M.C. Santos

Molecular determinants of Akt-induced keratinocyte transformation

The PI3K/PTEN/Akt signaling pathway has emerged in recent years as a main player in human cancers, increasing proliferation and decreasing apoptosis of transformed cells, and thus becoming a potential target for therapeutic intervention. Our previous data have demonstrated that Akt-mediated signaling is of a key relevance in the mouse skin carcinogenesis system, one of the best-known models of experimental carcinogenesis. Here, we investigated the involvement of several pathways as mediators of Akt-induced increased proliferation and tumorigenesis in keratinocytes. Tumors produced by subcutaneous injection of Akt-transformed keratinocytes showed increased Foxo3a phosphorylation, but no major alterations in p21Cip1/WAF1, p27Kip1 or mdm2 expression and/or localization. In contrast, we found increased expression and nuclear localization of ΔNp63, β-catenin and Lef1. Concomitantly, we also found increased expression of c-myc and CycD1, targets of the β-catenin/Tcf pathway. Such increase is associated with increased phosphorylation and stabilization of c-myc protein as well as increased translation of c-myc and CycD1 due to mTOR activation. Using immunohistochemistry approaches in samples of oral dysplasias and human head and neck squamous cell carcinomas, we confirmed that increased Akt activation significantly correlates with increased ΔNp63 and CycD expression, c-myc phosphorylation and nuclear accumulation of β-catenin. Collectively, these results demonstrate that Akt is able to transform keratinocytes by specific mechanisms involving transcriptional and post-transcriptional processes.

E2F1 loss induces spontaneous tumour development in Rb-deficient epidermis

The specific ablation of Rb1 gene in epidermis (RbF/F;K14cre) promotes proliferation and altered differentiation but does not produce spontaneous tumour development. These phenotypic changes are associated with increased expression of E2F members and E2F-dependent transcriptional activity. Here, we have focused on the possible dependence on E2F1 gene function. We have generated mice that lack Rb1 in epidermis in an inducible manner (RbF/F;K14creERTM). These mice are indistinguishable from those lacking pRb in this tissue in a constitutive manner (RbF/F;K14cre). In an E2F1-null background (RbF/F;K14creERTM; and E2F1−/− mice), the phenotype due to acute Rb1 loss is not ameliorated by E2F1 loss, but rather exacerbated, indicating that pRb functions in epidermis do not rely solely on E2F1. On the other hand, RbF/F;K14creERTM;E2F1−/− mice develop spontaneous epidermal tumours of hair follicle origin with high incidence. These tumours, which retain a functional p19arf/p53 axis, also show aberrant activation of β-catenin/Wnt pathway. Gene expression studies revealed that these tumours display relevant similarities with specific human tumours. These data demonstrate that the Rb/E2F1 axis exerts essential functions not only in maintaining epidermal homoeostasis, but also in suppressing tumour development in epidermis, and that the disruption of this pathway may induce tumour progression through specific alteration of developmental programs.

Altered T cell differentiation and Notch signaling induced by the ectopic expression of keratin K10 in the epithelial cells of the thymus

Transgenic mice expressing hK10 under the keratin K5 promoter display several alterations in the epidermis including decreased cell proliferation, and reduced susceptibility to tumor development. Given that K5 promoter is also active in the epithelial cells of the thymus, we explored the possible alterations of the thymus because of K10 transgene expression. We found severe thymic alterations, which affect not only the thymic epithelial cells (TEC), but also thymocytes. We observed altered architecture and premature thymus involution in the transgenic mice associated with increased apoptosis and reduced proliferation of the thymocytes. Interestingly, prior to the development of this detrimental phenotype, thymocytes of the transgenic mice also displayed altered differentiation, which is aggravated later on. Molecular characterization of this phenotype indicated that Akt activity is reduced in TEC, but not in thymocytes. In addition, we also observed altered expression of Notch family members and some of their ligands both in TEC and T cells. This produces reduced Notch activity in TEC but increased Notch activity in thymocytes, which is detectable prior to the disruption of the thymic architecture. In addition, we also detect altered Notch expression in the epidermis of bK5hK10 transgenic mice. Collectively the present data indicate that keratin K10 may induce severe alterations not only in a cell autonomous manner, but also in neighboring cells by the modulation of signals involved in cell–cell interactions.

Skin Tumors Rb(eing) Uncovered

The Rb1 gene was the first bona fide tumor suppressor identified and cloned more than 25u2009years ago. Since then, a plethora of studies have revealed the functions of pRb and the existence of a sophisticated and strictly regulated pathway that modulates such functional roles. An emerging paradox affecting Rb1 in cancer connects the relatively low number of mutations affecting Rb1 gene in specific human tumors, compared with the widely functional inactivation of pRb in most, if not in all, human cancers. The existence of a retinoblastoma family of proteins pRb, p107, and p130 and their potential unique and overlapping functions as master regulators of cell cycle progression and transcriptional modulation by similar processes, may provide potential clues to explain such conundrum. Here, we will review the development of different genetically engineered mouse models, in particular those affecting stratified epithelia, and how they have offered new avenues to understand the roles of the Rb family members and their targets in the context of tumor development and progression.

