).

Development of computer application for determination of chemical diffusion properties in biological tissues

The main objective of this work was to develop an application capable of determining the diffusion times and diffusion coefficients of optical clearing agents and water inside a known type of muscle. Different types of chemical agents can also be used with the method implemented, such as medications or metabolic products. Since the diffusion times can be calculated, it is possible to describe the dehydration mechanism that occurs in the muscle. The calculation of the diffusion time of an optical clearing agent allows to characterize the refractive index matching mechanism of optical clearing. By using both the diffusion times and diffusion of water and clearing agents not only the optical clearing mechanisms are characterized, but also information about optical clearing effect duration and magnitude is obtained. Such information is crucial to plan a clinical intervention in cooperation with optical clearing. The experimental method and equations implemented in the developed application are described in throughout this document, demonstrating its effectiveness. The application was developed in MATLAB code, but the method was personalized so it better fits the application needs. This process significantly improved the processing efficiency, reduced the time to obtain he results, multiple validations prevents common errors and some extra functionalities were added such as saving application progress or export information in different formats. Tests were made using glucose measurements in muscle. Some of the data, for testing purposes, was also intentionally changed in order to obtain different simulations and results from the application. The entire project was validated by comparing the calculated results with the ones found in literature, which are also described in this document.

Characterizing neural tissues with mass spectrometry imaging via enhanced computational approaches

Neuropeptides affect the activity of the myriad of neuronal circuits in the brain. They are under tight spatial and chemical control and the dynamics of their release and catabolism directly modify neuronal network activity. Understanding neuropeptide functioning requires approaches to determine their chemical and spatial heterogeneity within neural tissue, but most imaging techniques do not provide the complete information desired. To provide chemical information, most imaging techniques used to study the nervous system require preselection and labeling of the peptides of interest; however, mass spectrometry imaging (MSI) detects analytes across a broad mass range without the need to target a specific analyte. When used with matrix-assisted laser desorption/ionization (MALDI), MSI detects analytes in the mass range of neuropeptides. MALDI MSI simultaneously provides spatial and chemical information resulting in images that plot the spatial distributions of neuropeptides over the surface of a thin slice of neural tissue. Here a variety of approaches for neuropeptide characterization are developed. Specifically, several computational approaches are combined with MALDI MSI to create improved approaches that provide spatial distributions and neuropeptide characterizations. After successfully validating these MALDI MSI protocols, the methods are applied to characterize both known and unidentified neuropeptides from neural tissues. The methods are further adapted from tissue analysis to be able to perform tandem MS (MS/MS) imaging on neuronal cultures to enable the study of network formation. In addition, MALDI MSI has been carried out over the timecourse of nervous system regeneration in planarian flatworms resulting in the discovery of two novel neuropeptides that may be involved in planarian regeneration. In addition, several bioinformatic tools are developed to predict final neuropeptide structures and associated masses that can be compared to experimental MSI data in order to make assignments of neuropeptide identities. The integration of computational approaches into the experimental design of MALDI MSI has allowed improved instrument automation and enhanced data acquisition and analysis. These tools also make the methods versatile and adaptable to new sample types.

Canções de fogo: a apreensão de saberes através da performance da escuta no contexto da União do Vegetal

O ritual onde se utiliza a cocção conhecida por ayahuasca vem sendo ressignificado em contextos urbanos há mais de oito décadas, sobretudo após sua expansão iniciada pelos movimentos organizados. Essa experiência derivante dos povos da floresta amazônica, vem ultrapassando tanto fronteiras geográficas como culturais, sendo constantemente reinventada por diferentes agentes. Contemporaneamente, o fenômeno abarca indivíduos provenientes de várias partes do mundo incorporando legados doutrinários de mestres originários da América Latina. A linha doutrinária conhecida por União do Vegetal - UDV, na qual este trabalho se centra, vem ganhando espaço entre os circuitos europeus neo esotéricos. Atualmente o ritual é vivenciado por mais de 18 mil pessoas espalhadas pelo globo e tem como característica, após a ingestão da cocção, a utilização da música como uma das ferramentas mediadoras para a apreensão dos princípios doutrinários. Usualmente os mestres da UDV têm como característica a utilização de fonogramas de variadas categorias musicais que adquirem uma dimensão simbólica e funcional no momento do ritual. A comunidade estabelecida em Portugal - foco desta tese – e as comunidades digitais Musincante e Músicas do Alto, permitiram-me perceber, através do trabalho de campo e de observação participante, elementos constitutivos que abrangem toda a comunidade da UDV. O enfoque parte da perspectiva de observar essa doutrina como um lugar de conhecimento sistematizado e formal. No sentido de compreender os processos de construção desse saber, foi verificado o protagonismo do ato da escuta como elemento central para a apreensão da doutrina. Neste sentido, esta tese adota o conceito de performance da escuta como meio de explorar as formas como o indivíduo e o coletivo absorvem um saber doutrinário a partir da música e de como esta assume o papel de mediadora dos saberes transmitidos no contexto da UDV e de manutenção deste conhecimento num contexto extra ritual.

