Role of heme regulated eIF2α kinase in pancreatic beta cell function and viability

The eukaryotic translation initiation factor 2 alpha (eIF2α) is part of the initiation complex that drives the initiator amino acid methionine to the ribosome, a crucial step in protein translation. In stress conditions such as virus infection, endoplasmic reticulum (ER) stress, amino acid or heme deficiency eIF2α can be phosphorylated and thereby inhibit global protein synthesis. This adaptive mechanism prevents protein accumulation and consequent cytotoxic effects. Heme-regulated eIF2α kinase (HRI) is a member of the eIF2α kinase family that regulates protein translation in heme deficiency conditions. Although present in all tissues, HRI is predominantly expressed in erythroid cells where it remains inactive in the presence of normal heme concentrations. In response to heme deficiency, HRI is activated and phosphorylates eIF2α decreasing globin synthesis. This mechanism is important to prevent accumulation of heme-free globin chains which cause ER stress and apoptosis. RNA sequencing data from our group showed that in human islets and in primary rat beta cells HRI is the most expressed eIF2α kinase compared to the other family members. Despite its high expression levels, little is known about HRI function in beta cells. The aim of this project is to identify the role of HRI in pancreatic beta cells. This was investigated taking a loss-of-function approach. HRI knock down (KD) by RNA interference induced beta cell apoptosis in basal condition. HRI KD potentiated the apoptotic effects of palmitate or proinflammatory cytokines, two in vitro models for type 2 and type 1 diabetes, respectively. Increased cytokine-induced apoptosis was also observed in HRI-deficient primary rat beta cells. Unexpectedly, we observed a mild increase in eIF2α phosphorylation in HRI-deficient cells. The levels of mRNA or protein expression of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) were not modified. HRI KD cells have decreased spliced X-box binding protein 1 (XBP1s), an important branch of the ER stress response. However, overexpression of XBP1s by adenovirus in HRI KD cells did not protect from HRI siRNA-induced apoptosis. HRI deficiency decreased phosphorylation of Akt and its downstream targets glycogen synthase kinase 3 (GSK3), forkhead box protein O1 (FOXO1) and Bcl-2-associated death promoter (BAD). Overexpression of a constitutively active form of Akt by adenovirus in HRI-deficient beta cells partially decreased HRI KD-mediated apoptosis. Interestingly, BAD silencing protected from apoptosis caused by HRI deficiency. HRI silencing in beta cells also induced JNK activation. These results suggest an important role of HRI in beta cell survival through modulation of the Akt/BAD pathway. Thus, HRI may be an interesting target to modulate beta cell fate in diabetic conditions.

Self-standing curved crystals for X and Gamma rays focusing

The efficiency of a Laue lens for X and Gamma ray focusing in the energy range 60 ÷ 600 keV is closely linked to the diffraction efficiency of the single crystals composing the lens. A powerful focusing system is crucial for applications like medical imaging and X ray astronomy where wide beams must be focused. Mosaic crystals with a high density, such as Cu or Au, and bent crystals with curved diffracting planes (CDP) are considered for the realization of a focusing system for X rays, owing to their high diffraction efficiency. In this work, a comparison of the efficiency of CDP crystals and mosaic crystals was performed on the basis of the theory of X-ray diffraction. Si, GaAs and Ge CDP crystals with optimized thicknesses and moderate radii of curvature of several tens of metres demonstrate comparable or superior performance with respect to the higher atomic number mosaic crystals generally used. A simplified approach for calculating the integrated reflectivity of the crystals is applied. A bending technique used during this work to realize CDP crystals consists in a controlled surface damaging induced by a mechanical lapping process. A compressive strained layer of few micrometres in thickness is generated and causes the convex curvature of the damaged side of the crystal. Another new bending technique is developed and the main results are shown. The process consists on a film deposition of a selected bi-component epoxy resin on one side of crystal, made uniform in thickness by mean of a spin-coater. Choosing the speed of spin-coating, so changing the thickness of the film, a control of radius of curvature can be obtained. Moreover the possibility to combine the two bending technique to obtain CDP crystal with a stronger curvature in rather thick crystals was demonstrated. Detailed characterization of Si, and GaAs CDP crystals at low and high x-ray energies are performed on flat and bent crystals obtained with the damaging and the resin deposition technique. As expected an increase of diffraction efficiency in asymmetrical diffraction geometry in CDP crystals with respect to the flat ones is observed. On the other hand an unexpected increase of the integrated intensity in symmetrical geometry, not predicted by the theory, is observed in all the measurements performed with different set up. The experimental trend of the integrated reflectivity as a function of the radius of curvature is in a good agreement with that predicted by the theory of bent perfect crystals, so it is possible to conclude that the surface damage has a limited effect on the crystal reflectivity. A study of the integrated reflectivity in the energy range of interest (100÷350 keV) in CDP crystals realized with damaging and resin deposition technique at symmetrical and asymmetrical geometries was performed at ILL Institute. Also at these energies the diffraction efficiency of bent crystals was much larger (a 12 time increase is observed for bent crystals in asymmetrical 111 geometry) than that measured in flat crystals. The diffraction efficiency of CDP crystals realized with both techniques tends to coincide with that of flat crystals at very high energies (> 200 keV). This suggesting that also real flat perfect crystals can be considered as strongly bent or mosaic crystals at very high X ray energies.