Development of bionanotechnological strategies for signal enhancement in nucleic acids biosensors

Nucleic acid biosensors represent a powerful tool for clinical and environmental pathogens detection. For applications such as point-of-care biosensing, it is fundamental to develop sensors that should be automatic, inexpensive, portable and require a professional skill of the user that should be as low as possible. With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an amplification step must be implemented. Often this type of determinations should be performed with simple, automatic and inexpensive hardware: the use of a chemical (or nanotechnological) isothermal solution would be desirable. My Ph.D. project focused on the study and on the testing of four isothermal reactions which can be used to amplify the nucleic acid analyte before the binding event on the surface sensor or to amplify the signal after that the hybridization event with the probe. Recombinase polymerase amplification (RPA) and ligation-mediated rolling circle amplification (L-RCA) were investigated as methods for DNA and RNA amplification. Hybridization chain reaction (HCR) and Terminal deoxynucleotidil transferase-mediated amplification were investigated as strategies to achieve the enhancement of the signal after the surface hybridization event between target and probe. In conclusion, it can be said that only a small subset of the biochemical strategies that are proved to work in solution towards the amplification of nucleic acids does truly work in the context of amplifying the signal of a detection system for pathogens. Amongst those tested during my Ph.D. activity, recombinase polymerase amplification seems the best candidate for a useful implementation in diagnostic or environmental applications.

Advanced signal and receiver design for next generation OFDM systems

This thesis deal with the design of advanced OFDM systems. Both waveform and receiver design have been treated. The main scope of the Thesis is to study, create, and propose, ideas and novel design solutions able to cope with the weaknesses and crucial aspects of modern OFDM systems. Starting from the the transmitter side, the problem represented by low resilience to non-linear distortion has been assessed. A novel technique that considerably reduces the Peak-to-Average Power Ratio (PAPR) yielding a quasi constant signal envelope in the time domain (PAPR close to 1 dB) has been proposed.The proposed technique, named Rotation Invariant Subcarrier Mapping (RISM),is a novel scheme for subcarriers data mapping,where the symbols belonging to the modulation alphabet are not anchored, but maintain some degrees of freedom. In other words, a bit tuple is not mapped on a single point, rather it is mapped onto a geometrical locus, which is totally or partially rotation invariant. The final positions of the transmitted complex symbols are chosen by an iterative optimization process in order to minimize the PAPR of the resulting OFDM symbol. Numerical results confirm that RISM makes OFDM usable even in severe non-linear channels. Another well known problem which has been tackled is the vulnerability to synchronization errors. Indeed in OFDM system an accurate recovery of carrier frequency and symbol timing is crucial for the proper demodulation of the received packets. In general, timing and frequency synchronization is performed in two separate phases called PRE-FFT and POST-FFT synchronization. Regarding the PRE-FFT phase, a novel joint symbol timing and carrier frequency synchronization algorithm has been presented. The proposed algorithm is characterized by a very low hardware complexity, and, at the same time, it guarantees very good performance in in both AWGN and multipath channels. Regarding the POST-FFT phase, a novel approach for both pilot structure and receiver design has been presented. In particular, a novel pilot pattern has been introduced in order to minimize the occurrence of overlaps between two pattern shifted replicas. This allows to replace conventional pilots with nulls in the frequency domain, introducing the so called Silent Pilots. As a result, the optimal receiver turns out to be very robust against severe Rayleigh fading multipath and characterized by low complexity. Performance of this approach has been analytically and numerically evaluated. Comparing the proposed approach with state of the art alternatives, in both AWGN and multipath fading channels, considerable performance improvements have been obtained. The crucial problem of channel estimation has been thoroughly investigated, with particular emphasis on the decimation of the Channel Impulse Response (CIR) through the selection of the Most Significant Samples (MSSs). In this contest our contribution is twofold, from the theoretical side, we derived lower bounds on the estimation mean-square error (MSE) performance for any MSS selection strategy,from the receiver design we proposed novel MSS selection strategies which have been shown to approach these MSE lower bounds, and outperformed the state-of-the-art alternatives. Finally, the possibility of using of Single Carrier Frequency Division Multiple Access (SC-FDMA) in the Broadband Satellite Return Channel has been assessed. Notably, SC-FDMA is able to improve the physical layer spectral efficiency with respect to single carrier systems, which have been used so far in the Return Channel Satellite (RCS) standards. However, it requires a strict synchronization and it is also sensitive to phase noise of local radio frequency oscillators. For this reason, an effective pilot tone arrangement within the SC-FDMA frame, and a novel Joint Multi-User (JMU) estimation method for the SC-FDMA, has been proposed. As shown by numerical results, the proposed scheme manages to satisfy strict synchronization requirements and to guarantee a proper demodulation of the received signal.

