HIF1α regulates Mitochondrial biogenesis and Cellular senescence induced by Gamma Radiation

Cellular response to γ-rays is mediated by ATM-p53 axis. When p53 is phosphorylated, it can transactivate several genes to induce permanent cell cycle arrest (senescence) or apoptosis. Epithelial and mesenchymal cells are more resistant to radiation-induced apoptosis and respond mainly by activating senescence. Hence, tumor cells in a senescent state might remain as “dormant” malignant in fact through disruption of p53 function, cells may overcome growth arrest. Oncocytic features were acquired in the recurring neoplasia after radiation therapy in patient with colonrectal cancer. Oncocytic tumors are characterized by aberrant biogenesis and are mainly non-aggressive neoplasms. Their low proliferation degree can be explained by chronic destabilization of HIF1α, which presides to adaptation to hypoxia and also plays a pivotal role in hypoxia-related radio-resistance. The aim of the present thesis was to verify whether mitochondrial biogenesis can be induced following radiation treatment, in relation of HIF1α status and whether is predictive of a senescence response. In this study was demonstrate that mitochondrial biogenesis parameters like mitochondrial DNA copy number could be used for the prediction of hypoxic status of tissue after radiation treatment. γ-rays induce an increase of mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence. Mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a MDM2-mediated HIF1α degradation, leading to the release of PGC-1β inhibition by HIF1α. On the other hand, this protein blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally in vivo, post-radiotherapy mtDNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of senescence.

Characterization and cloning of chemically-induced mutants in barley (Hordeum vulgare L.)

Induced mutagenesis has been exploited for crop improvement and for investigating gene function and regulation. To unravel molecular mechanisms of stress resilience, we applied state-of-the-art genomics-based gene cloning methods to barley mutant lines showing altered root and shoot architecture and disease lesion mimic phenotypes. With a novel method that we named complementation by sequencing, we cloned NEC3, the causal gene for an orange-spotted disease lesion mimic phenotype. NEC3 belongs to the CYP71P1 gene family and it is involved in serotonin biosynthesis. By comparative phylogenetic analysis we showed that CYP71P1 emerged early in angiosperm evolution but was lost in some lineages including Arabidopsis thaliana. By BSA-Seq, we cloned the gene whose mutation increased leaf width, and we showed that the gene corresponded to the previously cloned BROADLEAF1. By BSA coupled to WGS sequencing, we cloned EGT1 and EGT2, two genes that regulate root gravitropic set point angle. EGT1 encodes a Tubby-like F-box protein and EGT2 encodes a Sterile Alpha Motive protein; EGT2 is phylogenetically related to AtSAM5 in Arabidopsis and to WEEP in peach where it regulates branch angle. Both EGT1 and EGT2 are conserved in wheat. We hypothesized that both participate to an anti-gravitropic offset mechanism since their disruption causes mutant roots to grow along the gravity vector. By the MutMap+ method, we cloned the causal gene of a short and semi-rigid root mutant and found that it encodes for an endoglucanase and is the ortholog of OsGLU3 in rice whose mutant has the same phenotype, suggesting that the gene is conserved in barley and rice. The mutants and the corresponding genes which were cloned in this work are involved in the response to stress and can potentially contribute to crop adaptation.