Bioimaging of animal embryogenesis: mathematical methods and computational algorithms

Some fundamental biological processes such as embryonic development have been preserved during evolution and are common to species belonging to different phylogenetic positions, but are nowadays largely unknown. The understanding of cell morphodynamics leading to the formation of organized spatial distribution of cells such as tissues and organs can be achieved through the reconstruction of cells shape and position during the development of a live animal embryo. We design in this work a chain of image processing methods to automatically segment and track cells nuclei and membranes during the development of a zebrafish embryo, which has been largely validates as model organism to understand vertebrate development, gene function and healingrepair mechanisms in vertebrates. The embryo is previously labeled through the ubiquitous expression of fluorescent proteins addressed to cells nuclei and membranes, and temporal sequences of volumetric images are acquired with laser scanning microscopy. Cells position is detected by processing nuclei images either through the generalized form of the Hough transform or identifying nuclei position with local maxima after a smoothing preprocessing step. Membranes and nuclei shapes are reconstructed by using PDEs based variational techniques such as the Subjective Surfaces and the Chan Vese method. Cells tracking is performed by combining informations previously detected on cells shape and position with biological regularization constraints. Our results are manually validated and reconstruct the formation of zebrafish brain at 7-8 somite stage with all the cells tracked starting from late sphere stage with less than 2% error for at least 6 hours. Our reconstruction opens the way to a systematic investigation of cellular behaviors, of clonal origin and clonal complexity of brain organs, as well as the contribution of cell proliferation modes and cell movements to the formation of local patterns and morphogenetic fields.