Progettazione e sviluppo di una versione distribuita di un algoritmo di subspace clustering

Il task del data mining si pone come obiettivo l'estrazione automatica di schemi significativi da grandi quantità di dati. Un esempio di schemi che possono essere cercati sono raggruppamenti significativi dei dati, si parla in questo caso di clustering. Gli algoritmi di clustering tradizionali mostrano grossi limiti in caso di dataset ad alta dimensionalità, composti cioè da oggetti descritti da un numero consistente di attributi. Di fronte a queste tipologie di dataset è necessario quindi adottare una diversa metodologia di analisi: il subspace clustering. Il subspace clustering consiste nella visita del reticolo di tutti i possibili sottospazi alla ricerca di gruppi signicativi (cluster). Una ricerca di questo tipo è un'operazione particolarmente costosa dal punto di vista computazionale. Diverse ottimizzazioni sono state proposte al fine di rendere gli algoritmi di subspace clustering più efficienti. In questo lavoro di tesi si è affrontato il problema da un punto di vista diverso: l'utilizzo della parallelizzazione al fine di ridurre il costo computazionale di un algoritmo di subspace clustering.

Analog Signal Acquisition and Conditioning for Near-Field Capacitive Communication and Active Combined EEG-EIT Monitoring

The work of the present thesis is focused on the implementation of microelectronic voltage sensing devices, with the purpose of transmitting and extracting analog information between devices of different nature at short distances or upon contact. Initally, chip-to-chip communication has been studied, and circuitry for 3D capacitive coupling has been implemented. Such circuits allow the communication between dies fabricated in different technologies. Due to their novelty, they are not standardized and currently not supported by standard CAD tools. In order to overcome such burden, a novel approach for the characterization of such communicating links has been proposed. This results in shorter design times and increased accuracy. Communication between an integrated circuit (IC) and a probe card has been extensively studied as well. Today wafer probing is a costly test procedure with many drawbacks, which could be overcome by a different communication approach such as capacitive coupling. For this reason wireless wafer probing has been investigated as an alternative approach to standard on-contact wafer probing. Interfaces between integrated circuits and biological systems have also been investigated. Active electrodes for simultaneous electroencephalography (EEG) and electrical impedance tomography (EIT) have been implemented for the first time in a 0.35 um process. Number of wires has been minimized by sharing the analog outputs and supply on a single wire, thus implementing electrodes that require only 4 wires for their operation. Minimization of wires reduces the cable weight and thus limits the patient's discomfort. The physical channel for communication between an IC and a biological medium is represented by the electrode itself. As this is a very crucial point for biopotential acquisitions, large efforts have been carried in order to investigate the different electrode technologies and geometries and an electromagnetic model is presented in order to characterize the properties of the electrode to skin interface.

