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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; Pardon, Els; Aumont-Nicaise, Magalie et al

Poster (2011)

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These ... [more ▼]

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. We have also shown that the binding of three variable domain of camelid antibodies or (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibrils inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non overlapping epitopes. We have demonstrated that five of these new VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as activity, stability, cooperativity and aggregation will be discussed. [less ▲]

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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; pardon, Els; Aumont-Nicaise, Magali et al

Poster (2012, June)

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These ... [more ▼]

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. We have also shown that the binding of three variable domain of camelid antibodies (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibril inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non-overlapping epitopes. We have demonstrated that five of these VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as stability, cooperativity and aggregation will be discussed. [1] Dumoulin, M., J.R. Kumita, and C.M. Dobson, Normal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants. Acc Chem Res, 2006, 39(9), 603-610. [2] Dumoulin, M., et al., A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature, 2003, 424, 783-788. [3] Chan, P.H., et al., Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils. Biochemistry, 2008, 47, 11041-11054. [less ▲]

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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme.
Dumont, Janice ULg; Menzer, Linda ULg; Pardon, Els et al

Poster (2010)

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These ... [more ▼]

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. The binding of three variable domain of camelid antibodies – also named nanobodies - (cAb-HuL 6 [2], cAb-HuL 5 and cAb-HuL 22 [3]) raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three nanobodies bind on different regions of lysozyme and act as Amyloid fibrils inhibitor through different mechanisms. On one hand, cAb-HuL 6 and cAb-HuL 22 stabilize the native state of the lysozyme variants thus restoring the global cooperativity characteristic of the wild-type protein. On the other, cAb-HuL 5 probably acts by binding soluble prefibrillar aggregates. In the present work, sixteen other nanobodies specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non overlapping epitopes. The effects of the binding of these nanobodies on the stability of the D67H variant of human lysozyme and on its aggregation into amyloid fibrils will be discussed. [less ▲]

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See detailNanobodies as tools to investigate the mechanism of aggregation of chimeric proteins made by the insertion of polyglutamine stretches into the beta-lactamase BlaP
Pain, Coralie ULg

Doctoral thesis (2014)

Among the neurodegenerative amyloidoses, ten disorders, referred to as polyglutamine (polyQ) diseases and including Huntington's disease and several spinocerebellar ataxias, are associated with ten ... [more ▼]

