Die Regulation der Ubiquitinligase CHIP durch das Cochaperon BAG2 im zellulären System und im Kontext der replikativen Seneszenz

Eine funktionierende Proteinqualitätskontrolle ist essenziell für die Vitalität einer Zelle. Das dynamische Gleichgewicht zwischen Proteinfaltung und -degradation wird von molekularen Chaperonen aufrechterhalten, deren Aktivität wiederum durch die Interaktion mit zahlreichen Cochaperonen moduliert wird. Das Cochaperon CHIP ist ein zentraler Faktor in Proteintriage-Entscheidungsprozessen, da es als Ubiquitinligase Chaperonsubstrate dem Abbau zuführt und somit die Chaperonmaschinerie direkt mit den Systemen der Proteindegradation verbindet. Um Polypeptide vor einem vorzeitigen Abbau zu schützen, wird die destruktive Aktivität von CHIP durch weitere Cochaperone reguliert. rnIn dieser Arbeit konnte die Hemmung der Ligaseaktivität von CHIP durch das Cochaperon BAG2 mechanistisch erstmals in einem zellulären System nachgewiesen werden. Dazu wurde die humane IMR-90 Fibroblasten Zelllinie verwendet. Die Ubiquitinierungsaktivität von CHIP wurde anhand von HSP72 als Modell-CHIP-Substrat untersucht. Durch die verringerte Ubiquitinierung, und damit dem reduzierten Abbau von HSP72, regulierte BAG2 dessen intrazelluläre Proteinspiegel, ohne dabei selbst eine Hitzeschockantwort zu induzieren. Überexprimiertes BAG2 wirkte sich trotz stabilisierter HSP72-Spiegel bei einem appliziertem Hitzestresses negativ auf die Zellvitalität aus, vermutlich da BAG2 durch die Inhibition von CHIP-vermittelter Ubiquitinierung massiv in das Gleichgewicht zwischen Substratfaltung und -degradation eingreift.rnDa sich die Mechanismen der Proteinqualitätskontrolle in der Alterung stark verändern und sich den wandelnden Bedingungen in der Zelle anpassen, wurde in einem zweiten Teil dieser Arbeit mit Hilfe des IMR-90 Zellsystems als etabliertes Modell zellulärer Seneszenz analysiert, inwieweit sich die Aktivität und die Regulation von CHIP durch BAG2 in der zellulären Alterung ändern. In seneszenten Zellen war HSP72 erheblich weniger ubiquitiniert als in jungen Fibroblasten, was auf eine reduzierte CHIP-Aktivität hinweist. Diese blieb jedoch durch BAG2 weiterhin modulierbar. Die Funktion von BAG2 als Inhibitor der Ubiquitinligase CHIP blieb demnach in seneszenten Zellen bestehen. In gealterten Fibroblasten regulierte BAG2 außerdem die Proteinspiegel des CHIP-Substrates und Seneszenzinitiators p53, was BAG2 eine mögliche Rolle in der Etablierung des Seneszenz-Phänotyps zuspricht. Weiterhin unterlagen die Proteinspiegel der beiden funktionell redundanten CHIP-Modulatoren BAG2 und HSPBP1 in der zellulären Alterung einer reziproken Regulation. In gealterten Mäusen trat die gegenläufige Veränderung der beiden Cochaperone gewebsspezifisch in der Lunge auf. Außerdem waren die BAG2-Proteinspiegel im Hippocampus gealterter Tiere signifikant erhöht.rnZusammenfassend konnte anhand der erzielten Ergebnisse die Funktion von BAG2 als Inhibitor von CHIP im zellulären System bestätigt werden. Außerdem durchlaufen die Aktivität und die Regulation von CHIP einen seneszenzspezifischen Adaptationsprozess, welcher für die Erhaltung der Proteostase in der Alterung relevant sein könnte und in welchem die Funktion von BAG2 als CHIP-Modulator möglicherweise eine wichtige Rolle spielt.rnZukünftige Studien könnten die komplexen Mechanismen weiterführend aufklären, mit denen CHIP-Aktivität reguliert wird. Dies kann helfen, der altersbedingten Abnahme an proteostatischer Kontrolle entgegenzuwirken und aberrante Proteinaggregation in altersassoziierten Erkrankungen vorzubeugen.rn

