16 research outputs found
DEVELOPMENT OF A NOVEL NANOSENSOR FOR THE STUDY OF BIOMOLECULAR INTERACTIONS
No full textThe evaluation and quantification of molecular interactions is of paramount importance in modern biology and molecular medicine. Therefore, there is a continuous exploration for new methodologies capable to detect and to measure binding affinities during reversible
molecular interactions. This work is devoted to explore a new tool based on the high
sensitivity that the measurement of the scattered light intensity offers when the binding
occurs on the surface of index-matched colloids.
Static light scattering is not a traditional technique to study molecular association because
the binding of insulated ligands and receptors in dilute solutions produce negligible
increment of the scattered light, while mesoscopic particles hosting multiple receptors or
ligands, including real bacteria, typically scatter too much light compared with the
contributions due to molecular adhesion on their surface. This difficulty can be overcome
by supporting the receptors on nano-scale latex spheres whose refractive index closely
matches the one of water.
As “Phantom Nanoparticles” (PNPs), we have used highly hydrophobic monodisperse
spherical fluoroelastomer colloids, with radius R = 39 Å} 1 nm, and whose refractive index,
at our working temperature (30ÅãC) and wavelength (633 nm), is np0 = 1.3248 (under the
same conditions the refractive index of water is nW = 1.3319). Surfactants added to a PNP
dispersion readily adsorb on their hydrophobic surfaces, generating a self-assembled
monolayer which can be easily equipped with molecular hydrophilic end groups of various
kind, including well-known receptors and/or ligands.
This label-free method has been assessed through the precise determination of the binding
constant of the antibiotic vancomycin with the tripeptide L-Lys-D-Ala-D-Ala and of the
vancomycin dimerization constant. We have enlightened the role of bidentate effect and
molecular hindrance in the activity of this glycopeptide.
After this success first result, an accurate determination of the optimal properties of
nanoparticles employed has been performed by comparative experiments and through
theoretical evaluation (CHAPETR 3). The effects of size, refractive index, electric charge,
and dilution on the reliability and accuracy of the method has been evaluated. Quite
surprisingly, perfect index matching and minimal size (i.e., maximum surface), which is
almost attained in one of the colloids here employed, do not represent the ideal conditions.
Rather, we show that a nanoparticle radius of 100 nm and a refractive index slightly below
that of water yields the best signal/background amplitude. We also show that repulsive
interactions can lead to artifacts in the adsorption isotherm, thus indicating that
electrostatic stabilization should be kept at a minimum.
Successively, the particles, already optically phantom, have also been made biologically
“invisible” through PEG coating and decorated by interacting proteins, thus providing a
mean to investigate the biological properties of proteins (CHAPETR 4). Avidin decorated
Phantom Nanoparticles have been prepared and were employed to detect interactions
between different kinds of biotinylated proteins. Using this approach, biotinylated protein
A was anchored on the surface of the nanoparticles, and were exploited as a functional
probe for the rapid, quantitative, picomolar detection of human IgG antibodies.
We used Phantom Nanoparticles to evaluate substrate recognition by Streptomyces PMF
Phospholipase D inactivated mutants (CHAPETR 5). The use of this specific technique
seems to have some peculiar advantages over other methods in the case of phospholipidsacting
enzymes. In fact, the substrate (or a substrate analog) can be organized onto the
surface of Phantom Nanoparticles at a desired concentration with optimal display of the
polar head group, while the hydrophobic chains result packed into the surfactant
monolayer, thus limiting the occurrence of non-specific interactions.
The last part of this work is focused on an attempt toward the stable functionalization of
Phantom Nanoparticles (CHAPETR 6). Diacetylene surfactants spontaneously adsorb on the
nanoparticles and then they are polymerized by exposure to UV light. So far, the stability
of the amphiphilic coating around the nanoparticle solely depended on weak hydrophobic
interactions. The attachment of the polymer to the particle surface, because of the
numerous contact points, is highly stable and can be improved further by crosslinking of
the polymer shell. Huns generating a quantifiable number of functional groups suitable for
covalent receptor anchorage.
All these observations, demonstrate the feasibility of this new technique, which makes it
possible to easily generate different synthetic receptors, and highlight this technique as a
versatile novel method to study, both qualitatively and quantitatively, of molecular
recognition processes.
The work described in this thesis is partially published in:
Morasso C., Bellini T., Monti D., Bassi M., Prosperi D., Riva S.:
Dispersed phantom scatterer technique reveals subtle differences in
substrate recognition by phospholipase D inactive mutant;
ChemBioChem. Submitted, currently under review
Prosperi D., Morasso C., Tortora P., Monti D., Bellini T.: Avidin decorated
core-shell nanoparticles for biorecognition studies by elastic light
scattering; ChemBioChem. 2007 (8): 1021-1028 (Impact factor: 3,446).
Prosperi D., Morasso C., Mantegazza F., Buscaglia M., Houg L., Bellini
T.: Phantom nanoparticles as probes of biomolecular interaction; Small.