Among the neurodegenerative amyloidoses, ten disorders, referred to as polyglutamine (polyQ) diseases and including Huntington's disease and several spinocerebellar ataxias, are associated with ten proteins within which a polyQ tract is expanded above a threshold of typically 35-45 glutamine residues. Such expanded polyQ tracts lead to the aggregation of the host protein into amyloid fibrils that accumulate in the nucleus of some populations of neurons; these aggregates or some of their precursors are thought to contribute to neuronal death. So far, no preventive or curative treatment exists for these devastating pathologies. While the expansion of the polyQ tract above the threshold is the determinant factor for aggregation, recent studies suggest that non-polyQ regions of these proteins can play a significant role, either preventative or facilitative, in the aggregation process. The general principles governing the complex interplay between the role of the expanded polyQ tract and the role of the non-polyQ regions in the aggregation process are not well understood yet. In order to develop therapeutic strategies, it is important to better understand this complex interplay. To contribute to this aim, we have engineered chimeric proteins via the insertion of polyQ repeats of various lengths (23, 30, 55 and 79Q) into two sites (197 and 216) of the BlaP beta-lactamase from Bacillus licheniformis 749/C. The properties of these chimeric proteins recapitulate the characteristic features of the disease-associated polyQ proteins, i.e. (i) there is a minimum number of inserted glutamines (threshold) required to trigger the aggregation of the chimeras into amyloid fibrils, and (ii) above the threshold, the longer the polyQ tract, the faster the aggregation. Interestingly, for the same polyQ length, the chimeras with insertions in position 216 have an increased propensity to form amyloid fibrils compared to their counterparts with insertions in position 197. These findings highlight the strong influence of the overall protein context on aggregation triggered by expanded polyQ tracts. This thesis addresses the use of the variable domains of camelid heavy-chain antibodies, referred to as nanobodies or VHHs, as structural and mechanistic probes to better understand the different aggregating properties of the two sets of BlaP-polyQ chimeras (197 and 216). We have also performed limited proteolysis experiments and transglutaminase-mediated reactions on the monomeric form of the BlaP-polyQ chimeras to further investigate the effects of the polyQ insertions on the structure and dynamics of the BlaP moiety, as well as the structure of the polyQ tract itself. From the blood of a llama immunised with BlaP197(Gln)55, we isolated more than 60 VHHs specific to the BlaP-polyQ chimeras. Twenty eight of them were produced, purified and characterised. These VHHs were found to be all specific to the BlaP moiety and could be classified into four different groups recognising distinct epitopes on the surface of BlaP. One representative VHH of each group (i.e. cAb-A3S, cAb-H7S, cAb-F11N and cAb-G10S) was selected as probe to investigate the mechanism of aggregation of the BlaP-polyQ chimeras. The epitope of three of them was determined by X-ray diffraction and/or by NMR spectroscopy. Although they recognise distinct epitopes and exhibit different affinities for BlaP, the binding of the four VHHs significantly slows down the aggregation of all the BlaP-polyQ chimeras investigated (i.e. BlaP197(Gln)55, BlaP197(Gln)79 and BlaP216(Gln)79). The extent of inhibition depends however on the chimera and on the experimental conditions. We show that the inhibition of the aggregation of BlaP197(Gln)55 and BlaP197(Gln)79 upon binding of the four VHHs is correlated with the stabilisation of their native state. In the case of BlaP216(Gln)79, the extent of inhibition could not be only correlated to the stabilisation of its native state; the location of the epitope of the VHH is instead also determinant. This observation demonstrates that the lower thermodynamic stability of BlaP216(Gln)79 is not the unique factor responsible for its increased aggregation propensity. It also further highlights the complexity of the aggregation mechanism of polyQ proteins and the strong influence of the non-polyQ regions on the amyloid fibril formation triggered by the expanded polyQ tract. All together our results suggest that antibodies or antibody fragments raised against the non-polyQ regions of polyQ proteins associated with diseases could constitute a relevant therapeutic strategy. They also further demonstrate the power of nanobodies as probes to get a deeper knowledge of the underlying mechanisms of amyloid fibril formation. The preliminary limited proteolysis and transglutamination experiments obtained suggest that the polyQ tracts are all flexible, except that of 23 glutamines inserted in position 197 of BlaP, which seems to be more rigid than the others. The results obtained confirm that, globally, the structure of BlaP is not significantly modified by the insertions while the 216 chimeras seem more dynamic than the 197 chimeras. [less ▲]

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See detailA Nanobody Binding to Non-amyloidogenic Regions of the Protein Human Lysozyme Enhances Partial Unfolding but Inhibits Amyloid Fibril Formation.
de Genst, EJ; Chan, PH; Pardon, Els et al

in Journal of Physical Chemistry B (2013)

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See detailNanocarriers tailored for the delivery of proteinaceous drugs
Grandfils, Christian ULg

Conference (2013, April 09)

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See detailNanocoatings of inorganic surfaces by molecular biomimetic
Vreuls, Christelle ULg; Genin, Alexis ULg; Zocchi, Germaine ULg et al

Poster (2010, June 30)

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See detailNanocoatings of inorganic surfaces by molecular biomimetic
Vreuls, Christelle ULg; Genin, Alexis ULg; Zocchi, Germaine ULg et al

Poster (2010, March 22)

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See detailNanocoatings of inorganic surfaces by the layer by layer (LbL) technology
Faure, Emilie ULg; Zocchi, Germaine ULg; Lenoir, Sandrine et al

Poster (2009, April 02)