Implementazione dell'algoritmo filtered back-projection (FBP) per architetture low-power di tipo systems-on-chip

Il presente lavoro di tesi, svolto presso i laboratori dell'X-ray Imaging Group del Dipartimento di Fisica e Astronomia dell'Università di Bologna e all'interno del progetto della V Commissione Scientifica Nazionale dell'INFN, COSA (Computing on SoC Architectures), ha come obiettivo il porting e l’analisi di un codice di ricostruzione tomografica su architetture GPU installate su System-On-Chip low-power, al fine di sviluppare un metodo portatile, economico e relativamente veloce. Dall'analisi computazionale sono state sviluppate tre diverse versioni del porting in CUDA C: nella prima ci si è limitati a trasporre la parte più onerosa del calcolo sulla scheda grafica, nella seconda si sfrutta la velocità del calcolo matriciale propria del coprocessore (facendo coincidere ogni pixel con una singola unità di calcolo parallelo), mentre la terza è un miglioramento della precedente versione ottimizzata ulteriormente. La terza versione è quella definitiva scelta perché è la più performante sia dal punto di vista del tempo di ricostruzione della singola slice sia a livello di risparmio energetico. Il porting sviluppato è stato confrontato con altre due parallelizzazioni in OpenMP ed MPI. Si è studiato quindi, sia su cluster HPC, sia su cluster SoC low-power (utilizzando in particolare la scheda quad-core Tegra K1), l’efficienza di ogni paradigma in funzione della velocità di calcolo e dell’energia impiegata. La soluzione da noi proposta prevede la combinazione del porting in OpenMP e di quello in CUDA C. Tre core CPU vengono riservati per l'esecuzione del codice in OpenMP, il quarto per gestire la GPU usando il porting in CUDA C. Questa doppia parallelizzazione ha la massima efficienza in funzione della potenza e dell’energia, mentre il cluster HPC ha la massima efficienza in velocità di calcolo. Il metodo proposto quindi permetterebbe di sfruttare quasi completamente le potenzialità della CPU e GPU con un costo molto contenuto. Una possibile ottimizzazione futura potrebbe prevedere la ricostruzione di due slice contemporaneamente sulla GPU, raddoppiando circa la velocità totale e sfruttando al meglio l’hardware. Questo studio ha dato risultati molto soddisfacenti, infatti, è possibile con solo tre schede TK1 eguagliare e forse a superare, in seguito, la potenza di calcolo di un server tradizionale con il vantaggio aggiunto di avere un sistema portatile, a basso consumo e costo. Questa ricerca si va a porre nell’ambito del computing come uno tra i primi studi effettivi su architetture SoC low-power e sul loro impiego in ambito scientifico, con risultati molto promettenti.

Surface-acoustic-wave counterflow micropumps for on-chip liquid motion control in two-dimensional microchannel arrays

Fully controlled liquid injection and flow in hydrophobic polydimethylsiloxane (PDMS) two-dimensional microchannel arrays based on on-chip integrated, low-voltage-driven micropumps are demonstrated. Our architecture exploits the surface-acoustic-wave (SAW) induced counterflow mechanism and the effect of nebulization anisotropies at crossing areas owing to lateral propagating SAWs. We show that by selectively exciting single or multiple SAWs, fluids can be drawn from their reservoirs and moved towards selected positions of a microchannel grid. Splitting of the main liquid flow is also demonstrated by exploiting multiple SAW beams. As a demonstrator, we show simultaneous filling of two orthogonal microchannels. The present results show that SAW micropumps are good candidates for truly integrated on-chip fluidic networks allowing liquid control in arbitrarily shaped two-dimensional microchannel arrays.

Using the Franck-Hertz Experiment To Illustrate Quantization Energy States of the Neon Atom by Electron Impact

na provide students with motivation for the study of quantum mechanics. That microscopic matter exists in quantized states can be demonstrated with modem versions of historic experiments: atomic line spectra (I), resonance potentials, and blackbody radiation. The resonance potentials of mercury were discovered by Franck and Hertz in 1914 (2). Their experiment consisted of bombarding atoms by electrons, and detecting the kinetic energy loss of the scattered electrons (3). Prior to the Franck-Hertz experiment, spectroscopic work bv Balmer and Rvdbere revealed that atoms emitted radiatibn at discrete ekergiis. The Franck-Hertz experiment showed directly that auantized enerm levels in an atom are real, not jist optiEal artifacts. atom can be raised to excited states by inelastic collisions with electrons as well as lowered from excited states by emission of photons. The classic Franck-Hertz experiment is carried out with mercury (4-7). Here we present an experiment for the study of resonance potentials using neon.

