Ornella Cavalleri

Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of β2-microglobulin (β2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of β2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes β2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7–99 truncated form of β2-m (ΔN6β2-m) that is a ubiquitous constituent of the natural β2-m fibrils. The formation of this β2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

High resolution X-ray photoelectron spectroscopy of L-cysteine self-assembled films

The increasing demand for heterogeneous materials prompts the interest for the physics and chemistry of organic–inorganic interfaces. Within this field of interest we have investigated the electronic states of L-cysteine (Cys) adsorbed on Au(111) from aqueous solution with synchrotron-based, high resolution X-ray photoemission spectroscopy. The analysis of the S 2p core level region in pristine samples prepared with purified Cys indicates that Cys adsorbs on gold as a thiolate. The absence of a signal related to physisorbed sulfur indicates that Cys forms a monomolecular layer. The high resolution allowed us to resolve two components in the C 1s spectral region related to carboxyl. The comparative analysis of C 1s, N 1s and O 1s core level regions indicates the presence of the neutral and ionic form for both the carboxyl and the amine groups. From the quantitative analysis of the C 1s and N 1s data we can infer that the zwitterionic form is the prevalent one, with a ratio of zwitterionic to neutral molecules of 3∶1. This value is quite different from that in the deposition solution. Prolonged exposure to the X-ray beam and thermal annealing induce the formation of atomic sulfur through S–C bond breaking and molecular fragment desorption. This response to X-ray irradiation differs from the results recently reported on long chain alkanethiols.

The two-fold aspect of the interplay of amyloidogenic proteins with lipid membranes

Investigating the pathways leading to the formation of amyloid protein aggregates and the mechanism of their cytotoxicity is fundamental for a deeper understanding of a broad range of human diseases. Increasing evidence indicates that early aggregates are responsible for the cytotoxic effects. This paper addresses the catalytic role of lipid surfaces in promoting aggregation of amyloid proteins and the permeability changes that these aggregates induce on lipid membranes. Effects of amyloid aggregates on model systems such as monolayers, vesicles, liposomes and supported lipid bilayers are reviewed. In particular, the relevance of atomic force microscopy in detecting both kinetics of amyloid formation and amyloid-membrane interactions is emphasized.

XPS measurements on l-cysteine and 1-octadecanethiol self-assembled films: a comparative study

Abstract The adsorption of l -cysteine on Au(111) surfaces from the solution phase has been investigated by X-ray photoelectron spectroscopy (XPS). To check the reliability of the XPS analysis, we performed measurements on l -cysteine/Au(111) and alkanethiol/Au(111) interfaces paying particular attention to spectra time evolution during X-ray exposure. In both systems, X-ray irradiation increases the Au4f intensity, but while in l -cysteine the line shape of the S2p spectra does not change, in 1-octadecanethiol it does. The analysis of the Au4f and S2p core level regions indicates the occurrence of a dissociative molecular chemisorption on the metal surface for both systems. In the case of l -cysteine, the S2p spectra indicate the presence of a minor sulphur component attributed to the presence of unbound cysteine molecules physisorbed on the first chemisorbed layer.

Ostwald ripening of vacancy islands at thiol covered Au(111)

Abstract The growth kinetics of vacancy islands at a Au(111) surface covered with self-assembled thiol monolayers has been investigated by in-situ scanning tunneling microscopy. The vacancy islands are found to coarsen by Ostwald ripening. The exponent characterizing the power-law time dependence of the coarsening is found to be n ≈ 1 2 independent of the thiol chain length ( Ch 3 ( CH 2 ) x SH , x = 5, 9, 17). This behavior is consistent with a model in which the mass transport is limited by the adsorption/desorption rate of vacancies at the hole edges.