Vibration as a method for muscular stimulation

Human reactions to vibration have been extensively investigated in the past. Vibration, as well as whole-body vibration (WBV), has been commonly considered as an occupational hazard for its detrimental effects on human condition and comfort. Although long term exposure to vibrations may produce undesirable side-effects, a great part of the literature is dedicated to the positive effects of WBV when used as method for muscular stimulation and as an exercise intervention. Whole body vibration training (WBVT) aims to mechanically activate muscles by eliciting neuromuscular activity (muscle reflexes) via the use of vibrations delivered to the whole body. The most mentioned mechanism to explain the neuromuscular outcomes of vibration is the elicited neuromuscular activation. Local tendon vibrations induce activity of the muscle spindle Ia fibers, mediated by monosynaptic and polysynaptic pathways: a reflex muscle contraction known as the Tonic Vibration Reflex (TVR) arises in response to such vibratory stimulus. In WBVT mechanical vibrations, in a range from 10 to 80 Hz and peak to peak displacements from 1 to 10 mm, are usually transmitted to the patient body by the use of oscillating platforms. Vibrations are then transferred from the platform to a specific muscle group through the subject body. To customize WBV treatments, surface electromyography (SEMG) signals are often used to reveal the best stimulation frequency for each subject. Use of SEMG concise parameters, such as root mean square values of the recordings, is also a common practice; frequently a preliminary session can take place in order to discover the more appropriate stimulation frequency. Soft tissues act as wobbling masses vibrating in a damped manner in response to mechanical excitation; Muscle Tuning hypothesis suggest that neuromuscular system works to damp the soft tissue oscillation that occurs in response to vibrations; muscles alters their activity to dampen the vibrations, preventing any resonance phenomenon. Muscle response to vibration is however a complex phenomenon as it depends on different parameters, like muscle-tension, muscle or segment-stiffness, amplitude and frequency of the mechanical vibration. Additionally, while in the TVR study the applied vibratory stimulus and the muscle conditions are completely characterised (a known vibration source is applied directly to a stretched/shortened muscle or tendon), in WBV study only the stimulus applied to a distal part of the body is known. Moreover, mechanical response changes in relation to the posture. The transmissibility of vibratory stimulus along the body segment strongly depends on the position held by the subject. The aim of this work was the investigation on the effects that the use of vibrations, in particular the effects of whole body vibrations, may have on muscular activity. A new approach to discover the more appropriate stimulus frequency, by the use of accelerometers, was also explored. Different subjects, not affected by any known neurological or musculoskeletal disorders, were voluntarily involved in the study and gave their informed, written consent to participate. The device used to deliver vibration to the subjects was a vibrating platform. Vibrations impressed by the platform were exclusively vertical; platform displacement was sinusoidal with an intensity (peak-to-peak displacement) set to 1.2 mm and with a frequency ranging from 10 to 80 Hz. All the subjects familiarized with the device and the proper positioning. Two different posture were explored in this study: position 1 - hack squat; position 2 - subject standing on toes with heels raised. SEMG signals from the Rectus Femoris (RF), Vastus Lateralis (VL) and Vastus medialis (VM) were recorded. SEMG signals were amplified using a multi-channel, isolated biomedical signal amplifier The gain was set to 1000 V/V and a band pass filter (-3dB frequency 10 - 500 Hz) was applied; no notch filters were used to suppress line interference. Tiny and lightweight (less than 10 g) three-axial MEMS accelerometers (Freescale semiconductors) were used to measure accelerations of onto patient’s skin, at EMG electrodes level. Accelerations signals provided information related to individuals’ RF, Biceps Femoris (BF) and Gastrocnemius Lateralis (GL) muscle belly oscillation; they were pre-processed in order to exclude influence of gravity. As demonstrated by our results, vibrations generate peculiar, not negligible motion artifact on skin electrodes. Artifact amplitude is generally unpredictable; it appeared in all the quadriceps muscles analysed, but in different amounts. Artifact harmonics extend throughout the EMG spectrum, making classic high-pass filters ineffective; however, their contribution was easy to filter out from the raw EMG signal with a series of sharp notch filters centred at the vibration frequency and its superior harmonics (1.5 Hz wide). However, use of these simple filters prevents the revelation of EMG power potential variation in the mentioned filtered bands. Moreover our experience suggests that the possibility of reducing motion artefact, by using particular electrodes and by accurately preparing the subject’s skin, is not easily viable; even though some small improvements were obtained, it was not possible to substantially decrease the artifact. Anyway, getting rid of those artifacts lead to some true EMG signal loss. Nevertheless, our preliminary results suggest that the use of notch filters at vibration frequency and its harmonics is suitable for motion artifacts filtering. In RF SEMG recordings during vibratory stimulation only a little EMG power increment should be contained in the mentioned filtered bands due to synchronous electromyographic activity of the muscle. Moreover, it is better to remove the artifact that, in our experience, was found to be more than 40% of the total signal power. In summary, many variables have to be taken into account: in addition to amplitude, frequency and duration of vibration treatment, other fundamental variables were found to be subject anatomy, individual physiological condition and subject’s positioning on the platform. Studies on WBV treatments that include surface EMG analysis to asses muscular activity during vibratory stimulation should take into account the presence of motion artifacts. Appropriate filtering of artifacts, to reveal the actual effect on muscle contraction elicited by vibration stimulus, is mandatory. However as a result of our preliminary study, a simple multi-band notch filtering may help to reduce randomness of the results. Muscle tuning hypothesis seemed to be confirmed. Our results suggested that the effects of WBV are linked to the actual muscle motion (displacement). The greater was the muscle belly displacement the higher was found the muscle activity. The maximum muscle activity has been found in correspondence with the local mechanical resonance, suggesting a more effective stimulation at the specific system resonance frequency. Holding the hypothesis that muscle activation is proportional to muscle displacement, treatment optimization could be obtained by simply monitoring local acceleration (resonance). However, our study revealed some short term effects of vibratory stimulus; prolonged studies should be assembled in order to consider the long term effectiveness of these results. Since local stimulus depends on the kinematic chain involved, WBV muscle stimulation has to take into account the transmissibility of the stimulus along the body segment in order to ensure that vibratory stimulation effectively reaches the target muscle. Combination of local resonance and muscle response should also be further investigated to prevent hazards to individuals undergoing WBV treatments.