Synthesis of Macrocyclic Polymers Formed via Intramolecular Radical Trap-Assisted Atom Transfer Radical Coupling

The synthesis of cyclic polystyrene (Pst) with an alkoxyamine functionality has been accomplished by intramolecular radical coupling in the presence of a nitroso radical trap Linear alpha,omega-dibrominated polystyrene, produced by the atom transfer radical polymerization (ATRP) of styrene using a dibrominated initiator, was subjected to chain-end activation via the atom transfer radical coupling (ATRC) process under pseudodilute conditions in the presence of 2-methyl-2-nitrosopropane (MNP). This radical trap-assisted, intramolecular ATRC (RTA-ATRC) produced cyclic polymers in greater than 90% yields possessing < G > values in the 0.8-0.9 range as determined by gel permeation chromatography (GPC). Thermal-induced opening of the cycles, made possible by the incorporated alkoxyamine, resulted in a return to the original apparent molecular weight, further supporting the formation of cyclic polymers in the RTA-ATRC reaction. Liquid chromatography-mass spectrometry (LC-MS) provided direct confirmation of the cyclic architecture and the incorporation of the nitroso group into the macrocycle RTA-ATRC cyclizations carried out with faster rates of polymer addition into the redox active solution and/or in the presence of a much larger excess of MNP (up to a 250:1 ratio of MNP:C-Br chain end) still yielded cyclic polymers that contained alkoxyamine functionality.

One Pot, Two Step Sequence Converting Atom Transfer Radical Polymerization Directly to Radical Trap-Assisted Atom Transfer Radical Coupling

Monobrominated polystyrene (PStBr) chains were prepared using standard atom transfer radical polymerization (ATRP) procedures at 80 °C in THF, with monomer conversions allowed to proceed to approximately 40%. At this time, additional copper catalyst, reducing agent, and ligand were added to the unpurified reaction mixture, and the reaction was allowed to proceed at 50 °C in an atom transfer radical coupling (ATRC) phase. During this phase, polymerization continued to occur as well as coupling; expected due to the substantial amount of residual monomer remaining. This was confirmed using gel permeation chromatography (GPC), which showed increases in molecular weight not matching a simple doubling of the PStBr formed during ATRP, and an increase in monomer conversion after the second phase. When the radical trap 2-methyl-2-nitrosopropane (MNP) was added to the ATRC phase, no further monomer conversion occurred and the resulting product showed a doubling of peak molecular weight (Mp), consistent with a radical trap-assisted ATRC (RTA-ATRC) reaction.

Selective Formation of Diblock Copolymers Using Radical Trap-Assisted Atom Transfer Radical Coupling

Polystyrene (PSt) radicals and poly(methyl acrylate) (PMA) radicals, derived from their monobrominated precursors prepared by atom transfer radical polymerization (ATRP), were formed in the presence of the radical trap 2-methyl-2-nitrosopropane (MNP), selectively forming PSt-PMA diblock copolymers with an alkoxyamine at the junction between the block segments. This radical trap-assisted, atom transfer radical coupling (RTA-ATRC) was performed in a single pot at low temperature (35 °C), while analogous traditional ATRC reactions at this temperature, which lacked the radical trap, resulted in no observed coupling and the PStBr and PMABr precursors were simply recovered. Selective formation of the diblock under RTA-ATRC conditions is consistent with the PStBr and PMABr having substantially different KATRP values, with PSt radicals initially being formed and trapped by the MNP and the PMA radicals being trapped by the in situ-formed nitroxide end-capped PSt. The midchain alkoxyamine functionality was confirmed by thermolysis of the diblock copolymer, resulting in recovery of the PSt segment and degradation of the PMA block at the relatively high temperatures (125 °C) required for thermal cleavage. A PSt-PMA diblock formed by chain extenstion ATRP using PStBr as the macroinitiator (thus lacking the alkoxyamine between the PSt-PMA segements) was inert to thermolysis. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3619–3626

