Development of a secreted cell-permeable NF-κB inhibitor to control inflammation

PhDRheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease, of unknown aetiology. Several disease-modulating approaches have been developed in the past years, however these are expensive, usually accompanied by unwanted side-effects and 30% of the patients fail to respond. The transcription factor NF-kB is a key factor in the development and perpetuation of the disease, as it regulates a number of inflammatory genes. The activity of certain signalling pathways can be modulated by delivering into cells inhibitors coupled to Protein Transduction Domains (PTDs). The aim of this study was to develop a secretable PTD-fusion NF-κΒ inhibitor that is produced and secreted by genetically engineered mammalian cells in sufficient amounts to subsequently transduce and regulate NF-κΒ activity in neighbouring cells. Such methodology could be useful for the management of RA by transplantation of engineered cells or directly using gene delivery into the synovial joints. In this study, PTD-fusion proteins were fused to the IL-2 secretion signal and their ability to be secreted from mammalian cells was explored. Secretable forms of TAT-IgG2A and TAT-eGFP were generated as control PTD-fusion proteins, and the TAT-srIκΒα (super repressor IκBα, a NF-κΒ inhibitor) was generated as an NF-κΒ PTD-fusion inhibitor. Western blotting analysis of supernatants from transiently transfected 293T cells revealed that TAT-IgG2A, TAT-eGFP and TAT-srIκΒα are secreted with variable efficiencies. When concentrated, PTD proteins were able to transduce mammalian cells, as demonstrated with Jurkat cells by confocal microscopy and western blotting analysis. The TAT PTD domain was replaced to a more stable, furin cleavage-resistant and less positively charged PTD domain, the TAT3 PTD domain, to ensure that PTDfusion proteins will be secreted more efficiently. This change of the PTD domain did not increase secretion levels of the srΙκBα. Subsequently, the Latent Associated Peptide (LAP) of TGFβ, was fused to the TAT3-srIκΒα inhibitor, via a matrix metalloproteinase (MMP) cleavage linker. This LAP-MMP-PTD-fusion NF-κΒ inhibitor was again poorly secreted. In turn, the srIκΒα inhibitor was replaced with a small synthetic NF-κΒ inhibitor, termed Nemo Binding Domain (NBD), in the form of LAP MMP-TAT3-NBD NF-κΒ inhibitor. Western blotting analysis of supernatants from transiently transfected 293T cells revealed that the LAP-MMP-TAT3-NBD was efficiently secreted. The ability of LAP-MMP-TAT3-NBD to inhibit NF-κΒ was tested in vitro with the use of a cell-assay based on HeLa cells that are permanently transfected with the luciferase gene driven by an NF-κB regulated promoter. In this assay, HeLa cells that were treated with the secreted LAP-MMP-TAT3-NBD, showed reduced levels of luciferase activity after IL-1β stimulation. Subsequently, using a replication-deficient lentiviral vector, genetically engeneered DBA/1 fibroblasts (DTF) able to produce the secreted LAP-MMP-TAT3-NBD were generated. The NF-κB inhibitory properties of the secreted LAP-MMP-TAT3-NBD were tested in vivo in the Carrageenan-induced paw oedema, Antigen Induced Arthritis and Air-Pouch acute inflammation models. Paws of mice that were treated with engineered cells or lentivirus encoding LAPMMP- TAT3-NBD demonstrated milder paw swelling, suggesting that LAP-MMPTAT3- NBD had a protective role in the induction of inflammation. However, the LAPMMP- TAT3-NBD did not demonstrate anti-inflammatory effects in the Air-Pouch model. In this study, I present a method to design PTD-fusion proteins that can be efficiently secreted from mammalian cells and I demonstrate a novel gene therapy approach for the local delivery of a therapeutic agent

In situ stress evolution during growth of transition metal nitride films and nanocomposites