Mathematical models of cognitive processes

The research activity carried out during the PhD course was focused on the development of mathematical models of some cognitive processes and their validation by means of data present in literature, with a double aim: i) to achieve a better interpretation and explanation of the great amount of data obtained on these processes from different methodologies (electrophysiological recordings on animals, neuropsychological, psychophysical and neuroimaging studies in humans), ii) to exploit model predictions and results to guide future research and experiments. In particular, the research activity has been focused on two different projects: 1) the first one concerns the development of neural oscillators networks, in order to investigate the mechanisms of synchronization of the neural oscillatory activity during cognitive processes, such as object recognition, memory, language, attention; 2) the second one concerns the mathematical modelling of multisensory integration processes (e.g. visual-acoustic), which occur in several cortical and subcortical regions (in particular in a subcortical structure named Superior Colliculus (SC)), and which are fundamental for orienting motor and attentive responses to external world stimuli. This activity has been realized in collaboration with the Center for Studies and Researches in Cognitive Neuroscience of the University of Bologna (in Cesena) and the Department of Neurobiology and Anatomy of the Wake Forest University School of Medicine (NC, USA). PART 1. Objects representation in a number of cognitive functions, like perception and recognition, foresees distribute processes in different cortical areas. One of the main neurophysiological question concerns how the correlation between these disparate areas is realized, in order to succeed in grouping together the characteristics of the same object (binding problem) and in maintaining segregated the properties belonging to different objects simultaneously present (segmentation problem). Different theories have been proposed to address these questions (Barlow, 1972). One of the most influential theory is the so called “assembly coding”, postulated by Singer (2003), according to which 1) an object is well described by a few fundamental properties, processing in different and distributed cortical areas; 2) the recognition of the object would be realized by means of the simultaneously activation of the cortical areas representing its different features; 3) groups of properties belonging to different objects would be kept separated in the time domain. In Chapter 1.1 and in Chapter 1.2 we present two neural network models for object recognition, based on the “assembly coding” hypothesis. These models are networks of Wilson-Cowan oscillators which exploit: i) two high-level “Gestalt Rules” (the similarity and previous knowledge rules), to realize the functional link between elements of different cortical areas representing properties of the same object (binding problem); 2) the synchronization of the neural oscillatory activity in the γ-band (30-100Hz), to segregate in time the representations of different objects simultaneously present (segmentation problem). These models are able to recognize and reconstruct multiple simultaneous external objects, even in difficult case (some wrong or lacking features, shared features, superimposed noise). In Chapter 1.3 the previous models are extended to realize a semantic memory, in which sensory-motor representations of objects are linked with words. To this aim, the network, previously developed, devoted to the representation of objects as a collection of sensory-motor features, is reciprocally linked with a second network devoted to the representation of words (lexical network) Synapses linking the two networks are trained via a time-dependent Hebbian rule, during a training period in which individual objects are presented together with the corresponding words. Simulation results demonstrate that, during the retrieval phase, the network can deal with the simultaneous presence of objects (from sensory-motor inputs) and words (from linguistic inputs), can correctly associate objects with words and segment objects even in the presence of incomplete information. Moreover, the network can realize some semantic links among words representing objects with some shared features. These results support the idea that semantic memory can be described as an integrated process, whose content is retrieved by the co-activation of different multimodal regions. In perspective, extended versions of this model may be used to test conceptual theories, and to provide a quantitative assessment of existing data (for instance concerning patients with neural deficits). PART 2. The ability of the brain to integrate information from different sensory channels is fundamental to perception of the external world (Stein et al, 1993). It is well documented that a number of extraprimary areas have neurons capable of such a task; one of the best known of these is the superior colliculus (SC). This midbrain structure receives auditory, visual and somatosensory inputs from different subcortical and cortical areas, and is involved in the control of orientation to external events (Wallace et al, 1993). SC neurons respond to each of these sensory inputs separately, but is also capable of integrating them (Stein et al, 1993) so that the response to the combined multisensory stimuli is greater than that to the individual component stimuli (enhancement). This enhancement is proportionately greater if the modality-specific paired stimuli are weaker (the principle of inverse effectiveness). Several studies have shown that the capability of SC neurons to engage in multisensory integration requires inputs from cortex; primarily the anterior ectosylvian sulcus (AES), but also the rostral lateral suprasylvian sulcus (rLS). If these cortical inputs are deactivated the response of SC neurons to cross-modal stimulation is no different from that evoked by the most effective of its individual component stimuli (Jiang et al 2001). This phenomenon can be better understood through mathematical models. The use of mathematical models and neural networks can place the mass of data that has been accumulated about this phenomenon and its underlying circuitry into a coherent theoretical structure. In Chapter 2.1 a simple neural network model of this structure is presented; this model is able to reproduce a large number of SC behaviours like multisensory enhancement, multisensory and unisensory depression, inverse effectiveness. In Chapter 2.2 this model was improved by incorporating more neurophysiological knowledge about the neural circuitry underlying SC multisensory integration, in order to suggest possible physiological mechanisms through which it is effected. This endeavour was realized in collaboration with Professor B.E. Stein and Doctor B. Rowland during the 6 months-period spent at the Department of Neurobiology and Anatomy of the Wake Forest University School of Medicine (NC, USA), within the Marco Polo Project. The model includes four distinct unisensory areas that are devoted to a topological representation of external stimuli. Two of them represent subregions of the AES (i.e., FAES, an auditory area, and AEV, a visual area) and send descending inputs to the ipsilateral SC; the other two represent subcortical areas (one auditory and one visual) projecting ascending inputs to the same SC. Different competitive mechanisms, realized by means of population of interneurons, are used in the model to reproduce the different behaviour of SC neurons in conditions of cortical activation and deactivation. The model, with a single set of parameters, is able to mimic the behaviour of SC multisensory neurons in response to very different stimulus conditions (multisensory enhancement, inverse effectiveness, within- and cross-modal suppression of spatially disparate stimuli), with cortex functional and cortex deactivated, and with a particular type of membrane receptors (NMDA receptors) active or inhibited. All these results agree with the data reported in Jiang et al. (2001) and in Binns and Salt (1996). The model suggests that non-linearities in neural responses and synaptic (excitatory and inhibitory) connections can explain the fundamental aspects of multisensory integration, and provides a biologically plausible hypothesis about the underlying circuitry.