Comparação de produtos de colagem de origem animal, vegetal e extratos de leveduras em vinhos tintos

No mercado têm surgido alternativas aos produtos de colagem de origem animal, nomeadamente as proteínas de origem vegetal e extratos de levedura. O objectivo deste trabalho foi a comparação de um elevado número de produtos de colagem de origem animal (gelatinas) e produtos alternativos de origem vegetal e levuriano. Inicialmente os produtos de colagem foram aplicados ao vinho em diferentes doses. Com base na avaliação sensorial, foram selecionados os produtos e as doses que revelaram melhor desempenho. Os produtos de colagem selecionados foram aplicados ao vinho e avaliado o seu efeito nas características sensoriais e físico-químicas. O parâmetro que apresentou diferenças mais significativas foi a turbidez. A análise estatística apenas revelou diferenças significativas para os atributos qualidade do sabor e nota final; apesar de não evidenciar diferenças significativas, as variações observadas para o atributo extração/secura foram muito importantes do ponto de vista prático. As gelatinas que mostraram melhor desempenho foram as POA 10 e POA 12 e as proteínas vegetais POV 6 (proteína de ervilha) e POV 10 (proteína de batata). Este estudo permitiu a recolha de informação para ser utilizada na seleção do produto de colagem mais apropriado para o perfil de vinho da adega e a alternativa mais apropriada às gelatinas.

Effects of silicon treatment and inoculation with Fusarium oxysporum f. sp. vasinfectum on cellular defences in root tissues of two cotton cultivars

BACKGROUND AND AIMS: Silicon has been shown to enhance the resistance of plants to fungal and bacterial pathogens. Here, the effect of potassium silicate was assessed on two cotton (Gossypium hirsutum) cultivars subsequently inoculated with Fusarium oxysporum f. sp. vasinfectum (Fov). Sicot 189 is moderately resistant whilst Sicot F-1 is the second most resistant commercial cultivar presently available in Australia. METHODS: Transmission and light microscopy were used to compare cellular modifications in root cells after these different treatments. The accumulation of phenolic compounds and lignin was measured. KEY RESULTS: Cellular alterations including the deposition of electron-dense material, degradation of fungal hyphae and occlusion of endodermal cells were more rapidly induced and more intense in endodermal and vascular regions of Sicot F-1 plants supplied with potassium silicate followed by inoculation with Fov than in similarly treated Sicot 189 plants or in silicate-treated plants of either cultivar not inoculated with Fov. Significantly more phenolic compounds were present at 7 d post-infection (dpi) in root extracts of Sicot F-1 plants treated with potassium silicate followed by inoculation with Fov compared with plants from all other treatments. The lignin concentration at 3 dpi in root material from Sicot F-1 treated with potassium silicate and inoculated with Fov was significantly higher than that from water-treated and inoculated plants. CONCLUSIONS: This study demonstrates that silicon treatment can affect cellular defence responses in cotton roots subsequently inoculated with Fov, particularly in Sicot F-1, a cultivar with greater inherent resistance to this pathogen. This suggests that silicon may interact with or initiate defence pathways faster in this cultivar than in the less resistant cultivar.

Studies on bovine oocytes and ovarian tissues cryopreservation : fundamental and applied aspects

Tese de Doutoramento, Ciências Agrárias (Reprodução Animal), 26 de Junho de 2013, Universidade dos Açores.

Mapa de la cobertura vegetal del volcán Chaparrastique, departamento de San Miguel, El Salvador

El mapa de la cobertura vegetal del volcán Chaparrastique de San Miguel se realizó con el propósito de describir las diferentes unidades de vegetación presentes en el Volcán, mediante la elaboración de un mapa temático; utilizando el sistema validado de clasificación de la UNESCO, 2010; los límites perimetrales para la zona de estudio del volcán Chaparrastique se establecieron en radio de 5.75 km a partir del centro del cráter del volcán, para el análisis y recolección de datos se apoyo en el procesamiento de imágenes del volcán basadas en satélite del programa Google Earth 5.2.1 de alta resolución; la identificación de las diferentes formaciones vegetales presentes en el volcán se realizo mediante la clasificación digital de imágenes; basado en el agrupamiento de valores visuales mediante el programa ArcView 3.2. Las etapas descritas dieron a conocer que el volcán Chaparrastique de San Miguel presenta cuatro tipos de ecosistemas naturales; 1.Bosque Tropical Semideciduo Latifoliado submontano. 2.Bosque Tropical Decíduo Latifoliado de tierras bajas. 3.Flujo de lava con escasa vegetación. y 4.Pradera o páramo altimontano.