Amyloid ß-fibrillumok nanomechanikája = Nanomechanics of amyloid ß-fibrils

Pályázatban amiloid béta (Aß) fibrillumokat, és ehhez kapcsolódóan hasonló természetű amiloid fibrillumokat és egyéb fibrilláris biomolekuláris rendszereket vizsgáltunk. A kísérletekben atomerőmikroszkóp segítségével vizsgáltuk a fibrillumok topográfiai szerkezetét és mechanikai erővezérelt szerkezeti átalakulásait. Különböző amiloid fibrillumok nanomechanikai ujjlenyomatát mértük meg. Felfedeztük, hogy az Aß peptid egy fragmentuma, az Aß25-35, trigonálisan orientált hálózatot hoz létre csillám felszínen. Ez nanotechnológiai alkalmazások lehetőségét veti fel, amellyel kapcsolatban szabadalmi bejelentést indítottunk el. Új mérési technológiákat fejesztettünk ki: térben és időben szinkronizált TIRF/AFM, illetve pásztázó próba kimográfia. A nanomechanikai módszereinket sikerrel adaptáltuk intermedier filamentumokra és miozin vastag filamentumokra. | In this grant proposal we have investigated te properties of amyloid beta (Aß) fibrils and other relevant amyoids and fibrillar biomolecular systems. In our experiments the topographical structure and mechanical force-driven structural changes of the fibrils were explored by using atomic force microscopy(AFM). We measured the nanomechanical fingerprint of various amyloid fibrils. We discovered that a toxic fragment of the full-length Aßpeptide, Aß25-35, forms a trigonally oriented network on mice. The phenomenon opens the possiblity towards nanotechnological applications. Based on this we filed a preliminary patent application (US 61/058,244). We developed novel methodologies: spatially and temporally resolved TIRF/AFM, scanning force kymography. Our nanomechanical methods were implemented on yet unexplored biomolecular systems: intermediate filaments and myosin thick filaments

Analysis of the transcription and overexpression of the mevalonate-isoprenoid biosynthesis pathway genes in mucor circinelloides

Zygomycetes have a great practical significance in industrial-biotechnological and agricultural fields and also as opportunistic pathogens. Mucor circinelloides is a carotenoid producing filamentous fungus, which has been used as a model organism in various genetic, biochemical and molecular studies. Terpenes are synthesised by a side-route of the general mevalonate-isoprenoid biosynthetic pathway in fungi. Terpene-type metabolites (such as sterols, carotenoids, hormones/pheromones, functional groups of different proteins, e.g. farnesylated or geranylgeranylated proteins) are involved in the formation of the structure of the cell membrane, morphogenesis, electron transport, signal transduction, apoptotic processes, protection against free radicals, cell differentiation, adaptation to environmental changes, etc. Today, ergosterol and its synthesis is a major target of antifungal therapy. Our aim is to reveal the function, regulation of the mevalonate-isoprenoid pathway genes in M. circinelloides. In this study, effects of cultivation time, light, salt stress, media, temperature, oxygen tension, and statin treatment on the transcription of six terpenoid pathway genes, encoding the the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (hmgS), mevalonate kinase (mvk), diphospho-mevalonate decarboxylase (dmd), isopentenyl-pyrophosphate isomerase (ipi), farnesyl-pyrophosphate synthase (isoA) and geranylgeranyl-pyrophosphate synthase (carG), were analysed. The nucleotide sequences of the genes and their regulatory regions, as well as the amino acid sequences of the encoded proteins were analysed. Autonomously replicating vectors, carrying one of the mevalonate-isoprenoid genes under the control of own and Mucor glyceraldehyde-3-phosphate dehydrogenase (gpd1) promoter and terminal sequences were constructed. The promoter of gpd1 is very effective and can be induced by glucose. PEG/CaCl2-mediated protoplast transformation with plasmids harbouring one of the isoprenoid genes (ipi, isoA and carG, respectively) was used to elevate the copy number of the examined genes in M. circinelloides. Viability and germination of spores, morphology, growth intensity and terpene production (e.g. carotenoid and ergosterol) of the resulting transformants were analysed. Investigation of the copy number of the introduced DNA, transcription of the overexpressed genes and effect of the elevated copy numbers on the transcription of the other terpenoid gene are in progress. Árpád Csernetics, Miklós Takó, Gábor Nagy and Csaba Vágvölgyi are supported by TÁMOP 4.2.4. A/2-11-1-2012-0001 „National Excellence Program – Elaborating and operating an inland student and researcher personal support system convergence program” The project was subsidized by the European Union and co-financed by the European Social Fund. The infrastructure and research equipment was supported by TÁMOP4.1.1.C- 12/1/KONV-2012-0014 and OTKA NN 106394

