Tumor-related splicing variant RAC1b in colorectal cancer: regulation and role in tumorigenesis

Purpose: Alternative splicing of the small GTPase RAC1 generates RAC1b, a hyperactivated variant that is overexpressed in a subtype of colorectal tumors. The objective of our studies is to understand the molecular regulation of this alternative splicing event and how it contributes to tumorigenesis. Experimental description: The regulation of the RAC1b splicing event in human colon cell lines was dissected using a transfected RAC1 minigene and the role of upstream regulating protein kinases through an RNA interference approach. The functional properties of the RAC1b protein were characterized by experimental modulation of Rac1b levels in colon cell lines. Results: The RAC1b protein results from an in-frame inclusion of an additional alternative exon encoding 19 amino acids that change the regulation and signaling properties of the protein. RAC1b is a hyperactive variant that exists predominantly in the GTP-bound active conformation in vivo and promotes cell cycle progression and cell survival through activation of the transcription factor NF-κB. RAC1b overexpression functionally cooperates with the oncogenic mutation in BRAF-V600E to sustain colorectal tumor cell survival. The splicing factor SRSF1 was identified to bind an exonic splice enhancer element in the alternative exon and acts as a prime regulator of Rac1b alternative splicing in colorectal cells. SRSF1 is controlled by upstream protein kinase SRPK1, the inhibition or depletion of which led to reduced SRSF1 phosphorylation and nuclear translocation with a concomitant reduction in RAC1b levels. As further SRSF1-regulating pathways we discovered kinase GSK3 and a cyclooxygenase independent effect of the non-steroidal anti-inflammatory drug ibuprofen. Conclusions: Expression of tumor-related RAC1b in colorectal cancer depends critically on SRSF1 for the observed deregulation of alternative splicing during tumorigenesis and is controlled by upstream protein kinases that can be pharmacologically targeted.

Nickel dependent carcinogenesis and infections: structural and biophysical characterization of NDRG1 and SgSrnR

In prokaryotic organisms, lower eukaryotes and plants, some important biological reactions are catalyzed by nickel-dependent enzymes, making this metal ion essential microelement for their life. On the other hand, excessive concentration of nickel into the cell, or prolonged exposure to nickel compounds, has toxic effects in living organisms. In addition, nickel has been classified by IARC as Group I human carcinogen, because of the correlation between its inhalation and increased incidence of nasal and lung cancers. The aim of this work was to investigate the nickel impact on human health, considering both its direct role on human cells and its indirect effect as essential element for human important bacteria. In humans, nickel induces N-myc downstream regulated gene 1 (NDRG1) expression, recently proposed as new target in cancer therapy. CD, light scattering and ITC were applied on the recombinant full-length protein and its C-terminal intrinsically disordered domain, for studying the NDRG1 structural and functional properties. In particular, the fold and dynamics of the C-terminal region were examined by NMR spectroscopy and site-directed spin labeling coupled to EPR, showing the features of an intrinsically disordered region. In nickel-dependent bacteria, nickel metabolism is strictly regulated, through the activity of different transcription factors. In Streptomyces griseus the expression of two superoxide dismutases (SODs) is antagonistically regulated by nickel thanks to the transcriptional complex SgSrnR/SgSrnQ. The SgSrnR protein was heterologously expressed and its activity as possible nickel sensor studied. DNaseI footprinting and β-galactosidase gene reporter assays revealed that SgSrnR functions as transcriptional activator, prompting the hypothesis of a new model to describe the activity of this complex. In addition, ITC, NMR and X-ray crystallography demonstrated that SgSrnR presents the fold typical of ArsR/SmtB transcription factors and low metal binding affinity, non compatible with a role as a nickel-sensor, function probably played by its partner SgSrnQ.