Nondestructive evaluation of concrete compression strength by means of Artificial Neural Network (ANN)

The evaluation of structural performance of existing concrete buildings, built according to standards and materials quite different to those available today, requires procedures and methods able to cover lack of data about mechanical material properties and reinforcement detailing. To this end detailed inspections and test on materials are required. As a consequence tests on drilled cores are required; on the other end, it is stated that non-destructive testing (NDT) cannot be used as the only mean to get structural information, but can be used in conjunction with destructive testing (DT) by a representative correlation between DT and NDT. The aim of this study is to verify the accuracy of some formulas of correlation available in literature between measured parameters, i.e. rebound index, ultrasonic pulse velocity and compressive strength (SonReb Method). To this end a relevant number of DT and NDT tests has been performed on many school buildings located in Cesena (Italy). The above relationships have been assessed on site correlating NDT results to strength of core drilled in adjacent locations. Nevertheless, concrete compressive strength assessed by means of NDT methods and evaluated with correlation formulas has the advantage of being able to be implemented and used for future applications in a much more simple way than other methods, even if its accuracy is strictly limited to the analysis of concretes having the same characteristics as those used for their calibration. This limitation warranted a search for a different evaluation method for the non-destructive parameters obtained on site. To this aim, the methodology of neural identification of compressive strength is presented. Artificial Neural Network (ANN) suitable for the specific analysis were chosen taking into account the development presented in the literature in this field. The networks were trained and tested in order to detect a more reliable strength identification methodology.

Efficient ultrasonic signal processing techniques for aided medical diagnostics

Ultrasound imaging is widely used in medical diagnostics as it is the fastest, least invasive, and least expensive imaging modality. However, ultrasound images are intrinsically difficult to be interpreted. In this scenario, Computer Aided Detection (CAD) systems can be used to support physicians during diagnosis providing them a second opinion. This thesis discusses efficient ultrasound processing techniques for computer aided medical diagnostics, focusing on two major topics: (i) Ultrasound Tissue Characterization (UTC), aimed at characterizing and differentiating between healthy and diseased tissue; (ii) Ultrasound Image Segmentation (UIS), aimed at detecting the boundaries of anatomical structures to automatically measure organ dimensions and compute clinically relevant functional indices. Research on UTC produced a CAD tool for Prostate Cancer detection to improve the biopsy protocol. In particular, this thesis contributes with: (i) the development of a robust classification system; (ii) the exploitation of parallel computing on GPU for real-time performance; (iii) the introduction of both an innovative Semi-Supervised Learning algorithm and a novel supervised/semi-supervised learning scheme for CAD system training that improve system performance reducing data collection effort and avoiding collected data wasting. The tool provides physicians a risk map highlighting suspect tissue areas, allowing them to perform a lesion-directed biopsy. Clinical validation demonstrated the system validity as a diagnostic support tool and its effectiveness at reducing the number of biopsy cores requested for an accurate diagnosis. For UIS the research developed a heart disease diagnostic tool based on Real-Time 3D Echocardiography. Thesis contributions to this application are: (i) the development of an automated GPU based level-set segmentation framework for 3D images; (ii) the application of this framework to the myocardium segmentation. Experimental results showed the high efficiency and flexibility of the proposed framework. Its effectiveness as a tool for quantitative analysis of 3D cardiac morphology and function was demonstrated through clinical validation.

Diagnosis and Fault detection in Electrical Machines and Drives based on Advanced Signal Processing Techniques

In the present thesis, a new methodology of diagnosis based on advanced use of time-frequency technique analysis is presented. More precisely, a new fault index that allows tracking individual fault components in a single frequency band is defined. More in detail, a frequency sliding is applied to the signals being analyzed (currents, voltages, vibration signals), so that each single fault frequency component is shifted into a prefixed single frequency band. Then, the discrete Wavelet Transform is applied to the resulting signal to extract the fault signature in the frequency band that has been chosen. Once the state of the machine has been qualitatively diagnosed, a quantitative evaluation of the fault degree is necessary. For this purpose, a fault index based on the energy calculation of approximation and/or detail signals resulting from wavelet decomposition has been introduced to quantify the fault extend. The main advantages of the developed new method over existing Diagnosis techniques are the following: - Capability of monitoring the fault evolution continuously over time under any transient operating condition; - Speed/slip measurement or estimation is not required; - Higher accuracy in filtering frequency components around the fundamental in case of rotor faults; - Reduction in the likelihood of false indications by avoiding confusion with other fault harmonics (the contribution of the most relevant fault frequency components under speed-varying conditions are clamped in a single frequency band); - Low memory requirement due to low sampling frequency; - Reduction in the latency of time processing (no requirement of repeated sampling operation).