Study of the thermal effects induced by magnetic resonance on endocardial leads: numerical models and experimental validation

Magnetic resonance imaging (MRI) is today precluded to patients bearing active implantable medical devices AIMDs). The great advantages related to this diagnostic modality, together with the increasing number of people benefiting from implantable devices, in particular pacemakers(PM)and carioverter/defibrillators (ICD), is prompting the scientific community the study the possibility to extend MRI also to implanted patients. The MRI induced specific absorption rate (SAR) and the consequent heating of biological tissues is one of the major concerns that makes patients bearing metallic structures contraindicated for MRI scans. To date, both in-vivo and in-vitro studies have demonstrated the potentially dangerous temperature increase caused by the radiofrequency (RF) field generated during MRI procedures in the tissues surrounding thin metallic implants. On the other side, the technical evolution of MRI scanners and of AIMDs together with published data on the lack of adverse events have reopened the interest in this field and suggest that, under given conditions, MRI can be safely performed also in implanted patients. With a better understanding of the hazards of performing MRI scans on implanted patients as well as the development of MRI safe devices, we may soon enter an era where the ability of this imaging modality may be more widely used to assist in the appropriate diagnosis of patients with devices. In this study both experimental measures and numerical analysis were performed. Aim of the study is to systematically investigate the effects of the MRI RF filed on implantable devices and to identify the elements that play a major role in the induced heating. Furthermore, we aimed at developing a realistic numerical model able to simulate the interactions between an RF coil for MRI and biological tissues implanted with a PM, and to predict the induced SAR as a function of the particular path of the PM lead. The methods developed and validated during the PhD program led to the design of an experimental framework for the accurate measure of PM lead heating induced by MRI systems. In addition, numerical models based on Finite-Differences Time-Domain (FDTD) simulations were validated to obtain a general tool for investigating the large number of parameters and factors involved in this complex phenomenon. The results obtained demonstrated that the MRI induced heating on metallic implants is a real risk that represents a contraindication in extending MRI scans also to patient bearing a PM, an ICD, or other thin metallic objects. On the other side, both experimental data and numerical results show that, under particular conditions, MRI procedures might be consider reasonably safe also for an implanted patient. The complexity and the large number of variables involved, make difficult to define a unique set of such conditions: when the benefits of a MRI investigation cannot be obtained using other imaging techniques, the possibility to perform the scan should not be immediately excluded, but some considerations are always needed.