Functional engineering of hybrid heterostructures for application in electronic, optical and optoelectronic nanostructured devices

Interfacing materials with different intrinsic chemical-physical characteristics allows for the generation of a new system with multifunctional features. Here, this original concept is implemented for tailoring the functional properties of bi-dimensional black phosphorus (2D bP or phosphorene) and organic light-emitting transistors (OLETs). Phosphorene is highly reactive under atmospheric conditions and its small-area/lab-scale deposition techniques have hampered the introduction of this material in real-world applications so far. The protection of 2D bP against the oxygen by means of functionalization with alkane molecules and pyrene derivatives, showed long-term stability with respect to the bare 2D bP by avoiding remarkable oxidation up to 6 months, paving the way towards ultra-sensitive oxygen chemo-sensors. A new approach of deposition-precipitation heterogeneous reaction was developed to decorate 2D bP with Au nanoparticles (NP)s, obtaining a “stabilizer-free” that may broaden the possible applications of the 2D bP/Au NPs interface in catalysis and biodiagnostics. Finally, 2D bP was deposited by electrospray technique, obtaining oxidized-phosphorous flakes as wide as hundreds of µm2 and providing for the first time a phosphorous-based bidimensional system responsive to electromechanical stimuli. The second part of the thesis focuses on the study of organic heterostructures in ambipolar OLET devices, intriguing optoelectronic devices that couple the micro-scaled light-emission with electrical switching. Initially, an ambipolar single-layer OLET based on a multifunctional organic semiconductor, is presented. The bias-depending light-emission shifted within the transistor channel, as expected in well-balanced ambipolar OLETs. However, the emitted optical power of the single layer-based device was unsatisfactory. To improve optoelectronic performance of the device, a multilayer organic architecture based on hole-transporting semiconductor, emissive donor-acceptor blend and electron-transporting semiconductor was optimized. We showed that the introduction of a suitable electron-injecting layer at the interface between the electron-transporting and light-emission layers may enable a ≈ 2× improvement of efficiency at reduced applied bias.

Polyhydroxyalkanoates production from salted food waste using mixed microbial cultures.

Plastic is an essential asset for the modern lifestyle, given its superiority as a material from the points of view of cost, processability and functional properties. However, plastic-related environmental pollution has become nowadays a very significant problem that can no longer be overlooked. For this reason, in recent decades, the research for new materials that could replace fossil fuel-based plastics has been focused on biopolymers with similar physicochemical properties to fossil fuel-based plastics, such as Polyhydroxyalkanoates (PHA). PHAs are a family of biodegradable polyesters synthesized by many microorganisms as carbon and energy reserves. PHA appears as a good candidate to substitute conventional petroleum-based plastics since it has similar properties, but with the advantage of being biobased and biodegradable, and has a wide range of applications (e.g., packaging). However, the PHA production cost is almost four times higher (€5/kg) than conventional plastic manufacturing. The PHA production by mixed microbial cultures (MMC) allows to reduce production costs as it does not require aseptic conditions and it enables the use of inexpensive by-products or waste streams as these cultures are more amenable to deal with complex feedstocks. Saline wastewaters (WWs), generated by several industries such as seafood, leather and dairy, are often rich in organic compounds and, due to a strong salt inhibition, the biological treatments are inefficient, and their disposal is expensive. These saline WWs are a potential feedstock for PHA production, as they are an inexpensive raw material. Moreover, saline WWs could allow the utilization of seawater in the process as dilution and cleaning agent, further decreasing the operational costs and the environmental burden of the process. The main goal of the current project is to assess and optimize the PHA production from a mixture of food waste and brine wastewater from the fishery industry by MMC.

Partitioning The Net Effect Of Host Diversity On An Emerging Amphibian Pathogen.

The 'dilution effect' (DE) hypothesis predicts that diverse host communities will show reduced disease. The underlying causes of pathogen dilution are complex, because they involve non-additive (driven by host interactions and differential habitat use) and additive (controlled by host species composition) mechanisms. Here, we used measures of complementarity and selection traditionally employed in the field of biodiversity-ecosystem function (BEF) to quantify the net effect of host diversity on disease dynamics of the amphibian-killing fungus Batrachochytrium dendrobatidis (Bd). Complementarity occurs when average infection load in diverse host assemblages departs from that of each component species in uniform populations. Selection measures the disproportionate impact of a particular species in diverse assemblages compared with its performance in uniform populations, and therefore has strong additive and non-additive properties. We experimentally infected tropical amphibian species of varying life histories, in single- and multi-host treatments, and measured individual Bd infection loads. Host diversity reduced Bd infection in amphibians through a mechanism analogous to complementarity (sensu BEF), potentially by reducing shared habitat use and transmission among hosts. Additionally, the selection component indicated that one particular terrestrial species showed reduced infection loads in diverse assemblages at the expense of neighbouring aquatic hosts becoming heavily infected. By partitioning components of diversity, our findings underscore the importance of additive and non-additive mechanisms underlying the DE.