Bottle microresonators for applications in quantum optics and all-optical signal processing

This thesis reports on the experimental realization, characterization and application of a novel microresonator design. The so-called “bottle microresonator” sustains whispering-gallery modes in which light fields are confined near the surface of the micron-sized silica structure by continuous total internal reflection. While whispering-gallery mode resonators in general exhibit outstanding properties in terms of both temporal and spatial confinement of light fields, their monolithic design makes tuning of their resonance frequency difficult. This impedes their use, e.g., in cavity quantum electrodynamics (CQED) experiments, which investigate the interaction of single quantum mechanical emitters of predetermined resonance frequency with a cavity mode. In contrast, the highly prolate shape of the bottle microresonators gives rise to a customizable mode structure, enabling full tunability. The thesis is organized as follows: In chapter I, I give a brief overview of different types of optical microresonators. Important quantities, such as the quality factor Q and the mode volume V, which characterize the temporal and spatial confinement of the light field are introduced. In chapter II, a wave equation calculation of the modes of a bottle microresonator is presented. The intensity distribution of different bottle modes is derived and their mode volume is calculated. A brief description of light propagation in ultra-thin optical fibers, which are used to couple light into and out of bottle modes, is given as well. The chapter concludes with a presentation of the fabrication techniques of both structures. Chapter III presents experimental results on highly efficient, nearly lossless coupling of light into bottle modes as well as their spatial and spectral characterization. Ultra-high intrinsic quality factors exceeding 360 million as well as full tunability are demonstrated. In chapter IV, the bottle microresonator in add-drop configuration, i.e., with two ultra-thin fibers coupled to one bottle mode, is discussed. The highly efficient, nearly lossless coupling characteristics of each fiber combined with the resonator's high intrinsic quality factor, enable resonant power transfers between both fibers with efficiencies exceeding 90%. Moreover, the favorable ratio of absorption and the nonlinear refractive index of silica yields optical Kerr bistability at record low powers on the order of 50 µW. Combined with the add-drop configuration, this allows one to route optical signals between the outputs of both ultra-thin fibers, simply by varying the input power, thereby enabling applications in all-optical signal processing. Finally, in chapter V, I discuss the potential of the bottle microresonator for CQED experiments with single atoms. Its Q/V-ratio, which determines the ratio of the atom-cavity coupling rate to the dissipative rates of the subsystems, aligns with the values obtained for state-of-the-art CQED microresonators. In combination with its full tunability and the possibility of highly efficient light transfer to and from the bottle mode, this makes the bottle microresonator a unique tool for quantum optics applications.

Antigen-dependent induction of a CD40 signal in peripheral B cells

Monoclonal antibodies have emerged as one of the most promising therapeutics in oncology over the last decades. The generation of fully human tumorantigen-specific antibodies suitable for anti-tumor therapy is laborious and difficult to achieve. Autoreactive B cells expressing those antibodies are detectable in cancer patients and represent a suitable source for human antibodies. However, the isolation and cultivation of this cell type is challenging. A novel method was established to identify antigen-specific B cells. The method is based on the conversion of the antigen independent CD40 signal into an antigen-specific one. For that, the artificial fusion proteins ABCos1 and ABCos2 (Antigen-specific B cell co-stimulator) were generated, which consist of an extracellular association-domain derived from the constant region of the human immunoglobulin (Ig) G1, a transmembrane fragment and an intracellular signal transducer domain derived of the cytoplasmic domain of the human CD40 receptor. By the association with endogenous Ig molecules the heterodimeric complex allows the antigen-specific stimulation of both the BCR and CD40. In this work the ability of the ABCos constructs to associate with endogenous IgG molecules was shown. Moreover, crosslinking of ABCos stimulates the activation of NF-κB in HEK293-lucNifty and induces proliferation in B cells. The stimulation of ABCos in transfected B cells results in an activation pattern different from that induced by the conventional CD40 signal. ABCos activated B cells show a mainly IgG isotype specific activation of memory B cells and are characterized by high proliferation and the differentiation into plasma cells. To validate the approach a model system was conducted: B cells were transfected with IVT-RNA encoding for anti-Plac1 B cell receptor (antigen-specific BCR), ABCos or both. The stimulation with the BCR specific Plac1 peptide induces proliferation only in the cotransfected B cell population. Moreover, we tested the method in human IgG+ memory B cells from CMV infected blood donors, in which the stimulation of ABCos transfected B cells with a CMV peptide induces antigen-specific expansion. These findings show that challenging ABCos transfected B cells with a specific antigen results in the activation and expansion of antigen-specific B cells and not only allows the identification but also cultivation of these B cells. The described method will help to identify antigen-specific B cells and can be used to characterize (tumor) autoantigen-specific B cells and allows the generation of fully human antibodies that can be used as diagnostic tool as well as in cancer therapy.

