Implementation of SELEX technique on Lab-on-Chip systems

The thesis presents the design and the experimental development of a compact and high sensitive Lab-on-Chip (LoC) system suitable for the implementation of SELEX technique. SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a combinatorial technique used in molecular biology to produce copies of the same nucleotide and to select a strand of DNA (aptamer) specific for a target molecule. The proposed Lab-On-Chip system includes the following functional units: an amplification module based on the PCR technique; a separation module able to obtain a single strand DNA from a double strand DNA and a selection module for the specific selection of the aptamer. These functionalities are implemented combining microfluidic components (micro-channels and micro-valves), electronic devices (amorphous silicon photosensors, thin film heaters, temperature sensors) and bioanalytical procedure

Integration of capillary and EWOD technologies for autonomous and low-power consumption micro-analytical systems

This work presents a miniaturized system combining, on the same microfluidic chip, capillarity and electrowetting-on-dielectric (EWOD) techniques for movement and control of fluids. The change in hydrophobicity occurring at the edge between a capillary channel and a hydrophobic layer is successfully exploited as a stop-and-go valve, whose operation is electronically controlled through the EWOD electrodes. Taking into account the variety of microfluidic operation resulting from the combination of the two handling techniques and their characteristic features, this work prompts the development of autonomous, compact and low-power consumption lab-on-chip systems

Integration of amorphous silicon balanced photodiodes and thin film heaters for biosensing application

This work presents the development and testing of an integrated system for on-chip detection of thermochemiluminescent biomolecules. The activation energy of the reaction is provided by a transparent structure of thin film heaters deposited on one side of a glass substrate. Light, passing through the substrate, reaches an array of amorphous silicon differential structure deposited on the opposite side of the glass substrate. The structure is designed to perform differential current measurements between a light- shielded diode, whose current is sensitive only to temperature, and a photosensor, sensitive to both incident light and temperature. The device therefore balances the thermal variations of the photodiode current and reduces the dark-current noise. These features make the presented system very appealing as highly miniaturized micro-analytical devices for biosensing applications

The lizard celestial compass detects linearly polarized light in the blue

SummaryThe present study first examined whether ruin lizards Podarcis sicula are able to orientate using plane polarized light produced by a LCD screen. Ruin lizards were trained and tested indoors, inside an hexagonal Morris water maze, positioned under the LCD screen producing white polarized light with a single E-vector, which provided an axial cue. White polarized light did not include wavelengths in the UV. Lizards orientated correctly either when tested with E-vector parallel to the training axis or after 90° rotation of the E-vector direction, and thus validating the apparatus. Further experiments examined whether in ruin lizards there is a preferential region of the light spectrum to perceive the E-vector direction of polarized light. For this purpose, lizards reaching learning criteria under white polarized light were subdivided into 4 experimental groups. Each group was respectively tested for orientation under a different spectrum of plane polarized light (named red, green, cyan and blue) with equalized photon flux density. Lizards tested under blue polarized light orientated correctly, whereas lizards tested under red polarized light were completely disoriented. Green polarized light was barely discernible by lizards, and thus insufficient for a correct functioning of their compass. When exposed to cyan polarized light, lizard orientation performances were optimal, indistinguishable from lizards detecting blue polarized light. Overall, the present results demonstrate that perception of linear polarization in the blue is necessary - and sufficient - for a proper functioning of the sky polarization compass of ruin lizards. This may be adaptively important, since detection of polarized light in the blue improves functioning of the polarization compass under cloudy skies, i.e. when the alternative celestial compass based on detection of the sun disk is rendered useless because the sun is obscured by clouds

Padina pavonica Extract Promotes In Vitro Differentiation and Functionality of Human Primary Osteoblasts