Surfactina: propriedades químicas, tecnológicas e funcionais para aplicações em alimentos

Surfactin, a lipopeptide produced by strains of Bacillus subtilis, has been proved to be a suitable biosurfactant in several applications. For many years, it has been investigated mainly for oil recovery and environmental usage. Its chemical, technological and functional characteristics turn surfactin into an attractive compound for several utilizations. In this review we emphasize some aspects of surfactin as a new food ingredient and its potential pharmaceutical and health applications.

Carotenóides: propriedades, aplicações e biotransformação para formação de compostos de aroma

Carotenoids are widely distributed in nature, providing yellow, orange or red color in a great number of vegetables, microorganisms and in some animals. Carotenoids act as biological antioxidants and seem to play an important role in human health by protecting cells and tissues from the damaging effects of free radicals and singlet oxygen. Several authors describe the oxidative cleavage of carotenoids in flavor compounds as occuring through chemical or photochemical degradations or through biotechnological processes. Biotransformation of carotenoids seems to be a reasonable alternative to produce flavor compounds since these compounds are considered 'natural' ingredients. In this work we describe the properties of some carotenoids, as well as biotechnological approaches to obtain its oxyfunctionalized derivatives.

Context tree selection: A unifying view

Context tree models have been introduced by Rissanen in [25] as a parsimonious generalization of Markov models. Since then, they have been widely used in applied probability and statistics. The present paper investigates non-asymptotic properties of two popular procedures of context tree estimation: Rissanen's algorithm Context and penalized maximum likelihood. First showing how they are related, we prove finite horizon bounds for the probability of over- and under-estimation. Concerning overestimation, no boundedness or loss-of-memory conditions are required: the proof relies on new deviation inequalities for empirical probabilities of independent interest. The under-estimation properties rely on classical hypotheses for processes of infinite memory. These results improve on and generalize the bounds obtained in Duarte et al. (2006) [12], Galves et al. (2008) [18], Galves and Leonardi (2008) [17], Leonardi (2010) [22], refining asymptotic results of Buhlmann and Wyner (1999) [4] and Csiszar and Talata (2006) [9]. (C) 2011 Elsevier B.V. All rights reserved.

Heat-moisture treatment and enzymatic digestibility of Peruvian carrot, sweet potato and ginger starches

The aim of this work was to study the effects of heat-moisture treatment (27% moisture, 100 degrees C, 16 h) and of enzymatic digestion (alpha-amylase and glucoamylase) on the properties of sweet potato (SP), Peruvian carrot (PC) and ginger (G) starches. The structural modification with heat-moisture treatment (HMT) affected crystallinity, enzyme susceptibility and viscosity profile. The changes in PC starch were the most pronounced, with a strong decrease of relative crystallinity (from 0.31 to 0.21) and a shift of X-ray pattern from B- to A-type. HMTof SP and G starch did not change the Xray pattern (A-type). The relative crystallinity of these starches changed only slightly, from 0.32 to 0.29 (SP) and from 0.33 to 0.32 (G). The extent of these structural changes (PC > SP > G) altered the susceptibility of the starches to enzymatic attack, but not in same order (PC > G > SP). HMT increased the starches digestion, probably due to rearrangement of disrupted crystallites, increasing accessible areas to attack of enzymes. The viscosity profiles and values changed significantly with HMT, resulting in higher pasting temperatures, decrease of viscosity values and no breakdown, i.e., stability at high temperatures and shear rates. Changes in pasting properties appeared to be more significant for PC and SP starch, whereas the changes for G starch were small. Setback was minimized following HMT in SP and G starches.