One Pot, Two Step Sequence Converting Atom Transfer Radical Polymerization Directly to Radical Trap-Assisted Atom Transfer Radical Coupling

Monobrominated polystyrene (PStBr) chains were prepared using standard atom transfer radical polymerization (ATRP) procedures at 80 degrees C in THF, with monomer conversions allowed to proceed to approximately 40%. At this time, additional copper catalyst, reducing agent, and ligand were added to the unpurified reaction mixture, and the reaction was allowed to proceed at 50 degrees C in an atom transfer radical coupling (ATRC) phase. During this phase, polymerization continued to occur as well as coupling; expected due to the substantial amount of residual monomer remaining. This was confirmed using gel permeation chromatography (GPC), which showed increases in molecular weight not matching a simple doubling of the PStBr formed during ATRP, and an increase in monomer conversion after the second phase. When the radical trap 2-methyl-2-nitrosopropane (MNP) was added to the ATRC phase, no further monomer conversion occurred and the resulting product showed a doubling of peak molecular weight (M-p), consistent with a radical trap-assisted ATRC (RTA-ATRC) reaction. (C) 2013 Elsevier Ltd. All rights reserved.

Selective Formation of Diblock Copolymers Using Radical Trap-Assisted Atom Transfer Radical Coupling

Polystyrene (PSt) radicals and poly(methyl acrylate) (PMA) radicals, derived from their monobrominated precursors prepared by atom transfer radical polymerization (ATRP), were formed in the presence of the radical trap 2-methyl-2-nitrosopropane (MNP), selectively forming PSt-PMA diblock copolymers with an alkoxyamine at the junction between the block segments. This radical trap-assisted, atom transfer radical coupling (RTA-ATRC) was performed in a single pot at low temperature (35 degrees C), while analogous traditional ATRC reactions at this temperature, which lacked the radical trap, resulted in no observed coupling and the PStBr and PMABr precursors were simply recovered. Selective formation of the diblock under RTA-ATRC conditions is consistent with the PStBr and PMABr having substantially different K-ATRP values, with PSt radicals initially being formed and trapped by the MNP and the PMA radicals being trapped by the in situ-formed nitroxide end-capped PSt. The midchain alkoxyamine functionality was confirmed by thermolysis of the diblock copolymer, resulting in recovery of the PSt segment and degradation of the PMA block at the relatively high temperatures (125 degrees C) required for thermal cleavage. A PSt-PMA diblock formed by chain extenstion ATRP using PStBr as the macroinitiator (thus lacking the alkoxyamine between the PSt-PMA segements) was inert to thermolysis. (c) 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3619-3626

Lung on Chip: In vitro HGF effects on injured alveolar A549 epithelial cells in microfluidic system

Microfluidic systems have become competitive tools in the invitro modelling of diseases and promising alternatives to animal studies. They allow obtaining more invivo like conditions for cellular assays. Research in idiopathic pulmonary fibrosis could benefit from this novel methodological approach to understand the pathophysiology of the disease & develop efficient therapies. The use of hepatocyte growth factor (HGF) for alveolar reepithelisation is a promising approach. In this study, we show a new microfluidic system to analyse the effects of HGF on injured alveolar epithelial cells. Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells (10,000 cells) were seeded and studied in these microfluidic systems with media perfusion (1μl/30min). Injury tests were made on the cells by the perfusion with media containing H2O2 or bleomycin. The degree of injury was then assessed by a metabolic and an apoptotic assays. Wound assays were also performed with a central laminar flow of trypsin. Monitoring of wound closure with HGF vs control media was assessed. The alveolar A549 epithelial cells grew and proliferated in the microfluidic system. In the wound closure assay, the degree of wound closure after 5 hours was (53.3±1.3%) with HGF compared to (9.8±2.4%) without HGF (P <0.001). We present a novel microfluidic model that allows culture, injury and wounding of A549 epithelial cells and represents the first step towards the development of an invitro reconstitution of the alveolar-capillary interface. We were also able to confirm that HGF increased alveolar epithelial repair in this system.

Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.