L'introduzione dell'e-commerce nel settore fieristico: piano di email marketing e studio di usabilità

L’obiettivo di questa relazione è presentare i risultati raggiunti dalle attività di marketing svolte al fine di promuovere e valutare la piattaforma e-commerce di BF Servizi, azienda che opera come intermediario nella fornitura di servizi all’interno del contesto fieristico di Bologna Fiere. A fine del 2009 BF Servizi ha commissionato a Gecod lo sviluppo di un sistema informativo per le gestione integrata di tutti i processi rilevanti per il business aziendale. A ottobre 2010 viene completato e presentato uno dei moduli previsti dall’intero progetto: la piattaforma e-commerce. Questa si differenzia dagli altri sistemi di commercio online in quanto è stata progettata specificatamente allo scopo di permettere la prenotazione di servizi da parte delle aziende che partecipano alle manifestazioni svolte presso i padiglioni di Bologna Fiere. Le sue particolarità sono quindi sia l’utenza, che configura la creazione di relazioni B2B, sia la tipologia di articoli offerti, non prodotti ma servizi, sia l’ambiente, le fiere svolte a Bologna. A seguito di questo rinnovamento BF Servizi ha ritenuto opportuno adeguare al nuovo contesto anche le proprie tecniche di marketing introducendo, come strumento da usare per la loro attuazione, il web. All’interno dell’organico dell’azienda però non era presente una figura che avesse le competenze per poter integrare, ai normali canali usati per le iniziative di marketing, le opportunità offerte da Internet e che quindi potesse utilizzarle per raggiungere gli obiettivi prefissati per tali attività e cioè la promozione della piattaforma e-commerce e la misurazione del suo gradimento da parte dell’utenza. Le mie conoscenze maturate in ambito universitario hanno apportato quel contributo necessario alla loro realizzazione. La prima decisione è stata la scelta di quale tra le tecniche di web marketing fosse la migliore per le promozione della piattaforma e-commerce di BF Servizi. La disponibilità di un voluminoso numero di indirizzi email dei loro clienti e prospect ed una generale bassa alfabetizzazione informatica degli stessi ha portato a individuare quale soluzione più adeguata la tecnica dell’email marketing. La comunicazione ai clienti per mezzo email era già prevista ma senza che vi fosse ne una pianificazione ne un'analisi dei risultati. Era quindi necessario formalizzare un vero e proprio piano di marketing e successivamente migliorarne le performance. L’assenza di strumenti adeguati alla sua realizzazione ha richiesto che prima venisse svolto un benchmark del settore basato sul confronto di funzionalità, costi e facilità d'uso. Il servizio ESP di Mailchimp è risultato il vincitore in quanto assicurava una maggiore tutela della privacy, una migliore gestione dello spam e strumenti più efficaci per la creazione e l'analisi delle campagne email. Per la valutazione del gradimento della piattaforma e-commerce da parte degli utenti è stato deciso di progettare un’apposita indagine sottoforma di Usability Test e Online Survey. Per il primo è stato necessario l’utilizzo di un apposito servizio per la condivisione dello schermo in quanto in era impossibile far ritrovare il test e l’osservatore nel medesimo luogo. Per l’Online Survey, grazie ad un piccolo Benchmark, è stato deciso di utilizzare SurveyGizmo. All’interno dell’organico dell’azienda non è presente una figura di importanza strategia quale il Web Marketing Manager che abbia la responsabilità di coordinare tutte le attività legate alla vendita o al lancio di un prodotto o di un servizio. L’attività di marketing viene svolta da una figura interna all’azienda (il Responsabile Marketing) che si avvale della collaborazione sia di Gecod, per quanto riguarda l'area informatica, sia di BitBang per quanto riguarda alcune attività di web marketing. La realizzazione delle due attività ha fortemente risentito di questa frammentazione dei compiti, è stato infatti opportuno adeguare le pianificazione alle esigenze di Gecod. Nonostante questo i dati conseguiti dall'attività di email marketing sono stati utili a capire l'efficacia e l'efficienza di questa tecnica nel contesto di BF Servizi. Anche le informazioni raccolte durante l'indagine sono state sufficienti per poter dare un valore all'usabilità della piattaforma e-commerce. La pianificazione iniziale di un piano di email marketing richiede che vengano svolte varie verifiche per valutare la qualità ed inoltre richiede periodo medio-lungo per poter essere reso ottimale. Era quindi già chiaro sin dall'inizio come uno degli obiettivi fosse quello di progettare una buona base su cui apportare successive modifiche. Le prime campagne realizzate sarebbero state utili a ricavare le informazioni necessarie per correggere il piano di marketing. La tipologia di relazione che si instaura tra BF Servizi e i suoi clienti è particolare, la gran parte di essi infatti non vi mantiene un rapporto continuativo durante l’anno ma lo esaurisce in un unico momento poco prima del manifestarsi dell’evento fieristico. L'attenzione dei clienti verso le comunicazioni di BF Servizi è elevata solo in questo periodo e la pianificazione delle campagne ha dovuto tener conto di questo aspetto. Sono state realizzate le campagne solo per gli espositori che partecipavano agli eventi fieristici di Marca 2011, Artefiera 2011 e Fishing Show 2011, cioè le manifestazioni che si sono svolte durante il mio periodo di lavoro in azienda. Le performance delle campagne sono state buone e i dati raccolti hanno permesso di identificare quali siano gli aspetti da migliorare per poter renderle ancora migliori.