Penetration of hydrolysed soy protein-added brine and its effect on yield and pH of beef steaks from the biceps femoris muscle

This study was aimed to evaluate the penetration behaviour of different brines with tumbled beef steaks from the biceps femoris muscle, specifically their interactions with pH and effects on yield. Six muscles from different animals, divided into origin (OP) and insertion (IP) portions, were cut into 60 steaks of 2.5 cm thickness and tumbled for 30 or 60 min. The steaks were tumbled with two brines, with (WTB/HSP) or without (WTB) hydrolysed soy protein (HSP), and steaks that were not tumbled with brine or water were used as controls. Brine penetration was verified by measuring the amount of dye-containing brine (absorbance at 627 nm) recovered from homogenates of four thin (2 mm) slices from the surface of the beef steaks after tumbling. The WTB/HSP steaks exhibited greater (P < 0.05) brine penetration when tumbled for 60 min than for 30 min. The OP steaks showed greater yield and lower pH (P < 0.05) than IP steaks. HSP-added brine increased the water absorption and retention in the first slices of the steaks, and its efficiency was increased with a longer tumbling time. The portion of the biceps femoris muscle used influenced brine absorption and retention, impacting meat yield. (C) 2010 Elsevier Ltd. All rights reserved.

Effect of the Partial Substitution of Soy Proteins by Highly Methyl-esterified Pectin on Chemical and Sensory Characteristics of Sausages

Pectin can be used as a natural emulsifier in food formulations. In this study, textured soybean protein (TSP), used as an emulsifier in commercial sausages, was partially replaced by a mixture containing pectin and isolated soybean proteins, which were either extruded (EXT) or not extruded (MIX), and the chemical and sensory characteristics of samples were evaluated after 60 days of storage at 4 degrees C. Responses such as oxidation measured by PV and TBARS, hardness, color, pH and sensory characteristics were compared with those of a commercial sausage (CON). The mixture containing highly methyl-esterified pectin, textured soybean proteins and isolated soybean proteins, as emulsifier agent, reduced the hardness (EXT: 21.69 +/- 0.98 and MIX: 20.17 +/- 2.76 N) and the pH (EXT: 5.46 +/- 0.03 and MIX: 5.29 +/- 0.01) of the samples and increased the concentration of peroxides (EXT: 0.10 +/- 0.01 and MIX: 0.15 +/- 0.01 meq/kg) when compared with samples formulated only with TSP (28.57 +/- 2.54 N, pH of 6.92 +/- 0.04 and PV = 0.07 +/- 0.01 meq/kg). These effects were likely caused by the anionic character of the emulsifier. However, no sensory difference was observed between the sausages containing highly methyl-esterified pectin, textured soybean proteins and isolated soybean proteins submitted to the extrusion process (EXT) and the control sausages, suggesting that the formulation proposed in this study can be a potential alternative for the further development of sausages that have functional properties or are free of artificial additives.

Brazil nut ingestion increased plasma selenium but had minimal effects on lipids, apolipoproteins, and high-density lipoprotein function in human subjects

The Brazil nut (Bertholletia excelsa) of the Amazon region is consumed worldwide. It is rich in both monounsaturated fatty acids and polyunsaturated fatty acids and is known for its high selenium content. This study tested the hypothesis whether the consumption of this nut could affect the plasma lipids and apolipoproteins and some functional properties of the antiatherogenic high-density lipoprotein (HDL). Fifteen normolipidemic subjects aged 27.3 +/- 3.9 years and with body mass index of 23.8 +/- 2.8 kg/m(2) consumed 45 g of Brazil nuts per day during a 15-day period. On days 0 and 15, blood was collected for biochemical analysis, determination of HDL particle size, paraoxonase 1 activity, and lipid transfer from a lipoprotein-like nanoparticle to the HDL fraction. Brazil nut ingestion did not alter HDL, low-density lipoprotein cholesterol, triacylglycerols, apolipoprotein A-1, or apolipoprotein B concentrations. HDL particle diameter and the activity of antioxidative paraoxonase 1, mostly found in the HDL fraction, Were also unaffected. Supplementation increased the reception of cholesteryl esters (P <.05) by the HDL yet did not alter the reception of phospholipids, free cholesterol, or triacylglycerols. As expected, plasma selenium was significantly increased. However, the consumption of Brazil nuts for short duration by normolipidemic subjects in comparable amounts to those tested for other nuts did not alter serum lipid profile. The only alteration in HDL function was the increase in cholesteryl ester transfer. This latter finding may be beneficial because it would improve the nonatherogenic reverse cholesterol transport pathway. (c) 2008 Elsevier Inc. All rights reserved.

