DEVELOPMENT OF NOVEL HIGH PERFORMANCE PROTEIN MICROARRAYS FOR DIAGNOSTIC APPLICATIONS

Several application of protein microarray technology in diagnostics have been published and a limited number of protein microarrays is currently available on the In Vitro Diagnostics (IVD) market. Albeit several advantages, related to the miniaturization, the multiplexing capability and the possibility of integrating the immunoassays in biosensing devices, microarrays may still lack of specificity or sensitivity. To overcome these limitations and expand the use of protein microarray platform in diagnostics, the present PhD research aimed at developing innovative approaches to increase the assay specificity and sensitivity, reaching very low detection limits, that are compatible with the use of the proposed devices in diagnostics. Furthermore, the use of protein microarrays has been applied to the characterization of emerging biomarkers: exosomes. First of all, surface immobilized hydrogels have been investigated as reagent reservoir for microarray reagents. They have been demonstrated to store reagents in a dry form, stable over days, in a format easy to transport and to preserve. Moreover, they also acted as chambers able to physically separate analytes or reagents which may cross-react with proteins on the printed arrays. In this way the solution was prevented from spreading over the surface and the assays provided sentitive performances, comparable to standard static incubations. In further studies, the complementarity of information provided by fluorescence-based, label-free IRIS and SP-IRIS microarray platforms has been applied to develop immunoassays useful in the diagnostics of Neurodegenerative Disorders. Specifically, two different assay formats have been exploited. The first part of the work focused on the development of a classical sandwich immunoassay able to detect physiological concentrations of Amyloid-beta peptides, biomarkers for Alzheimer\u2019s disease, in both artificial cerebrospinal fluid and real human samples. The second study was aimed at extending the concept of protein microarrays to extracellular vesicles (i.e., exosomes) detection through surface antigen-antibodies recognition. In this innovative application, the nanoparticles were detected with label-free IRIS (total biomass measurements) and SP-IRIS (particle counting and size distribution). In addition, individual particles were incubated with gold-labeled antibodies to identify biomarkers expressed on their surface

Simplified high-order Volterra series transfer function for optical transmission links

We develop a simplified high-order multi-span Volterra series transfer function (SHMS- VSTF), basing our derivation on the well-known third-order Volterra series transfer function (VSTF). We notice that when applying an approach based on a recursive method and considering the phased-array factor, the order of the expression for the transfer function grows as 3 raised to the number of considered spans. By imposing a frequency-flat approximation to the higher-order terms that are usually neglected in the commonly used VSTF approach, we are able to reduce the overall expression order to the typical third-order plus a complex correction factor. We carry on performance comparisons between the purposed SH-MS-VSTF, the well-known split-step Fourier method (SSFM), and the third-order VSTF. The SH-MS-VSTF exhibits a uniform improvement of about two orders of magnitude in the normalized mean squared deviation with respect to the other methods. This can be translated in a reduction of the overall number of steps required to fully analyze the transmission link up to 99.75% with respect to the SSFM, and 98.75% with respect to the third-order VSTF, respectively, for the same numerical accuracy

Development of the Prevent for Work questionnaire (P4Wq) for assessment of musculoskeletal risk in the workplace: part 1-literature review and domains selection

Objective This study aims to define appropriate domains and items for the development of a self-administered questionnaire to assess the risk of developing work-related musculoskeletal disorder (WMSD) and the risk of its progression to chronicity. Design Literature review and survey study. Setting and participants A literature review and a two-round interview with 15 experts in musculoskeletal pain were performed to identify the available domains for WMSD assessment. Interventions and outcome To ensure quality, only validated questionnaires were included for the Delphi process. A three-round Delphi method, with three round steps, was used to select the most pertinent and relevant domains and items. Results Nine questionnaires were identified through the expert discussion and literature review, comprising 38 candidate domains and 504 items. In the first round of the Delphi group, 17 domains reached more than 70% agreement and were selected. In the second round, 10 domains were rejected, while 11 were selected to complete the pool of domains. In the third and final round, 89 items belonging to 28 domains were defined as significant to develop a WMSDs risk assessment questionnaire. Conclusions No specific risk assessment questionnaires for WMSDs were identified from the literature. WMSD risk of presence and chronicity can be defined by an assessment tool based on the biopsychosocial model and the fear-avoidance components of chronic pain. The present study provides the formulation and operationalisation of the constructs in domains and items needed for developing and validating the questionnaire

Membrane-binding peptides for extracellular vesicles on-chip analysis

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis

BPSL1626 : Reverse and Structural Vaccinology Reveal a Novel Candidate for Vaccine Design Against Burkholderia pseudomallei

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 angstrom resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules

Molecular modeling and docking of food-derived Angiotensin I-converting enzyme inhibiting peptides

This study evaluated insecticidal and repellent effects of Vitex cymosa and Eschweilera pedicellata extracts against Sitophilus zeamais adults. Contact on filter paper discs and contaminated grain ingestion assays were performed. The repellent effect was evaluated with the "preferential area" method. The extracts provided good results by ingestion and as repellents, but not by contact. V. cymosa branches methanol extract was the best, killing nearly 70% of the individuals at its highest concentration, followed by V. cymosa flowers dichloromethane extract and E. pedicellata branches aqueous extract. Among these, only V. cymosa leaves dichloromethane extract did not reduce the number of individuals in F1. Analyzing the repellent effect, when the variable concentration was taken into account, no extract was dose-dependent, and the intensity of response varied with the time interval. Among the extracts tested, V. cymosa branches methanol extract is the most promising one, which negative effect on parental resulted in F1 decrease number and the ingestion way was the most efficient

[Evolution of choledochus prostheses using lyophilised and siliconated dura mater. Final results].

Some research into choledochic prosthesis with lyophilised and siliconated dura mater has been concluded. Cholangiographic and histological controls one year after intervention are reported