Spinophilin expression determines cellular growth, cancer stemness and 5-flourouracil resistance in colorectal cancer

The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC

Low spinophilin expression enhances aggressive biological behavior of breast cancer

Spinophilin, a putative tumor suppressor gene, has been shown to be involved in the pathogenesis of certain types of cancer, but its role has never been systematically explored in breast cancer. In this study, we determined for the first time the expression pattern of spinophilin in human breast cancer molecular subtypes (n = 489) and correlated it with survival (n = 921). We stably reduced spinophilin expression in breast cancer cells and measured effects on cellular growth, apoptosis, anchorage-independent growth, migration, invasion and self-renewal capacity in vitro and metastases formation in vivo. Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells. Spinophilin expression was significantly lower in basal-like breast cancer (p<0.001) and an independent poor prognostic factor in breast cancer patients (hazard ratio = 1.93, 95% confidence interval: 1.24-3.03; p = 0.004) A reduction of spinophilin levels increased cellular growth in breast cancer cells (p<0.05), without influencing activation of apoptosis. Anchorage-independent growth, migration and self-renewal capacity in vitro and metastatic potential in vivo were also significantly increased in spinophilin-silenced cells (p<0.05). Finally, we identified several differentially expressed genes in spinophilin-silenced cells. According to our data, low levels of spinophilin are associated with aggressive behavior of breast cancer

Micro electrochemical sensors and PCR systems: cellular and molecular tools for wine yeast analysis

Nowadays, exciting bioanalytical microsystems are currently receiving increasing attention in biology since they can comply with the considerable demand for reliable, sensitive and low-cost analysis tools. Small reagents volumes, low power consumption, portability, fast analysis, high throughput and systems integration are the key aspects that make these systems more and more appealing within both the academic and industrial communities. In the last years, many microdevices were developed for a wide range of biological applications, particularly dedicated to cellu-lar or molecular analysis. Many efforts were devoted to the realization of Cell-Based Biosensors (CBBs) to monitor the dynamic behaviour of cell cultures for pharmacological screening and basic research. Other researchers focused their interests in the development of so-called Lab-on-a-Chip (LOC) systems for DNA analysis mostly applied to clinical diagnosis. This thesis deals with the investigation of two miniaturized devices – a cell-based biosensor and a DNA amplification system – for the cellular and molecular analysis of wine yeasts, respectively. The first device consists of integrated electrochemical sensors – Ion-Sensitive Field-Effect Transistor (ISFET), impedimetric and temperature sensors – for the real time evaluation of pH and cell settling of yeasts under batch culture conditions. The assessment of yeast performance and robustness has been focused on ethanol tolerance, as it is one of the main stress factors acting in wine, and thus, one of the major causes of stuck fermentations. A good agreement between extracellular acidification and cell growth trends at different ethanol concentration has been demonstrated, significantly reducing the time of the traditional assays. Moreover, resistivity measurements have shown the possibility to follow progressive settling of the cell suspension. Concerning the second system, a Polymerase Chain Reaction (PCR) microdevice has been biologically validated by successfully amplifying yeast genomic DNA fragments. Additionally, the outcome of PCR has been positively assessed with diluted samples and boiled yeast cultures, demonstrating the possibility to skip the time-consuming purification process for potential LOC applications with very little or no pre-PCR sample manipulations. The encouraging results from both microsystems have demonstrated their suitability for wine yeast analysis, aimed at quality improvements of the winemaking process

A micro Polymerase Chain Reaction (uPCR) module for integrated and portable DNA analysis systems

This work deals with the design, fabrication and characterization of a disposable miniaturized Polymerase Chain Reaction (PCR) module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane) chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC). This PLCC is also useful avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nano-powder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy

Design and Fabrication of a Micro PCR Module for POC Applications

This work deals with the development of a disposable miniaturized Polymerase Chain Reaction (PCR) module that will be integrated in an innovative Lab on a Chip (LOC) as Point of Care Testing (POCT) platform to detect the susceptibility of complex diseases with genetic profiling. The amplification system consists of a micro-chamber reactor with a hybrid silicon-polymer structure. The temperature control system has been implemented by means of Platinum microheaters and thermometers integrated on a Silicon substrate and the reaction chamber has been completely made of Polydimethylsiloxane (PDMS) since it is biocompatible, transparent and easily moldable. To assure a perfect sealing, an oxygen plasma bonding process step has been implemented. The device design has been supported by analytical and finite elements simulations in such a way to evaluate the thermal requirements. A first PCR chamber prototype has been fabricated and packaged

Development and characterization of a multiparametric microsensor for yeast cell growth monitoring

In this work, the development and testing of a microfabricated multiparametric sensor for rapid cell growth monitoring is described, especially focused on yeast quality assessment for wine applications. The device consists of two integrated microsensors (pH, impedance), able to monitor extracellular metabolism. Microbial growth has been performed both in standard culture conditions and in presence of ethanol (12% v/v) in order to carry out a common screening of wine yeast strains. Cell growth tests can be performed in just three hours, providing a fast, reliable, sensitive and low cost analysis with respect to the conventional procedures

A multiparametric electrochemical microsensor for wine yeast quality assessment

This work is aimed at the realization of an integrated platform for high-throughput screening of wine yeast strains in order to improve the overall quality and productivity of wine making process. The approach is based on a multiparametric sensors integrated with a non-standard fabrication process derived from a 4µm Al-gate CMOS technology, allowing the on-line monitoring of pH, temperature and impedance of yeast cultures for the characterization of ethanol resistance of yeasts

Recent Proton and Co60 Radiation Test Data from a Newly Developed European Optocoupler Source for space Application

The permanent degradation introduced in the main electrical parameters of a new optocoupler type is described, as a function of proton fluence and Co-60 total ionizing dose. These results refer to Optoi’s devices assembled in Leadless Chip Carrier packages, coded OIER10 and developed in the framework of an ECI2 project for ESA, aimed at the ESCC component qualification