Spatially and Temporally Synchronized Atomic Force and Total Internal Reflection Fluorescence Microscopy for Imaging and Manipulating Cells and Biomolecules

AbstractThe atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution

Citoszkeletális fehérjék szerkezete, dinamikája, mechanikája és kölcsönhatásai: egyedi molekuláktól szupramolekuláris rendszerekig = Structure, dynamics, mechanics and interactions of cytoskeletal proteins: from single molecules to supramolecular systems

Pályázatunkban a citoszkeletális fehérjék szerkezetét, dinamikáját, mechanikáját és kölcsönhatásait vizsgáltuk elsősorban a harántcsíkolt izom különböző molekuláris rendszerein. Szintetikus miozin vastag filamentumok szerkezetét és nanomechanikáját atomerőmikroszkóppal (AFM) mértük. A titin Z-lemez horgonyzó komplex mechanikai stabilitását erőspektroszkópiával vizsgáltuk. Natív vázizom titin rugalmasságának és erővezérelt szerkezetváltozásainak vizsgálatára erővisszacsatolt lézercsipeszt fejlesztettünk. A titin PEVK domén konformációs dinamikáját FRET spektroszkópiával vizsgáltuk. A dezmin intermedier filamentumok és protofibrillumok szerkezetét és nanomechanikáját ugyancsak AFM-mel mértük meg. A szívizom típusú miozin-kötő C-fehérje molekuláris mechanikáját Monte-Carlo módszerrel szimuláltuk. Az aktomiozin motilitás pontosabb térbeli változásainak mérésére fluoreszcencia interferencia kontraszt (FLIC) mikroszkópia fejlesztését kezdtük meg. Az izommechanika organizmusban való mérésére speciális, AFM-alapú módszert dolgoztunk ki C. elegans rendszeren. A pályázat közvetlen támogatásával nyolc eredeti közlemény és egy könyvfejezet került publikálásra. | In our project we investigated the structure, dynamics, mechanics and interactions of cytoskeletal proteins mainly on muscle-derived molecular systems. The structure and nanomechanics of myosin thick filaments were explored by using atomic force microscopy (AFM). The mechanical stability of the Z-disc titin-anchoring complex was measured with single-molecule force spectroscopy. In order to measure the elasticity and force-driven structural changes in native titin molecules with high resolution, we developed a fast force-clamp optical tweezers apparatus. The structural dynamics of titin PEVK domain fragments was measured by using FRET spectroscopy. The structure and nanomechanical behavior of desmin intermediate filaments and protofibrils were also measured with AFM. For the simulation of the force versus extension of cardiac myosin-binding protein-C we used Monte-Carlo methods. To reveal greater spatial detail in the in vitro actomyosin motility we began developing a fluorescence interference contrast (FLIC) microscope system. In order to investigate the actomyosin mechanics within an organism, we developed a novel, AFM-based detection method based on C. elegans. With the direct support of the grant eight original papers and one book chapter were published

A Szépművészeti Múzeum Antik Gyűjteményének szakkatalógusai VI = Catalogues of the Classical Collection of the Museum of Fine Arts, VI