Frequency-dependent response of snow in uniaxial compression and comparison with numerical simulation

La determinazione del modulo di Young è fondamentale nello studio della propagazione di fratture prima del rilascio di una valanga e per lo sviluppo di affidabili modelli di stabilità della neve. Il confronto tra simulazioni numeriche del modulo di Young e i valori sperimentali mostra che questi ultimi sono tre ordini di grandezza inferiori a quelli simulati (Reuter et al. 2013)⁠. Lo scopo di questo lavoro è stimare il modulo di elasticità studiando la dipendenza dalla frequenza della risposta di diversi tipi di neve a bassa densità, 140-280 kg m-3. Ciò è stato fatto applicando una compressione dinamica uniassiale a -15°C nel range 1-250 Hz utilizzando il Young's modulus device (YMD), prototipo di cycling loading device progettato all'Istituto per lo studio della neve e delle valanghe (SLF). Una risposta viscoelastica della neve è stata identificata a tutte le frequenze considerate, la teoria della viscoelasticità è stata applicata assumendo valida l'ipotesi di risposta lineare della neve. Il valore dello storage modulus, E', a 100 Hz è stato identificato come ragionevolmente rappresentativo del modulo di Young di ciascun campione neve. Il comportamento viscoso è stato valutato considerando la loss tangent e la viscosità ricavata dai modelli di Voigt e Maxwell. Il passaggio da un comportamento più viscoso ad uno più elastico è stato trovato a 40 Hz (~1.1•10-2 s-1). Il maggior contributo alla dissipazione è nel range 1-10 Hz. Infine, le simulazioni numeriche del modulo di Young sono state ottenute nello stesso modo di Reuter et al.. La differenza tra le simulazioni ed i valori sperimentali di E' sono, al massimo, di un fattore 5; invece, in Reuter et al.⁠, era di 3 ordini di grandezza. Pertanto, i nostri valori sperimentali e numerici corrispondono meglio, indicando che il metodo qui utilizzato ha portato ad un miglioramento significativo.

Finite element modeling of flow/compression-induced deformation of alginate scaffolds for bone tissue engineering

Trauma or degenerative diseases such as osteonecrosis may determine bone loss whose recover is promised by a "tissue engineering“ approach. This strategy involves the use of stem cells, grown onboard of adequate biocompatible/bioreabsorbable hosting templates (usually defined as scaffolds) and cultured in specific dynamic environments afforded by differentiation-inducing actuators (usually defined as bioreactors) to produce implantable tissue constructs. The purpose of this thesis is to evaluate, by finite element modeling of flow/compression-induced deformation, alginate scaffolds intended for bone tissue engineering. This work was conducted at the Biomechanics Laboratory of the Institute of Biomedical and Neural Engineering of the Reykjavik University of Iceland. In this respect, Comsol Multiphysics 5.1 simulations were carried out to approximate the loads over alginate 3D matrices under perfusion, compression and perfusion+compression, when varyingalginate pore size and flow/compression regimen. The results of the simulations show that the shear forces in the matrix of the scaffold increase coherently with the increase in flow and load, and decrease with the increase of the pore size. Flow and load rates suggested for proper osteogenic cell differentiation are reported.