Marine algae have gained much importance in the development of nutraceutical products due to their high content of bioactive compounds. In this work, we investigated the activity of Padina pavonica with the aim to demonstrate the pro-osteogenic ability of its extract on human primary osteoblast (HOb). Our data indicated that the acetonic extract of P. pavonica (EPP) is a safe product as it did not show any effect on osteoblast viability. At the same time, EPP showed to possess a beneficial effect on HOb functionality, triggering their differentiation and mineralization abilities. In particular EPP enhanced the expression of the earlier differentiation stage markers: a 5.4-fold increase in collagen type I alpha 1 chain (COL1A1), and a 2.3-fold increase in alkaline phosphatase (ALPL), as well as those involved in the late differentiation stage: a 3.7-fold increase in osteocalcin (BGLAP) expression and a 2.8-fold in osteoprotegerin (TNFRSF11B). These findings were corroborated by the enhancement in ALPL enzymatic activity (1.7-fold increase) and by the reduction of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) ratio (0.6-fold decrease). Moreover, EPP demonstrated the capacity to enhance the bone nodules formation by 3.2-fold in 4 weeks treated HOb. Therefore, EPP showed a significant capability of promoting osteoblast phenotype. Given its positive effect on bone homeostasis, EPP could be used as a useful nutraceutical product that, in addition to a healthy lifestyle and diet, can be able to contrast and prevent bone diseases, especially those connected with ageing, such as osteoporosis (OP)

Lab-on-chip system combining a microfluidic-ELISA with an array of amorphous silicon photosensors for the detection of celiac disease epitopes

This work presents a lab-on-chip system, which combines a glass-polydimethilsiloxane microfluidic network and an array of amorphous silicon photosensors for the diagnosis and follow-up of Celiac disease. The microfluidic chip implements an on-chip enzyme-linked immunosorbent assay (ELISA), relying on a sandwich immunoassay between antibodies against gliadin peptides (GPs) and a secondary antibody marked with horseradish peroxidase (Ig-HRP). This enzyme catalyzes a chemiluminescent reaction, whose light intensity is detected by the amorphous silicon photosensors and transduced into an electrical signal that can be processed to recognize the presence of antibodies against GPs in the serum of people affected by Celiac syndrome.The correct operation of the developed lab-on-chip has been demonstrated using rabbit serum in the microfluidic ELISA. In particular, optimizing the dilution factors of both sera and Ig-HRP samples in the flowing solutions, the specific and non-specific antibodies against GPs can be successfully distinguished, showing the suitability of the presented device to effectively screen celiac disease epitopes. Keywords: Lab-on-chip, Celiac disease, Microfluidics, On-chip detection, ELISA, Amorphous silicon photosensor

Relapsed/Refractory Diffuse Large B-Cell Lymphoma (R/R DLBCL) Patients: A Retrospective Analysis of Eligibility Criteria for CAR-T Cell Therapy

Patients (pts) with diffuse large B-cell lymphoma (DLBCL) refractory to second-line therapy or relapsed after an autologous stem cell transplant (ASCT) have a very poor clinical outcome with a median overall survival (OS) of 5 and 8-10 months, respectively. Autologous anti-CD19 chimeric antigen receptor (CD19 CAR) T cells have been associated with sustained complete remissions and long-term survivals in a large proportion of pts with R/R DLBCL by the two pivotal clinical trials Zuma1 and Juliet. This has led to the rapid approval by FDA and then by EMA of CAR-T cells for the third-line treatment of R/R DLBCL. Despite being a potentially revolutionary treatment for pts with advanced disease, the costs are much greater than any previously approved cancer therapy and this may become a substantial economic challenge for the health care system. The definition of inclusion and exclusion criteria capable of identifying more precisely pts who can successfully undergo CAR-T cell therapy, minimizing the severity of the toxicity, still remains a matter of discussion. Moreover, some eligible pts run the risk of becoming ineligible because of poor disease control. Indeed, one of the major obstacles to the successful use of CAR-T cells is the 4-5 week period so far required for the manufacturing and transfer of CAR-T cells. To address this issue, we have examined data of R/R DLBCL pts managed between 2010 and 2018 at our Center in order to: 1) better identify the characteristics and outcome of a cohort of R/R DLBCL pts potentially eligible, according to the approval criteria, for CAR-T cell therapy; 2) define factors influencing CAR-T cell eligibility; 3) make a realistic estimate of pts eligible for CAR-T cells. In this retrospective real-life cohort of R/R DLBCLs, 82/480 pts (17%) were R/R tosecond-line treatment including ASCT. Considering Juliet's inclusion/exclusion criteria for CAR-T cell therapy, only 50 pts (10.4%) would be eligible for CAR-T cells. Our analysis suggests that elevated LDH plus ECOG ≥2 have to be considered the two most significant features of very rapid disease progression. These variables should be taken in account in order to better select DLBCL pts potentially eligible to CAR-T therapy