COPPER ELECTRODEPOSITION ON ALKANETHIOLATE COVERED GOLD ELECTRODES

We have investigated the structure and thermal dynamics of alkanethiolate layers on Au(lll) with variable temperature scanning tunnelling microscopy (STM), X-ray photoelectron spectroscopy (XPS) and voltammetry. The results build the basis for a study of electrodeposition of copper on alkanethiolate-covered Au(lll). Electrodeposition has been studied as a function of the thiolate chain length, the deposition potential and the temperature. Time-resolved in situ STM and voltammetry, both at temperatures up to 345 K, and XPS of emersed samples showed that copper can

Spectro-ellipsometry on cadmium stearate Langmuir-Blodgett films

Abstract We have studied cadmium stearate Langmuir–Blodgett (LB) films by spectroscopic ellipsometry (SE) and we have compared the results with those obtained by using X-ray reflectivity and atomic force microscopy. The measurements on cadmium stearate LB films deposited on silicon wafers capped by a native oxide layer provide sound results for films formed by 1–25 molecular layers. The experimental data are well fitted by a Cauchy-type model over the whole examined wavelength range (245–725 nm). The model, which takes into account the anisotropy of the film, uses the film thickness of several mono- and multi-layers films, together with the in-plane and the out-of-plane components of the refraction index as free parameters. The obtained mean thickness of the monolayer (ML), 2.50±0.02 nm, very well agrees with X-rays reflectivity measurements and with literature data.

Electrochemical Cu deposition on thiol covered Au(111) surfaces

Abstract The electrochemical deposition of Cu on Au(111) surfaces covered by self-assembled thiol monolayers has been followed in situ by electrochemical STM. Monolayers formed by thiols of different chain length have been chemisorbed on gold electrodes. The thiol molecules are organized in a quasi-crystalline structure characterized by a multi-domain ordering. The presence of the organic layer strongly influences the Cu deposition process. Cyclovoltammetry shows the absence of the underpotential deposition peaks observed on bare gold as well as a decrease of the electrochemical current in the overpotential regime. Independently of the chain length, in the UPD region we observe the formation of Cu nanoparticles 2–5 nm in diameter, one Cu atomic layer in height, uniformly distributed at the surface. The Cu cluster density reaches its maximum in the UPD regime. In the OPD region a chain length dependent behaviour is observed. Long thiol monolayers prevent any further growth of already existing clusters while on short thiol covered surfaces an almost 2D growth of Cu is observed.

Optical properties of Yeast Cytochrome c monolayer on gold: an in situ spectroscopic ellipsometry investigation.

The adsorption of Yeast Cytochrome c (YCC) on well defined, flat gold substrates has been studied by Spectroscopic Ellipsometry (SE) in the 245-1000 nm wavelength range. The investigation has been performed in aqueous ambient at room temperature, focusing on monolayer-thick films. In situ δΨ and δΔ difference spectra have shown reproducibly well-defined features related to molecular optical absorptions typical of the so-called heme group. The data have been reproduced quantitatively by a simple isotropic optical model, accounting for the molecular absorption spectrum and film-substrate interface effects. The simulations allowed a reliable estimate of the film thickness and the determination of the position and the shape of the so-called Soret absorption peak that, within the experimental uncertainty, is the same found for molecules in liquid. These findings suggest that YCC preserves its native structure upon adsorption. The same optical model was able to reproduce also ex situ results on rinsed and dried samples, dominated by the spectral features associated to the polypeptide chain that tend to overwhelm the heme absorption features.

Encapsulated yeast cells inside Paramecium primaurelia: a model system for protection capability of polyelectrolyte shells

One of the most promising applications of encapsulated living cells is their use as protected transplanted tissue into the human body. A suitable system for the protection of living cells is the use of nano‐ or microcapsules of polyelectrolytes. These shells can be deposited easily on top of the cells by means of a layer‐by‐layer technique. An interesting feature of the capsules is the possibility to control their properties on a nanometre level, tuning their wall texture via the preparation conditions. Here we introduce a model system to test the protection ability of polyelectrolyte capsules. Common bakery yeast cells were encapsulated. They were coated with a fluorescently labelled shell at conditions known to guarantee cell survival, and the cell interior was stained with DAPI. The protozoan Paramecium primaurelia was incubated with this double‐stained living yeast and visualized by means of two‐photon excitation fluorescence microscopy. Cross‐sections of the dye‐stained material as well as autofluorescence of the fixed protozoan allowed us to follow the digestion of the coated yeast with time. Our investigation reveals that capsules prepared under these deposition conditions are permeable to lysosomal enzymes, leading to degradation of the yeast inside the intact capsules. Our preliminary results indicate the suitability of the introduced model as a test system of this permeability.