Ease of Application of Medical Compression-stocking Systems for the Treatment of Venous Ulcers

OBJECTIVE: To evaluate the ease of application of two-piece, graduated, compression systems for the treatment of venous ulcers. METHODS: Four kits used to provide limb compression in the management of venous ulcers were evaluated. These have been proven to be non-inferior to various types of bandages in clinical trials. The interface pressure exerted above the ankle by the under-stocking and the complete compression system and the force required to pull the over-stocking off were assessed in vitro. Ease of application of the four kits was evaluated in four sessions by five nurses who put stockings on their own legs in a blinded manner. They expressed their assessment of the stockings using a series of visual analogue scales (VASs). RESULTS: The Sigvaris Ulcer X((R)) kit provided a mean interface pressure of 46 mmHg and required a force in the range of 60-90 N to remove it. The Mediven((R)) ulcer kit exerted the same pressure but required force in the range of 150-190 N to remove it. Two kits (SurePress((R)) Comfort and VenoTrain((R)) Ulcertec) exerted a mean pressure of only 25 mmHg and needed a force in the range of 100-160 N to remove them. Nurses judged the Ulcer X and SurePress kits easiest to apply. Application of the VenoTrain kit was found slightly more difficult. The Mediven kit was judged to be difficult to use. CONCLUSIONS: Comparison of ease of application of compression-stocking kits in normal legs revealed marked differences between them. Only one system exerted a high pressure and was easy to apply. Direct comparison of these compression kits in leg-ulcer patients is required to assess whether our laboratory findings correlate with patient compliance and ulcer healing.

Comparison of low-strength compression stockings with bandages for the treatment of recalcitrant venous ulcers

OBJECTIVE: To compare the proportion and rate of healing, pain, and quality of life of low-strength medical compression stockings (MCS) with traditional bandages applied for the treatment of recalcitrant venous leg ulcers. METHODS: A single-center, randomized, open-label study was performed with consecutive patients. Sigvaris prototype MCS providing 15 mm Hg-25 mm Hg at the ankle were compared with multi-layer short-stretch bandages. In both groups, pads were placed above incompetent perforating veins in the ulcer area. The initial static pressure between the dressing-covered ulcer and the pad was 29 mm Hg and 49 mm Hg with MCS and bandages, respectively. Dynamic pressure measurements showed no difference. Compression was maintained day and night and changed every week. The primary endpoint was healing within 90 days. Secondary endpoints were healing within 180 days, time to healing, pain (weekly Likert scales), and monthly quality of life (ChronIc Venous Insufficiency Quality of Life [CIVIQ] questionnaire). RESULTS: Of 74 patients screened, 60 fulfilled the selection criteria and 55 completed the study; 28 in the MCS and 27 in the bandage group. Ulcers were recurrent (48%), long lasting (mean, 27 months), and large (mean, 13 cm2). All but one patient had deep venous reflux and/or incompetent perforating veins in addition to trunk varices. Characteristics of patients and ulcers were evenly distributed (exception: more edema in the MCS group; P = .019). Healing within 90 days was observed in 36% with MCS and in 48% with bandages (P = .350). Healing within 180 days was documented in 50% with MCS and in 67% with bandages (P = .210). Time to healing was identical. Pain scored 44 and 46 initially (on a scale in which 100 referred to maximum and 0 to no pain) and decreased within the first week to 20 and 28 in the MCS and bandage groups, respectively (P < .001 vs .010). Quality of life showed no difference between the treatment groups. In both groups, pain at 90 days had decreased by half, independent of completion of healing. Physical, social, and psychic impairment improved significantly in patients with healed ulcers only. CONCLUSION: Our study illustrates the difficulty of bringing large and long-standing venous ulcers to heal. The effect of compression with MCS was not different from that of compression with bandages. Both treatments alleviated pain promptly. Quality of life was improved only in patients whose ulcers had healed.