Intracoronary ST-Segment Shift Soon After Elective Percutaneous Coronary Intervention Accurately Predicts Periprocedural Myocardial Injury

Background— Elevation of cardiac biomarkers after coronary angioplasty (percutaneous coronary intervention [PCI]) reflects periprocedural myocardial damage and is associated with adverse cardiac events. We assessed whether periprocedural myocardial damage that occurs despite successful PCI could be rapidly and easily identified by intracoronary ST-segment recording with the use of a catheter guidewire. Methods and Results— In 108 consecutive stable patients undergoing elective single-vessel PCI, we recorded unipolar ECG from the intracoronary guidewire in the distal coronary before PCI and 2 minutes after the last balloon inflation. After PCI, intracoronary ST-segment shift ≥1 mm from baseline was considered significant. Troponin I levels were measured at baseline and at 8 and 24 hours after intervention, and myocardial damage was defined as troponin I increase above the upper normal value after intervention. All patients had normal cardiac marker values before PCI, and PCI was successful in all (residual stenosis <20%, Thrombolysis in Myocardial Infarction grade 3 flow). After PCI, long-term follow-up data were collected; myocardial damage was detected in 50 patients (46%), although abnormal creatine kinase-MB values were documented in only 11 (10%). Significant intracoronary ST-segment shift after PCI was present in 40 patients (37%; group A) and absent in the remaining 68 (63%; group B). Procedural myocardial damage was documented in 37 group A patients (93%) and in 13 group B patients (19%; P <0.001); significant ECG changes were found on standard ECG after intervention in only 5 patients (13%) and 1 patient (1%) ( P <0.05). Sensitivity of intracoronary ST-segment shift for predicting myocardial damage was 74%, and specificity was 95%, with positive and negative predictive values of 93% and 81%, respectively. On multivariate analysis, intracoronary ST-segment shift was the sole independent predictor of myocardial damage (odds ratio, 54.1; 95% confidence interval, 12.1 to 240; P <0.0001). At a median follow-up of 12±5 months, major coronary event–free survival was significantly worse in group A patients (log-rank test χ 2 =4.0; P <0.05). Conclusions— After successful single-vessel PCI, intracoronary ST-segment shift allows the prompt and inexpensive identification of patients developing myocardial injury, who may require adjunctive therapy and longer in-hospital stay

MICA-129 dimorphism and soluble MICA are associated with the progression of multiple myeloma

Natural killer (NK) cells are immune innate effectors playing a pivotal role in the immunosurveillance of multiple myeloma (MM) since they are able to directly recognize and kill MM cells. In this regard, among activating receptors expressed by NK cells, NKG2D represents an important receptor for the recognition of MM cells, being its ligands expressed by tumor cells, and being able to trigger NK cell cytotoxicity. The MHC class I-related molecule A (MICA) is one of the NKG2D ligands; it is encoded by highly polymorphic genes and exists as membrane-bound and soluble isoforms. Soluble MICA (sMICA) is overexpressed in the serum of MM patients, and its levels correlate with tumor progression. Interestingly, a methionine (Met) to valine (Val) substitution at position 129 of the α2 heavy chain domain classifies the MICA alleles into strong (MICA-129Met) and weak (MICA-129Val) binders to NKG2D receptor. We addressed whether the genetic polymorphisms in the MICA-129 alleles could affect MICA release during MM progression. The frequencies of Val/Val, Val/Met, and Met/Met MICA-129 genotypes in a cohort of 137 MM patients were 36, 43, and 22%, respectively. Interestingly, patients characterized by a Val/Val genotype exhibited the highest levels of sMICA in the sera. In addition, analysis of the frequencies of MICA-129 genotypes among different MM disease states revealed that Val/Val patients had a significant higher frequency of relapse. Interestingly, NKG2D was downmodulated in NK cells derived from MICA-129Met/Met MM patients. Results obtained by structural modeling analysis suggested that the Met to Val dimorphism could affect the capacity of MICA to form an optimal template for NKG2D recognition. In conclusion, our findings indicate that the MICA-129Val/Val variant is associated with significantly higher levels of sMICA and the progression of MM, strongly suggesting that the usage of soluble MICA as prognostic marker has to be definitely combined with the patient MICA genotype