2006 (8-9):1060-1067 (Impact factor: 6,408)
Preoperative Systemic Inflammatory Biomarkers Are Independent Predictors of Disease Recurrence in ER+ HER2- Early Breast Cancer
No full textThe host’s immune system plays a crucial role in determining the clinical outcome of many cancers, including breast cancer. Peripheral blood neutrophils and lymphocytes counts may be surrogate markers of systemic inflammation and potentially reflect survival outcomes. The aim of the present study is to assess the role of preoperative systemic inflammatory biomarkers to predict local or distant relapse in breast cancer. In particular we investigated ER+ HER2- early breast cancer, considering its challenging risk stratification. A total of 1,763 breast cancer patients treated at tertiary referral Breast Unit were reviewed. Neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte (LMR) ratios were assessed from the preoperative blood counts. Multivariate analyses for 5-years locoregional recurrence-free (LRRFS), distant metastases-free (DMFS) and disease-free survivals (DFS) were performed, taking into account both blood inflammatory biomarkers and clinical-pathological variables. Low NLR and high LMR were independent predictors of longer LRRFS, DMFS and DFS, and low PLR was predictive of better LRRFS and DMFS in the study population. In 999 ER+ HER2- early breast cancers, high PLR was predictive of worse LRRFS (HR 0.42, p=0.009), while high LMR was predictive of improved LRRFS (HR 2.20, p=0.02) and DFS (HR 2.10, p=0.01). NLR was not an independent factor of 5-years survival in this patients’ subset. Inflammatory blood biomarkers and current clinical assessment of the disease were not in agreement in terms of estimate of relapse risk (K-Cohen from -0.03 to 0.02). In conclusion, preoperative lymphocyte ratios, in particular PLR and LMR, showed prognostic relevance in ER+ HER2- early breast cancer. Therefore, they may be used in risk stratification and therapy escalation/de-escalation in patients with this type of tumor
Towards a Universal Method for the Stable and Clean Functionalization of Inert Perfluoropolymer Nanoparticles : Exploiting Photopolymerizable Amphiphilic Diacetylenes
No full textHighly fluorinated materials are being widely investigated due to a number of peculiar properties, which are potentially useful for various applications, including use as lubricants, anti-adhesive films, and substitutes for biological fluids for biomedical utilization. However, at present such potential is still poorly exploited. One of the major drawbacks that hampers the rapid development of nanoscale fluoro-hybrid devices is the remarkable inertness of perfluoropolymeric materials that lack reactive functionalities, as they do not offer any functional groups that can be employed to covalently anchor organic molecules on their surface. In this paper, a convenient method for the stable biofunctionalization of strongly unreactive perfluoropolymer nanoparticles (PnPs) is reported. PnPs are easily coated with newly synthesized asymmetric diacetylenic monomer compounds (ADMs), thanks to PnP's high propensity to interact with hydrophobic moieties. Once monomerically adsorbed onto PnPs, such suitably designed ADMs enable the formation of a robust polymeric shell around the perfluoroelastomer core via a clean UV-promoted localized photopolymerization. Given the peculiar optical characteristics of PnPs, the coating of the particles can be monitored step by step using light scattering, which also allows estimation of the fraction of reacted monomers by competitive adsorption with smaller particles. The potential of this method for the biofunctionalization of PnPs is demonstrated with representative proteins and carbohydrates. Among them, the extension to avidin-biotin technology may broaden the scope and applicability of this strategy to potentially a large number of molecules of biomedical interest. Making the unreactive reactive: A smart method for the biofunctionalization of strongly inert perfluoropolymer nanoparticles (PnPs) is presented, using a stable coating with novel diacetylenic compounds followed by clean UV photopolymerization to generate reactive functionalities on the PnP surface. This method further allows fine tuning of the amount of conjugated biomolecules, which can be sensitively and straightforwardly quantified
Raman spectroscopy reveals biochemical differences in plasma derived extracellular vesicles from sporadic amyotrophic lateral sclerosis patients
No full textSporadic Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease for which there is no validated blood based biomarker. Extracellular vesicles (EVs) have the potential to solve this unmet clinical need. However, due to their heterogeneity and complex chemical composition, EVs are difficult to study. Raman spectroscopy (RS) is an optical method that seems particularly well suited to address this task. In fact, RS provides an overview of the biochemical composition of EVs quickly and virtually without any sample preparation. In this work, we studied by RS small extracellular vesicles (sEVs), large extracellular vesicles (lEVs) and blood plasma of sporadic ALS patients and of a matched cohort of healthy controls. The obtained results highlighted lEVs as a particularly promising biomarker for ALS. In fact, their Raman spectra show that sporadic ALS patients have a different lipid content and less intense bands relative to the aromatic aminoacid phenylalanine
Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells
No full textExtracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves
Commentary on Uses of Arguments From Definition in Children’s Argumentation
No full textConference Papers and Commentary.This paper presents an analysis of the reasoning of two 5-year old children’s use of argument from definition. The author uses the Argumentum Model of Topics (AMT; Rigotti & Greco-Morasso, 2009) to accomplish this task. A brief history of the “locus of definition” is presented, as well as a description of how and where the data were collected. More specifically, the data come from a study of students conducted for over 30 years in Switzerland. Two examples are discussed where an adult experimenter examined these children’s responses to conservation of liquid and number tasks. The two examples of children’s responses were analyzed using the locus from definition. Results show that children are capable of providing responses indicating their beliefs and support for them that are beyond Piaget’s description of a preoperational child’s reasoning abilities
On least-cost path for realistic simulation of human motion
No full textWe are interested in "human-like" automatic motion simulation with applications in ergonomics.