Crittoanalisi Logica di DES

La crittografia ha sempre rivestito un ruolo primario nella storia del genere umano, dagli albori ai giorni nostri, e il periodo in cui viviamo non fa certo eccezione. Al giorno d'oggi, molti dei gesti che vengono compiuti anche solo come abitudine (operazioni bancarie, apertura automatica dell'auto, accedere a Facebook, ecc.), celano al loro interno la costante presenza di sofisticati sistemi crittografici. Proprio a causa di questo fatto, è importante che gli algoritmi utilizzati siano in qualche modo certificati come ragionevolmente sicuri e che la ricerca in questo campo proceda costantemente, sia dal punto di vista dei possibili nuovi exploit per forzare gli algoritmi usati, sia introducendo nuovi e sempre più complessi sistemi di sicurezza. In questa tesi viene proposto una possibile implementazione di un particolare tipo di attacco crittoanalitico, introdotto nel 2000 da due ricercatori dell'Università "La Sapienza" di Roma, e conosciuto come "Crittoanalisi Logica". L'algoritmo su cui è incentrato il lavoro è il Data Encryption Standard (DES), ostico standard crittografico caduto in disuso nel 1999 a causa delle dimensioni ridotte della chiave, seppur tuttora sia algebricamente inviolato. Il testo è strutturato nel seguente modo: il primo capitolo è dedicato ad una breve descrizione di DES e della sua storia, introducendo i concetti fondamentali con cui si avrà a che fare per l'intera dissertazione Nel secondo capitolo viene introdotta la Crittoanalisi Logica e viene fornita una definizione della stessa, accennando ai concetti matematici necessari alla comprensione dei capitoli seguenti. Nel capitolo 3 viene presentato il primo dei due software sviluppati per rendere possibile l'attuazione di questo attacco crittoanalitico, una libreria per la rappresentazione e la manipolazione di formule logiche scritta in Java. Il quarto ed ultimo capitolo descrive il programma che, utilizzando la libreria descritta nel capitolo 3, elabora in maniera automatica un insieme di proposizioni logiche semanticamente equivalenti a DES, la cui verifica di soddisfacibilità, effettuata tramite appositi tools (SAT solvers) equivale ad effettuare un attacco di tipo known-plaintext su tale algoritmo.