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See detailNanocoatings of steel surfaces by molecular biomimetic
Vreuls, Christelle ULg; Charlot, Aurelia; Farina, Fabrice ULg et al

Scientific conference (2007, June 01)

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See detailNanocomposite fiber reinforced mortars
Coppola, Bartolomeo; Di Maio, Luciano; Courard, Luc ULg et al

in Proceedings of the International Conference on Composites/Nano Engineering (2014)

The use of fibers to reinforce a brittle material is an extensively studied application. In the field of cementitious materials a wide range of fibers have been investigated, from natural to synthetic ... [more ▼]

The use of fibers to reinforce a brittle material is an extensively studied application. In the field of cementitious materials a wide range of fibers have been investigated, from natural to synthetic fiber (wood, cellulose, carbon, glass, polypropylene) in order to achieve several purposes. Nowadays there is a continuing effort to take advantage of recent advances in nanotechnology, in the polymer and fiber industry. Nanoclays are some of the most affordable materials that have shown promising results in nanocomposite polymers. They are characterized by a “platelet” structure with average dimension of 1 nm thick and 70 to 150 nm wide. This work is aimed at studying the different behavior of fiber reinforced mortars, containing nanocomposite polymeric fibers. [less ▲]

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See detailNanoindentation and scanning force microscopy as a novel method for the characterization of tribological transfer films
Randall, N. X.; Bozet, Jean-Luc ULg

in Wear (1997), 212(1), 18-24

In conventional pin-on-disk testing of the tribological characteristics of two different materials in sliding contact, the main parameters of interest are notably the friction and wear properties of the ... [more ▼]

In conventional pin-on-disk testing of the tribological characteristics of two different materials in sliding contact, the main parameters of interest are notably the friction and wear properties of the material pair. However, when two bodies consisting of hard and soft materials respectively are subjected to such testing, the appearance of a transfer film, or third body, which can be a composite mixture of the two, is often observed. Until now the characterization of transfer films in terms of their mechanical properties has been hampered by their nonhomogeneous distribution across a tested surface, their small size, low thickness and the difficulty in accurately positioning a test probe such that the film properties can be measured independently from those of the substrate. In this paper a new method is introduced, consisting of nanoindentation and scanning force microscopy (SFM), which is capable of highly localized indentation testing of a specified sample site with high resolution imaging of the area prior to and after indentation. In this way the hardness and modulus of a transfer film can be obtained, as well as valuable surface topographical information concerning the material response to the indentation. Measurements are presented for the material pair A286/polyimide after testing on a pin-on-disk tribometer in ambient air and liquid nitrogen. Distinct variations in hardness between the transfer films and their contacting bodies have een observed and correlated to the wear behaviour and testing environment. (C) 1997 Elsevier Science S.A. [less ▲]

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See detailNanoindentation investigation of Ti/TiN multilayers films
Ben Daia, M.; Aubert, P.; Labdi, S. et al

in Journal of Applied Physics (2000), 87(11), 7753-7757

The hardness of Ti/TiN nanolaminated films is investigated in this study. Monolithic Ti and TiN films and Ti/TiN multilayers were deposited on silicon substrates by radio-frequency sputtering. The period ... [more ▼]

The hardness of Ti/TiN nanolaminated films is investigated in this study. Monolithic Ti and TiN films and Ti/TiN multilayers were deposited on silicon substrates by radio-frequency sputtering. The period thickness of multilayers was decreased from 20 to 2.5 nm. Grazing x-ray reflectometry showed that the modulation of composition of Ti/TiN multilayers exists for all the period thickness considered. From nanoindentation measurements, we determined the hardness and Young's modulus of multilayers. Hardness increased with decreasing period thickness to go beyond the rule-of-mixture value for samples with period thickness of Lambda less than or equal to 5 nm. The maximum hardness, 1.6 times higher than the value obtained by the rule of mixture, is obtained for Lambda=2.5 nm. Our results are compared to a dislocation-based model previously introduced by Lehoczky. (C) 2000 American Institute of Physics. [S0021-8979(00)09411-1]. [less ▲]

