Intracellular signaling and trafficking in cancer: Role of Rab5-GTPases in migration and invasion of breast cancer cells

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. Steroid hormones, such as estrogen, and growth factors, which include insulin growth factor I/II (IGF-1/IGF-2) therapy has been associated with most if not all of the features of metastasis. It has been determined that IGF-1 increases cell survival of cancer cells and potentiate the effect of E2 and other ligand growth factors on breast cancer cells. However not much information is available that comprehensively expounds on the roles of insulin growth factor receptor (IGFR) and Rab GTPases may play in breast cancer. The latter, Rab GTPases, are small signaling molecules and critical in the regulation of many cellular processes including cell migration, growth via the endocytic pathway. This research involves the role of Rab GTPases, specifically Rab5 and its guanine exchange factors (GEFs), in the promotion of cancer cell migration and invasion. Two important questions abound: Are IGFR stimulation and downstream effect involved the endocytic pathway in carcinogenesis? What role does Rab5 play in cell migration and invasion of cancer cells? The hypothesis is that growth factor signaling is dependent on Rab5 activity in mediating the aggressiveness of cancer cells. The goal is to demonstrate that IGF-1 signaling is dependent on Rab5 function in breast cancer progression. Here, the results thus far, have shown that while activation of Rab5 may mediate increased cell proliferation, migration and invasion in breast cancer cells, the Rab5 GEF, RIN1 interacts with the IGFR thereby facilitating migration and invasion activities in breast cells. Furthermore, endocytosis of the IGFR in breast cancer cells seems to be caveolin dependent as the data has shown. This taken together, the data shows that IGF-1 signaling in breast cancer cells relies on IGF-1R phosphorylation, caveolae internalization and sequestration to the early endosome RIN1 function and Rab5 activation.^

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Revisión sistemática utilidad de asa y calcio con o sin vitamina d en la prevención de cáncer colorrectal y la recurrencia de pólipos adenomatosos en paciente con riesgo de padecer esta patología

Introducción: El cáncer colorrectal es una patología con alto impacto en la salud pública, debido a su prevalencia, incidencia, severidad, costo e impacto en la salud mental y física del individuo y la familia. Ensayos clínicos realizados en pacientes con antecedente de infarto al miocardio que consumían ácido acetil salicílico (asa), calcio con y sin vitamina D, mostraron asociación entre el consumo de estos medicamentos y disminución en la incidencia en cáncer colorrectal y pólipos adenomatosos. Objetivo: Evaluar la literatura sobre el uso de asa, calcio con y sin vitamina D con relación a su impacto en la prevención del cáncer colorrectal y pólipos adenomatosos. Métodos: Se realizó revisión sistemática buscando ensayos clínicos realizados en pacientes con factores de riesgo para cáncer colorrectal y pólipos adenomatosos que usaron asa, calcio con y sin vitamina D fueron incluidos. Resultados: se escogieron 105 para la revisión sistemática. Conclusiones: Es necesario desarrollar más estudios que lleven a evaluar el efecto protector de la aspirina, calcio y vitamina D. En los artículos revisados la aspirina a dosis de 81 a 325 mg día se correlaciona con reducción de riesgo de aparición de CRC aunque la dosis ideal, el tiempo de inicio y la duración de la ingesta continua no son claros. Hacen falta estudios que comparen poblaciones con ingesta de asa a diferentes dosis.

Estudio molecular en dos hermanas afectadas por ictiosis congénita autosómica recesiva : descripción de una nueva mutación causal en TGM1

Se describe la variante homocigota c.320-2A>G de TGM1 en dos hermanas con ictiosis congénita autosómica recesiva. El clonaje de los transcritos generados por esta variante permitió identificar tres mecanismos moleculares de splicing alternativos.

