Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells

Deciphering the regulation of perforin-2-mediated endocytic escape in dendritic cells

Dendritic cells (DCs) are important players in the initiation of cytotoxic CD8+ T cell-mediated immune responses against pathogens and tumours. DCs continuously sample their environment for antigens and load them on their MHC class I molecules to prime naïve CD8+ T cells in a process known as cross-presentation. Unlike most cell types, cross-presenting DCs have leaky endocytic compartments from where internalised proteins can escape into the cytosol for proteasome-mediated generation of MHC-I-binding peptides. Prior to my project, the Kozik lab had identified the pore-forming protein perforin-2 as a dedicated effector of endocytic escape that allows cytosolic entry of exogenous antigen during cross-presentation. Perforin-2 is a member of the membrane attack complex and perforin superfamily (MACPF) and, similar to perforin and complement, can form oligomeric pores on liposomes *in vitro*. However, the regulation of perforin-2 pore formation in the context of endocytic escape *in vivo* is still poorly understood. The aim of my PhD project was to decipher the mechanisms that enable DCs to assemble perforin-2 pores on their own membranes for endocytic escape without causing uncontrolled lysis of intracellular compartments. Analysis of its steady-state distribution showed that perforin-2 was proteolytically processed with full-length and cleaved forms present in early and late endocytic compartments, respectively. Since perforin-2 was recruited to antigen-containing phagosomes, I studied its stepwise proteolytic processing during phagosome maturation. Flow cytometry analysis of isolated phagosomes revealed that, in line with previous *in vitro* studies, perforin-2 processing was dependent on acidification. However, by developing an assay to monitor perforin-2 activity at the phagosome, I discovered that inhibiting acidification had no impact on the efficiency of perforin-2-mediated escape. In addition, perforin-2 activity had no detectable effect on the luminal pH or degradative potential of phagosomes. To better understand the role of pH-dependent processing of perforin-2 during phagosome maturation, I studied the proteases involved. The analysis showed that the pore-forming ectodomain was cleaved off the transmembrane domain by cysteine proteases and further processed by asparagine endopeptidase while the remaining membrane stub was removed by γ-secretase. Specific inhibition of these proteases had no impact on perforin-2-mediated endocytic escape suggesting that they are not directly required for pore formation. However, analysis of a perforin-2 trafficking mutant suggested that a currently unknown processing step may be necessary for efficient perforin-2 activity at the phagosome. In summary, this thesis offers novel insights into the regulation of perforin-2 which potentially releases antigens for cross-presentation early during phagosome maturation

Experimental determination of the PTW 60019 microDiamond dosimeter active area and volume

Purpose: Small field output correction factors have been studied by several research groups for the PTW 60019 micro Diamond (MD) dosimeter, by comparing the response of such a device with both reference dosimeters and Monte Carlo simulations. A general good agreement is observed for field sizes down to about 1 cm. However, evident inconsistencies can be noticed when comparing some experimental results and Monte Carlo simulations obtained for smaller irradiation fields. This issue was tentatively attributed by some authors to unintentional large variations of the MD active surface area. The aim of the present study is a nondestructive experimental determination of the MD active surface area and active volume. Methods: Ten MD dosimeters, one MD prototype, and three synthetic diamond samples were investigated in the present work. 2D maps of the MD response were recorded under scanned soft x-ray microbeam irradiation, leading to an experimental determination of the device active surface area. Profiles of the device responses were measured as well. In order to evaluate the MD active volume, the thickness of the diamond sensing layer was independently evaluated by capacitance measurements and alpha particle detection experiments. The MD sensitivity, measured at the PTW calibration laboratory, was also used to calculate the device active volume thickness. Results: An average active surface area diameter of (2.19±0.02) mm was evaluated by 2D maps and response profiles of all the MDS. Average active volume thicknesses of (1.01±0.13) μm and (0.97±0.14) μm were derived by capacitance and sensitivity measurements, respectively. The obtained results are well in agreement with the nominal values reported in the manufacturer dosimeter specifications. A homogeneous response was observed over the whole device active area. Besides the one from the device active volume, no contributions from other components of the housing nor from encapsulation materials were observed in the 2D response maps. Conclusions: The obtained results demonstrate the high reproducibility of the MD fabrication process. The observed discrepancies among the output correction factors reported by several authors for MD response in very small fields are very unlikely to be ascribed to unintentional variations of the device active surface area and volume. It is the opinion of the authors that the role of the volume averaging as well as of other perturbation effects should be separately investigated instead, both experimentally and by Monte Carlo simulations, in order to better clarify the behaviour of the MD response in very small fields. © 2016 American Association of Physicists in Medicine

Species diversification – which species should we use?

Large detector systems for particle and astroparticle physics; Particle tracking detectors; Gaseous detectors; Calorimeters; Cherenkov detectors; Particle identification methods; Photon detectors for UV. visible and IR photons; Detector alignment and calibration methods; Detector cooling and thermo-stabilization; Detector design and construction technologies and materials. The LHCb experiment is dedicated to precision measurements of CP violation and rare decays of B hadrons at the Large Hadron Collider (LHC) at CERN (Geneva). The initial configuration and expected performance of the detector and associated systems. as established by test beam measurements and simulation studies. is described. © 2008 IOP Publishing Ltd and SISSA