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See detailNANOMATERIAL BEHAVIOUR OF A GOLD MICROCANTILEVER SUBJECTED TO PLASTIC DEFORMATIONS
Pustan, Marius ULg

in Digest Journal of Nanomaterials & Biostructures [=DJNB] (2011), 6(1), 285-290

The nanomechanical material behaviour of a gold microcantilever subjected to plastic deformations is presented in this paper. Using an atomic force microscope, experimental investigations are performed in ... [more ▼]

The nanomechanical material behaviour of a gold microcantilever subjected to plastic deformations is presented in this paper. Using an atomic force microscope, experimental investigations are performed in order to determine the dependence between bending deflections of sample versus applied forces and to estimate the maximum stress in the beam structure. During testing, the force has successive positions on microcantilever, starting from the beam free-end and moving toward to the anchor. The plastic deformation of microcantilever occurs when the force is applied close to the beam anchor and performed large deflections. Finite element analysis is used to visualize the deflection of microcantilever and to estimate the maximum stress. [less ▲]

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See detailNanomechanical and nanotribological characterization of microelectromechanical system
Pustan, Marius; Muller, Raluca; Golinval, Jean-Claude ULg

in Journal of Optoelectronics and Advanced Materials [= JOAM] (2012), 14(3-4), 401-412

Investigations of the mechanical and tribological properties of microelectromechanical system (MEMS) components on nanoscale can provide insights into failure mechanism of material. The main goal of this ... [more ▼]

Investigations of the mechanical and tribological properties of microelectromechanical system (MEMS) components on nanoscale can provide insights into failure mechanism of material. The main goal of this paper is focused on the mechanical and tribological characterizations of MEMS mechanical components in order to improve their reliability design. The mechanical properties of interests are stiffness, modulus of elasticity, stress, strain. Dynamical investigations are performed to analyze the resonant frequency response, velocity and amplitude of oscillations of electrostatically actuated microcomponents and to estimate the quality factor. Finite element analysis is used to validate the experimental results of mechanical properties and to simulate the dynamical behaviour of investigated microcomponents. Tribological investigations are developed to estimate the stiction and friction. Testing and the individual characterization of MEMS materials and structures, performed using advanced equipments such as atomic force microscope and optical vibrometer analyzer are presented. [less ▲]

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See detailNanomechanical properties of sensing microcomponents
Pustan, Marius ULg; Golinval, Jean-Claude ULg; Rochus, Véronique ULg

Scientific conference (2009)

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See detailNanometer scale organization of mixed surfactin/phosphatidylcholine monolayers
Deleu, Magali ULg; Paquot, Michel ULg; Jacques, P. et al

in Biophysical Journal (1999), 77(4), 2304-2310

Mixed monolayers of the surface-active lipopeptide surfactin-C-15 and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic ... [more ▼]

Mixed monolayers of the surface-active lipopeptide surfactin-C-15 and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM topographic images revealed phase separation for mixed monolayers prepared at 0.1, 0.25, and 0.5 surfactin molar ratios. This was in agreement with the monolayer properties at the air-water interface indicating a tendency of the two compounds to form bidimensional domains in the mixed systems. The step height measured between the surfactin and the DPPC domains was 1.2 +/- 0.1 nm, pointing to a difference in molecular orientation: while DPPC had a vertical orientation, the large peptide ring of surfactin was lying on the mica surface. The N/C atom concentration ratios obtained by XPS for pure monolayers were compatible with two distinct geometric models: a random layer for surfactin and for DPPC, a layer of vertically-oriented molecules in which the polar headgroups are in contact with mica. XPS data for mixed systems were accounted for by a combination of the two pure monolayers, considering respective surface coverages that were in excellent agreement with those measured by AFM. These results illustrate the complementarity of AFM and XPS to directly probe the molecular organization of multicomponent monolayers. [less ▲]

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