The issue of stress evolution during growth of hard transition metal nitride (TMN) based coatings is of vital importance to understand origin of intrinsic stress development and to control stress level in order to avoid mechanical failure of coated components and devices. By using in situ and real-time wafer curvature measurements based on a multiple- beam optical stress sensor (MOSS), basic insights on the atomistic mechanisms at the origin of stress development and stress relaxation can be obtained. In the present paper, a review of recent advances on stress development during reactive magnetron sputter-deposition of binary TMN films (TiN, ZrN, TaN) as wells as ternary systems (TiZrN, TiTaN) will be presented. The influence of growth energetics on the build-up of compressive stress will be addressed. A correlation between stress, texture and film morphology is demonstrated. Finally, illustration will be given for quaternary TiZrAlN nanocomposites. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/2074

Laser induced ultrafast combustion synthesis of solution-based AlOx for thin film transistors

Solution processing of amorphous metal oxides using excimer laser annealing (ELA) has been lately used as a viable option to implement large-area electronics, offering high quality materials at a reduced associated cost and process time. However, the research has been focused on semiconductor and transparent conductive oxide layers rather than on the insulator layer. In this work we present amorphous aluminum oxide (AlOx) thin films produced at low temperature (≤150 °C) via combustion synthesis triggered by ELA, for oxide thin film transistors (TFTs) suitable for manufacturing flexible electronics. The study showed that combining ELA and combustion synthesis leads to an improvement in the dielectric thin film's densification in a shorter time (≤15 min). Optimized dielectric layers were obtained combining a short drying cycle at 150 °C followed by ELA treatment. High breakdown voltage (4 MV cm−1) and optimal dielectric constant (9) was attained. In general, TFT devices comprising the AlOx fabricated with a drying cycle of 15 min followed by ELA presented great TFT properties, a high saturation mobility (20.4 ± 0.9 cm2 V−1 s−1), a small subthreshold slope (0.10 ± 0.01 V dec−1) and a turn-on voltage ∼0 V. ELA is shown to provide excellent quality solution-based high-κ AlOx dielectric, that surpass other methods, like hot plate annealing and deep ultraviolet (DUV) curing. The results achieved are promising and expected to be of high value to the printed electronic industry due to the ultra-fast film densification and the surface/area selective nature of ELA