Az OTKA által immáron hatodik ciklusban támogatott pályázat során három szakkatalógus elkészítését tűztük ki célul (kilenc megkezdett kutatás közül). Megjelent a CVA Hongrie 2 kötete (Szilágyi János György, etruszk és és campaniai vázák; a kézirat még az előző ciklusban készült el). Közvetlenül a leadás előtti fázisig jutott a CVA Hongrie 3 kötet a bronzkori, geometrikus és korinthosi kerámia feldolgozása (Márton András, Lebegyev Judit). Lényegében elkészült a CVA Hongrie 4 kötet a délitáliai kerámiáról (Vandlik Katalin) és a délitáliai terrakották feldolgozása (Bencze Ágnes). Mindkettő közvetlenül kapcsolódik a két kutató doktori dolgozatához (Bencze Ágnesé sajtó alatt van a nápolyi Centre Jean Bérard gondozásában, Vandlik Katalinét pedig már kitűzték védésre). Az előzőkhöz hasonlóan ezek a kötetek is a római L'Erma di Brettschneider kiadónál fognak megjelenni. Valamennyi szerző a klasszika archaeológia fiatal nemzedékéhez tartozik. A projekt lehetővé tette egy új anyagcsoport, a daktyliothékák rendezését és internetes publikációjuk megalapozását. Az OTKA támogatásnak ebben a ciklusban is döntő szerepe volt abban, hogy fenn tudott maradni a magyarországi klasszika archaeológia múzeumi bázisa. | Through the grant provided by OTKA now for the sixth project cycle, we aimed at preparing three reference catalogues (out of nine ongoing research projects). The volume of CVA 2 has been published (János György Szilágyi: Etruscan and Campanian Vases; the manuscript was completed in the previous project cycle). The volume of CVA 3 treating Bronze Age, Geometric and Corinthian pottery (András Márton, Judit Lebegyev) is ready to be laid out for publication. The volume of CVA 4 on Southern Italian pottery (Katalin Vandlik) and the treatment of Southern Italian terracottas (Ágnes Bencze) is practically also finished. Both works are directly connected with the PhD dissertations of the two researchers. Similarly to previous catalogues these volumes will also be published by L’Erma di Brettschneider in Rome. All authors belong to the younger generation of classical archaeologists. The project has also facilitated the classification of a new group of objects, that of dactyliotheques, and has also provided a basis for their publication through the internet. As in the previous projects cycles, the OTKa support has played a crucial role in the survival of the museological basis of classical archaeology in Hungary

Investigation of the mechanical and chemical characteristics of nanotubular and nano-pitted anodic films on grade 2 titanium dental implant materials

Abstract Objective The objective of this study was to investigate the reproducibility, mechanical integrity, surface characteristics and corrosion behavior of nanotubular (NT) titanium oxide arrays in comparison with a novel nano-pitted (NP) anodic film. Methods Surface treatment processes were developed to grow homogenous NT and NP anodic films on the surface of grade 2 titanium discs and dental implants. The effect of process parameters on the surface characteristics and reproducibility of the anodic films was investigated and optimized. The mechanical integrity of the NT and NP anodic films were investigated by scanning electron microscopy, surface roughness measurement, scratch resistance and screwing tests, while the chemical and physicochemical properties were investigated in corrosion tests, contact angle measurement and X-ray photoelectron spectroscopy (XPS). Results and discussion The growth of NT anodic films was highly affected by process parameters, especially by temperature, and they were apt to corrosion and exfoliation. In contrast, the anodic growth of NP film showed high reproducibility even on the surface of 3-dimensional screw dental implants and they did not show signs of corrosion and exfoliation. The underlying reason of the difference in the tendency for exfoliation of the NT and NP anodic films is unclear; however the XPS analysis revealed fluorine dopants in a magnitude larger concentration on NT anodic film than on NP surface, which was identified as a possible causative. Concerning other surface characteristics that are supposed to affect the biological behavior of titanium implants, surface roughness values were found to be similar, whereas considerable differences were revealed in the wettability of the NT and NP anodic films. Conclusion Our findings suggest that the applicability of NT anodic films on the surface of titanium bone implants may be limited because of mechanical considerations. In contrast, it is worth to consider the applicability of nano-pitted anodic films over nanotubular arrays for the enhancement of the biological properties of titanium implants