The apparent redundancy of the humanoid wrt its explicit tasks leads to the problem of choosing a plausible movement in the framework of redundant kinematics.
Some results have been obtained in the human motion literature for reach motion that involves the position of the hands. We discuss these results and a motion generation scheme associated. When orientation is also explicitly required, very few works are available and even the methods for analysis are not defined.
We discuss the choice for metrics adapted to the orientation, and also the problems encountered in defining a proper metric in both position and orientation. Motion capture and simulations are provided in both cases.
The main goals of this paper are: to provide a survey on human motion features at task level for both position and orientation, to propose a kinematic control scheme based on these features, to define properly the error between motion capture and automatic motion simulation
Retórica ¿de la reacción?: Reconceptualizando las tesis de Hirschman a partir de las nociones de topos, máxima y actantes
No full textIn The Rhetoric of Reaction (2020), Albert Hirschman defines three classes of reactionary theses: the “perversity thesis”, the “futility thesis” and the “risk thesis”. The author seeks to describe the argumentative compulsions found in reactionary discourse throughout modern Western political history. We will claim here that these thesis acquire greater explanatory capacity if they are dis-sociated from the contingent values that fill them with local and historical meanings. To achieve such a conceptual operation, we propose to think of them as argumentative topoi, identifiable from the definitions provided by Ruth Amossy (2017), or as maxims, following the Argumentum Model of Topics of the Lugano school, represented by Sara Greco Morasso and Eddo Rigotti. This conceptual operation leads us to discriminate between “actors” and “actants”, as formal positions in a discussion and identified by Plantin (2001) as the “proposer”, the “opponent” and the “third party” in his dialogic model. Standing on this theoretical framework, the objective of this article is to find a more precise conceptualization of the thesis described by Hirschman to understand the feature of universality that makes them capable of operating effectively and indistinctly at both ends of the political-ideological arc.En The Rhetoric of Reaction (2020), Albert Hirschman define tres clases de tesis reaccionarias: la “tesis de la perversidad”, la “tesis de la futilidad” y la “tesis del riesgo”. El autor busca describir las compulsiones argumentativas que pueden hallarse en el discurso reaccionario a lo largo de la historia política moderna de occidente. Sostendremos aquí que estas tesis adquieren mayor capacidad explicativa si son disociadas de los valores contingentes que las llenan de sentidos locales e históricos plenos. Para lograr tal operación de vaciamiento semántico, proponemos pensarlas como topoi argumentativos, identificables a partir de las definiciones provistas por Ruth Amossy (2017), o, alternativamente, como máximas, siguiendo el Argumentum Model of Topics de la escuela de Lugano, representada fundamentalmente por Sara Greco Morasso y Eddo Rigotti. La operación conceptual propuesta nos conduce a discriminar entre “actores” y “actantes”, como posiciones formales en una discusión e identificados por Plantin (2001) como el “proponente”, el “oponente” y el “tercero” en su modelo dialogal. Partiendo de este marco teórico de cuño discursivista, el objetivo de este trabajo consiste en dar con una conceptualización más precisa de las tesis descriptas por Hirschman para entender el carácter universal que las hace capaces de operar eficaz e indistintamente en ambos extremos del arco político-ideológic
A singular value approach for humanoid motion analysis and simulation
No full textWe are interested in human motion characterization and automatic motion simulation. The apparent redundancy of the humanoid w.r.t its explicit tasks lead to the problem of choosing a plausible movement in the framework of redundant kinematics. This work explores the intrinsic relationships between singular value decomposition at kinematic level and optimization principles at task level and joint level. Two task-based schemes devoted to simulation of human motion are then proposed and analyzed. These results are illustrated by motion captures, analyses and task-based simulations. Pattern of singular values serve as a basis for a discussion concerning the similarity of simulated and real motions
On singular values decomposition and patterns for human motion analysis and simulation
No full textWe are interested in human motion characterization and automatic motion simulation. The apparent redun- dancy of the humanoid w.r.t its explicit tasks lead to the problem of choosing a plausible movement in the framework of redun- dant kinematics. This work explores the intrinsic relationships between singular value decomposition at kinematic level and optimization principles at task level and joint level. Two task- based schemes devoted to simulation of human motion are then proposed and analyzed. These results are illustrated by motion captures, analyses and task-based simulations. Pattern of singular values serve as a basis for a discussion concerning the similarity of simulated and real motions