Ανάπτυξη, δομή και ηλεκτρονικές ιδιότητες τριαδικών υμενίων μεταβατικών μετάλλων

The unique combination of electrical conductivity, chemical and metallurgical stability, refractory character and lattice constants close to those of III-nitrides, make the transition metal nitrides (TMNs) of groups IVb-VIb promising candidates for electronics and device applications. In this thesis, we extensively studied the structure, stability and microstructure of binary and ternary transition metal nitrides in thin films. This study includes many binary systems and the widest range of ternary nitrides reported ever. Additionally, we examined also the electronic structure, the optical properties and the mechanical behavior of these materials in order to accomplish a complete study about these materials. This joined PhD thesis, is the result of a close collaboration between two institutes, the University of Poitiers and the University of Ioannina in the field of growth, structure and functional properties of complex transition metal nitrides. The growth of samples and the analysis has been performed at both institutes, and the combination of knowledge and the transfer of know-how, strengthen the results and raises the quality of this work. Three different deposition techniques, pulsed laser deposition (PLD), magnetron sputtering (MS) and ion beam sputtering were used to deposit the samples. X-ray diffraction (XRD), optical reflectance spectroscopy (ORS), in situ and real time stress measurements, hardness and computational data are some techniques, among others, used to cover this wide study of transition metal nitrides. Special emphasis has been given to the acquisition of accurate experimental data of various structural and functional properties of a wide variety of binary and ternary TMNs, in an effort to establish a database for these materials.Ο σπάνιος συνδυασμός της ηλεκτρικής αγωγιμότητας, της χημικής και μεταλλουργικής σταθερότητας, ο πυρίμαχος χαρακτήρας και η τιμή της κρυσταλλική σταθεράς, που είναι κοντά στα νιτρίδια της ομάδας ΙΙΙ, έχουν καθιερώσει τα νιτρίδια μεταβατικών μετάλλων των ομάδων IVb-VIb σαν υποψήφια υλικά για εφαρμογές στην ηλεκτρονική και σε διατάξεις. Στην παρούσα διδακτορική διατριβή, μελετήσαμε εκτενώς την δομή, την σταθερότητα και την μικροδομή υμενίων δυαδικών και τριαδικών νιτριδίων μεταβατικών μετάλλων. Η μελέτη αυτή περιλαμβάνει πολλά δυαδικά συστήματα καθώς και την εκτενέστερη συλλογή από τριαδικά που έχουν αναφερθεί ποτέ. Επιπρόσθετα, μελετήσαμε την ηλεκτρονική δομή, τις οπτικές ιδιότητες και την μηχανική συμπεριφορά αυτών των υλικών έτσι ώστε να φέρουμε εις πέρας μια ολοκληρωμένη μελέτη από κάθε άποψη σχετικά με αυτά τα υλικά. Η παρούσα κοινή διδακτορική διατριβή, είναι το αποτέλεσμα μια στενής συνεργασίας μεταξύ δύο ιδρυμάτων, του Πανεπιστημίου του Πουατιέ (University of Poitiers) και του πανεπιστημίου Ιωαννίνων, στον τομέα της ανάπτυξης, των δομικών και λειτουργικών ιδιοτήτων περίπλοκων νιτριδίων μεταβατικών μετάλλων. Η ανάπτυξη και η μελέτη των δειγμάτων έγιναν και στα δύο ιδρύματα, και ο συνδυασμός γνώσης και η μεταφορά τεχνογνωσίας ενίσχυσαν τα αποτελέσματα και ανέβασαν το επίπεδο της παρούσας μελέτης. Τρεις διαφορετικές τεχνικές εναπόθεσης υμενίων, εναπόθεση με παλμικό λέηζερ (Pulsed Laser Deposition, PLD), μαγνητική ιοντοβολή (magnetron sputtering, MS) και ιοντοβολή με δέσμη ιόντων (ion beam sputtering) χρησιμοποιήθηκαν για την παρασκευή των δειγμάτων. Περίθλαση ακτίνων Χ, οπτική ανακλαστικότητα, μετρήσεις τάσεων σε πραγματικό χρόνο και in situ και υπολογιστικά δεδομένα, μεταξύ άλλων, χρησιμοποιήθηκαν για να καλύψουν την παρούσα ευρεία μελέτη των νιτριδίων μεταβατικών μετάλλων. Ειδική έμφαση δόθηκε στην συγκέντρωση με ακρίβεια, πειραματικών δεδομένων διαφόρων δομικών και λειτουργικών ιδιοτήτων σε μια μεγάλη ποικιλία δυαδικών και τριαδικών νιτριδίων μεταβατικών μετάλλων, σε μια προσπάθεια να θεμελιωθεί μια βάση δεδομένων για αυτά τα υλικά

Intrinsic Stress in ZrN Thin Films: Evaluation of Grain Boundary Contribution From in Situ Wafer Curvature and Ex Situ x-ray Diffraction Techniques

Low-mobility materials, like transition metal nitrides, usually undergo large residual stress when sputter-deposited as thin films. While the origin of stress development has been an active area of research for high-mobility materials, atomistic processes are less understood for low-mobility systems. In the present work, the contribution of grain boundary to intrinsic stress in reactively magnetron-sputtered ZrN films is evaluated by combining in situ wafer curvature measurements, providing information on the overall biaxial stress, and ex situ x-ray diffraction, giving information on elastic strain (and related stress) inside crystallites. The thermal stress contribution was also determined from the in situ stress evolution during cooling down, after deposition was stopped. The stress data are correlated with variations in film microstructure and growth energetics, in the 0.13-0.42 Pa working pressure range investigated, and discussed based on existing stress models. At low pressure (high energetic bombardment conditions), a large compressive stress is observed due to atomic peening, which induces defects inside crystallites but also promotes incorporation of excess atoms in the grain boundary. Above 0.3-0.4 Pa, the adatom surface mobility is reduced, leading to the build-up of tensile stress resulting from attractive forces between under-dense neighbouring column boundary and possible void formation, while crystallites can still remain under compressive stres