Influence of Lactobacillus paracasei and inulin on instrumental texture and sensory evaluation of fresh cream cheese

The influence of the addition of a potential probiotic culture of Lactobacillus paracasei and of the prebiotic fiber inulin on the texture profile and on the sensory evaluation of probiotic and synbiotic fresh cream-cheeses was monitored. Three cheese-making trials were prepared in quintuplicate, all supplemented with a Streptococcus thermophilus starter culture (T1, T2 and T3). L. paracasei subsp. paracasei was added to T1 and T2, and inulin, to T2. The instrumental texture profile was determined after 1, 7, 14 and 21 days of storage of the cheeses. Sensory evaluation was performed after 7 days of storage. The presence of Lactobacillus paracasei in cheeses T1 and T2 and of inulin in cheeses T2 did not alter the texture profile significantly. Cheeses T1 were the least preferred in the sensory evaluation and differed signifcantly from T2 and T3, due to acidic taste, according to panelists. On the other hand, T2 was the most preferred one, though not significantly different from T3. The addition of the prebiotic ingredient inulin to fresh cream cheese processed with a potentially probiotic Lactobacillus paracasei strain resulted in a product with appropriate features and with aggregated functional properties.

Governing coordination: Behavioural principles and neural correlates

The coordination of movement is governed by a coalition of constraints. The expression of these constraints ranges from the concrete—the restricted range of motion offered by the mechanical configuration of our muscles and joints; to the abstract—the difficulty that we experience in combining simple movements into complex rhythms. We seek to illustrate that the various constraints on coordination are complementary and inclusive, and the means by which their expression and interaction are mediated systematically by the integrative action of the central nervous system (CNS). Beyond identifying the general principles at the behavioural level that govern the mutual interplay of constraints, we attempt to demonstrate that these principles have as their foundation specific functional properties of the cortical motor systems. We propose that regions of the brain upstream of the motor cortex may play a significant role in mediating interactions between the functional representations of muscles engaged in sensorimotor coordination tasks. We also argue that activity in these ldquosupramotorrdquo regions may mediate the stabilising role of augmented sensory feedback.

The sites of neural adaptation induced by resistance training in humans

Although it has long been supposed that resistance training causes adaptive changes in the CNS, the sites and nature of these adaptations have not previously been identified. In order to determine whether the neural adaptations to resistance training occur to a greater extent at cortical or subcortical sites in the CNS, we compared the effects of resistance training on the electromyographic (EMG) responses to transcranial magnetic (TMS) and electrical (TES) stimulation. Motor evoked potentials (MEPs) were recorded from the first dorsal interosseous muscle of 16 individuals before and after 4 weeks of resistance training for the index finger abductors (n = 8), or training involving finger abduction-adduction without external resistance (n = 8). TMS was delivered at rest at intensities from 5 % below the passive threshold to the maximal output of the stimulator. TMS and TES were also delivered at the active threshold intensity while the participants exerted torques ranging from 5 to 60 % of their maximum voluntary contraction (MVC) torque. The average latency of MEPs elicited by TES was significantly shorter than that of TMS MEPs (TES latency = 21.5 ± 1.4 ms; TMS latency = 23.4 ± 1.4 ms; P < 0.05), which indicates that the site of activation differed between the two forms of stimulation. Training resulted in a significant increase in MVC torque for the resistance-training group, but not the control group. There were no statistically significant changes in the corticospinal properties measured at rest for either group. For the active trials involving both TMS and TES, however, the slope of the relationship between MEP size and the torque exerted was significantly lower after training for the resistance-training group (P < 0.05). Thus, for a specific level of muscle activity, the magnitude of the EMG responses to both forms of transcranial stimulation were smaller following resistance training. These results suggest that resistance training changes the functional properties of spinal cord circuitry in humans, but does not substantially affect the organisation of the motor cortex.