Fractional Distillation of Organic Liquid Compounds Produced by Catalytic Cracking of Fats, Oils, and Grease

This work aims to investigate the fractional distillation of organic liquid products (OLP) obtained by catalytic cracking of palm oil (Elaeis guineensis Jacq.) at 450°C, 1.0 atm, with 5, 10, and 15% (wt) Na 2 CO 3 , using a stirred tank reactor of 143 L. The fractional distil‐ lations of OLP were carried out in laboratory scale with and without reflux using col‐ umns of different heights, and a pilot‐packed distillation column with internal reflux. OLP and distillation fractions (gasoline, kerosene, light diesel, and heavy diesel) were physicochemically characterized for density, kinematic viscosity, acid value, saponi‐ fication value, refractive index, flash point, and copper strip corrosion. The OLP and light diesel fractions were analyzed by Fourier transform infrared spectroscopy (FT‐IR) and gas chromatography‐mass spectrometry (GC‐MS). For the experiments in labora‐ tory scale, the yields of distillates decrease along with column height, with and without reflux, while those of bottoms products increase. The yields of distillates and gas increase with increasing Na 2 CO 3 content, while those of bottoms products decrease. The densities of gasoline, kerosene, and light diesel produced in laboratory scale with reflux superpose exactly those of kerosene, light diesel, and heavy diesel produced in laboratory scale without reflux. The kinematic viscosity decreases with increasing column height for the experiments in laboratory scale. The acid values of distillation fractions decrease along with the column height for the experiments with and without reflux. The FT‐IR of distil‐ lation fractions in pilot and laboratory scales identified the presence of aliphatic hydro‐ carbons and oxygenates. The GC‐MS analysis identified OLP composition of 92.84% (area) hydrocarbons and 7.16% (area) oxygenates. The light diesel fraction contains 100% hydrocarbons with an acid value of 0.34 mg KOH/g, proving the technical feasibility of OLP de‐acidification by the fractional distillation process.

Production and Characterization of Energy Materials with Adsorbent Properties by Hydrothermal Processing of Corn Stover with Subcritical H2O

This work aims to investigate the effect of temperature on the process performance of hydrothermal processing (HTC) of corn Stover with subcritical H 2 O and on the morphology of solid products. The experiments were carried out at 200, 225 and 250 ÂoC, reaction time of 240 minutes, heating rate of 2.0 ÂoC/min, and biomass to water ratio of 1:10, using a pilot scale stirred tank reactor (STR) of 5 gallon, operating in batch mode. The process performance analyzed by computing the yields of solid and liquid reaction products (RLP). The aqueous phase (H 2 O + RLP) was physicochemical analyzed for pH and total carboxylic acids, expressed as total acetic acid content. The chemical compositions of carboxylic acids, furfural, and hydroxymethylfurfural (HMF) in the aqueous phase determined by GC-MS and HPLC. The results showed solid yields ranging from 57.39 to 35.82% (wt.), and liquid reaction products (RLP) yields ranging from 39.53 to 54.59% (wt.). The solid phase products were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). The chemically activated (2.0 M NaOH) solid phase energy material obtained by HTC at 250°C, applied as adsorbent to investigate the capacity and/or efficiency to adsorb acetic acid from 1.0 to 4.0 g/L model solutions at 25 °C. The solid phase yield decreases along with the temperature, showing an inflection region between 200 and 225 °C, whereas a drastic change takes place, while that of liquid phase increases, showing also a drastic change between 200 and 225 °C. The total acetic acid content of aqueous phase varied from 4064 to 5387 mg/L, while the pH from 3.77 to 3.91. The GC analysis identified the presence of volatile carboxylic acids, particularly acetic acid, in concentrations between 4020 and 5040 mg/L. HPLC identified the presence of furfural and hydroxymethylfurfural, whose concentrations decrease exponentially and linearly along with the temperature between 686.7 and 0.0, and 443.9 and 0.0 mg/L, respectively, being both compounds not detectable at 250 °C. The elemental/ultimate analysis of solid products shows that carbon content increases, while the oxygen and hydrogen contents decrease, along with the temperature. The H/C and O/C ratios decrease linearly as process temperature increases, and the high heating value (HHV) of solid reaction products, an energy densified material, changes sharply between 200 and 225 °C, showing an increase with temperature. The SEM, EDX, and XDR indicates a change on the morphology and mineralogical phases present in solid reaction products with temperature, particularly at 250 °C. The activated solid phase has proven to be very selective to adsorb acetic acid, showing that recovery of acetic acid from hydrothermal carbonization/liquefaction aqueous solutions is feasible by using a multistage-stage adsorption process in series.