Poliamines en estrès abiòtic i mecanismes moleculars

Les poliamines (PAs) putrescina (Put), espermidina (Spd) i espermina (Spm) són mol•lècules policatiòniques de baix pes molecular, presents en els microorganismes, animals i plantes. Les PAs han estat implicades en diversos processos cel•lulars importants, incloent la resposta de les plantes a l'estrès. No obstant això, el seu mode d'acció està es desconeix. En les plantes, es van acumulant evidències de que les PAs interactuen amb macromolècules i estructures cel•lulars, com ara proteïnes de membrana, i la seva possible participació en transducció de senyals s'ha convertit en una creixent i interessant àrea d'estudi. En aquesta tesi, la possible interacció entre les poliamines i les vies de senyalització de fosfolípids és investigada. Resultats previs, han posat de manifest que alteracions en els nivells endògens de poliamines (PAs), per sobreexpressió gènica o pèrdua de funció de gens de biosíntesi, redueixen o milloren, respectivament, la capacitat de les plantes d'Arabidopsis per tolerar agressions per estrès abiòtic, produint en alguns casos notables alteracions en el desenvolupament. En aquestes plantes amb nivells alterats de PAs s'han detectat canvis importants en l'expressió gènica i s'ha trobat una connexió entre el contingut de PAs ii la biosíntesi / senyalització d'àcid abscísic (ABA). La hipòtesi actual de treball és que aquestes alteracions en l'expressió gènica poden estar mediades, si més no en part, pel catabolisme de PAs, i l’acció directa o indirecta de les espècies reactives d'oxigen (ROS) que se’n deriven.

Assessment of insecticide resistance in eggs and neonate larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Spanish Cydia pomonella (L.) field populations have developed resistance to several insecticide groups. Diagnostic concentrations were established as the LC90 calculated on a susceptible strain (S_Spain) for five and seven insecticides and tested on eggs and neonate larvae field populations, respectively. The three most relevant enzymatic detoxification systems (mixed-function oxidases (MFO), glutathione S-tranferases (GST) and esterases (EST)) were studied for neonate larvae. In eggs, 96% of the field populations showed a significantly lower efficacy when compared with the susceptible strain (S_Spain) and the most effective insecticides were fenoxycarb and thiacloprid. In neonate larvae, a significant loss of susceptibility to the insecticides was detected. Flufenoxuron, azinphos-methyl and phosmet showed the lowest efficacy, while lambda-cyhalothrin, alpha-cypermethrin and chlorpyrifos-ethyl showed the highest. Biochemical assays showed that the most important enzymatic system involved in insecticide detoxification was MFO, with highest enzymatic activity ratios (5.1–16.6 for neonates from nine field populations). An enhanced GST and EST activities was detected in one field population, with enzymatic activity ratios of threefold and fivefold for GST and EST, respectively, when compared with the susceptible strain. The insecticide bioassays showed that the LC90 used were effective as diagnostic concentrations. Measures of MFO activity alongside bioassays with insecticide diagnostic concentrations could be used as tools for monitoring insecticide resistance in neonate larvae of C. pomonella.

NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.