Remoção de ibuprofeno de águas por oxidação catalítica

A ocorrência e destino de fármacos no ambiente aquático tem vindo a ser reconhecido como um problema emergente em química ambiental. Alguns compostos são resistentes à degradação nas estações de tratamento de águas residuais, ETARs, enquanto que outros, ainda que sofram degradação parcial, continuam a ser lançados nos meios aquáticos em quantidades apreciáveis. O Ibuprofeno, IB, um dos anti­ inflamatórios mais consumidos por todo o mundo, é um dos fármacos mais detectados no meio hídrico. Apesar dos sistemas de tratamento convencionais utilizados nas ETARs removerem até 90% do IB das águas residuais, é frequente o efluente descarregado conter ainda quantidades significativas deste poluente. A presença destes compostos no ambiente deve ser avaliada dado que possuem actividade biológica, mesmo a baixas concentrações. Os processos avançados de oxidação com peróxido de hidrogénio, na presença de catalisadores heterogéneos, permitem melhorar significativamente a remoção deste tipo de compostos em águas. Assim, foi objectivo deste trabalho o estudo da utilização de peróxido de hidrogénio como agente oxidante na remoção de IB em soluções aquosas, na presença de complexo de acetilacetonato de Ni (II) disperso em PDMS ou encapsulado em zeólitos NaY. Para o doseamento do fármaco em solução foi necessário desenvolver um método analítico consistindo de separação cromatográfica por HPLC e detecção e quantificação por UV-Vis. Não houve necessidade de recorrer a um passo de pré­ concentração de amostras por extracção em fase sólida (SPE) devido ao facto das concentrações de IB medidas ao longo do trabalho se terem sempre encontrado acima do LOQ (811 g L-1) do método analítico por injecção directa. Deste estudo pode concluir-se que o catalisador que apresentou melhor actividade catalítica e consequentemente maior remoção do IB em solução, foi o complexo de acetilacetonato de Ni (II), disperso em PDMS. Foi avaliada a influência, na conversão do IB, de diferentes parâmetros como a concentração inicial de peróxido de hidrogénio adicionada, quantidade de catalisador utilizada na mistura reaccional e temperatura. Os resultados permitiram concluir que os aumentos destes parâmetros conduzem a um aumento da actividade catalítica da reacção. A estabilidade catalítica do acetilacetonato de Ni (II)/PDMS, foi avaliada em ensaios consecutivos com a mesma amostra e nas mesmas condições, tendo-se observado que, após 8 utilizações, o catalisador perde ligeiramente a actividade (cerca de 11% do seu valor inicial). ABSTRACT: The presence and fate of pharmaceuticals in the aquatic environment is an emergent issue in environmental chemistry. Some compounds are poorly removed in wastewater treatment plants (WWTPs) while others, in spite of being partially removed, are still present in the WWTPs effluents and discharged in the receiving water bodies. Ibuprofen, IB, a non-steroid anti-inflammatory drug, is one of the most used and also one of the most frequently detected pharmaceutical contaminants in aquifers worldwide. Its removal by conventional wastewater treatment processes used in most WWTPs is usually high (up to 90% of incoming IB may be removed), but duet the high loads present in the influents, still significant amounts of IB usually leave the WWTPs in the treated effluents. The presence of these compounds in the environment must be evaluated considering that they may have some biological activity even at low concentrations. Advanced oxidation processes using hydrogen peroxide, in the presence of heterogeneous catalysts, provide a significantly improved removal of this type of substances from waters. Therefore, it was the aim of this work to study the use of hydrogen peroxide as an oxidizing agent in the removal of IB from aqueous solutions, in the presence of the catalyst nickel (II) acetylacetonate dispersed in PDMS or encapsulated in the NaY zeolite. For the quantification of the pharmaceutical in aqueous solution it was necessary to develop an analytical methodology based in chromatographic separation by HPLC and with UV-Vis detection and quantification. There was no need for a pre­concentration step of the samples by solid phase extraction (SPE) as the IB concentrations measured were always above the limit of quantification (811 bL1 of) the analytical method. The results from this study have shown that the catalyst which presented the best catalytic activity and the highest IB removal in solution was nickel (II) acetylacetonate dispersed in PDMS.