Analisi della connettività e della dinamica di crescita di cellule di Purkinje tramite imaging in vitro ed algoritmi computazionali

L’obiettivo di questa tesi è lo studio della connettività e della morfologia neuronale in vitro al fine di comprendere come la patologia dell’autismo alteri lo sviluppo cerebrale e cerebellare. L’autismo è un disturbo neuronale che danneggia la capacità di una persona di pensare, di provare emozioni, di comunicare e di relazionarsi in maniera appropriata al mondo esterno. Le ricerche su questa patologia sono iniziate negli anni ’60 quando gli studi neurologici hanno dimostrato che il cervello di persone affette da autismo presenta delle anormalità. I risultati di questi studi si basano su evidenze macroscopiche. Quello che manca nello stato dell’arte è uno studio dell’evoluzione delle anormalità neuro-strutturali su scala microscopica. Questo lavoro di tesi si colloca nell’ambito di un progetto che ha lo scopo di affrontare il problema del disordine cerebrale che causa l’autismo a livello microscopico cioè a livello di reti e di singoli neuroni. Gli studi realizzati vengono effettuati in dinamico cioè seguendo lo sviluppo dei neuroni coltivati in vitro nel tempo. Il lavoro si basa sulla realizzazione di foto di neuroni primari di topo in vitro a diversi intervalli di tempo e sulla successiva analisi di tali immagini attraverso l’utilizzo di appositi algoritmi implementati in Matlab. Lo scopo è quello di estrarre, dallo studio dei dati ottenuti con l’applicazione del Software, delle caratteristiche metriche e topologiche generali e di vedere come esse variano nel tempo. Il presente lavoro si è articolato in due fasi. Inizialmente ci si è occupati di proseguire un precedente lavoro di tesi in cui è stato messo a punto un modello in vitro per il mappaggio dei circuiti neurali durante le prime fasi dello sviluppo del cervello. La seconda fase del lavoro, è quella più consistente ed è la parte centrale della tesi. Basandosi sullo stesso principio di estrazione di variabili nel tempo di cellule primarie, sono state seguite due colture di cellule di Purkinje. La scelta di questo tipo di cellule è dovuta al fatto che, come visto sopra, esse risultano essere danneggiate nei soggetti autistici e quindi molto diverse dalla norma. I dati ottenuti sono stati poi analizzati per cercare di estrarre delle informazioni di carattere generale comuni allo sviluppo di tutte le cellule. Per il momento sono stati presi in considerazione solamente neuroni normali. Solo dopo sarà effettivamente possibile, esaminando lo sviluppo e la morfologia di neuroni ottenuti da dei modelli animali dell’autismo, sulla base di un confronto con i risultati che otterremo in questa tesi, rilevare quali possano essere le alterazioni morfologiche e di connettività alla base dell’autismo e comprendere come queste influenzino l’iniziale sviluppo anormale del cervello

Olfactory Testing in Frontotemporal Dementia: A Literature Review

Frontotemporal dementia (FTD) is a heterogeneous disorder featuring language impairment, personality changes, and executive defects, often due to the frontotemporal lobar degeneration (FTLD). Both FTD and FTLD are often associated with olfactory impairment, early biomarker for neurodegeneration, which can be evaluated with different techniques, among which low-cost olfactory tests are widely used. Therefore, we conducted a review of the literature focusing on papers published between January 1, 2007, and June 12, 2017, investigating the usefulness of olfactory testing in FTD/FTLD. A general decrease in the olfactory identification ability was seen in most of the articles and, taken together with a preserved odor discrimination, reveals a higher order impairment, possibly linked to cognitive decrease or language impairments, and not to a specific deficit of the olfactory system. This evidence could represent a useful add-on to the current literature, increasing the diagnostic value of olfactory assessment, particularly in cases where differential diagnosis is difficult

Novel methodologies and technologies for the multiscale and multimodal study of Autism Spectrum Disorders (ASDs)

The aim of this PhD thesis was the development of novel bioengineering tools and methodologies that provide a support in the study of ASDs. ASDs are very heterogeneous disturbs and their abnormalities are present both at local and global level. For this reason a multimodal and multiscale approach was followed. The analysis of microstructure was executed on single Purkinje neurons in culture and on organotypic slices extracted from cerebella of GFP wild-type mice and animal models of ASDs. A methodology for the non-invasive imaging of neurons during their growth was set up and a software called NEMO (NEuron MOrphological analysis tool) for the automatic analysis of morphology and connectivity was developed. Microstructure properties can be inferred also in vivo through the quite recent technique of Diffusion Tensor Imaging (DTI). DTI studies in ASDs are based on the hypothesis that the disorder involves aberrant brain connectivity and disruption of white matter tracts between regions implicated in social functioning. In this study DTI was used to investigate structural abnormalities in the white matter structure of young children with ASDs. Moreover the neurostructural bases of echolalia were investigated. The functionality of the brain was analyzed through Functional Magnetic Resonance Imaging (fMRI) using a novel task based on face processing of human, android and robotic faces. A case-control study was performed in order to study how the face processing network is altered in ASDs and how robots are differently processed in ASDs and control groups. Measurements characterizing physiology and behavior of ASD children were also collected using an innovative platform called FACE-T (FACE-Therapy). FACE-T consists of a specially equipped room in which the child, wearing unobtrusive devices for recording physiological and behavioral data as well as gaze information, can interact with an android (FACE, Facial Automaton for Conveying Emotions) and a therapist. The focus was on ECG, as from the analysis of power spectrum density of ECG it is possible to extract features related to the autonomic nervous system that is correlated with brain functionality. These studies give new insights in the study of ASDs exploring aspects not yet addressed. Moreover the methodologies and tools developed could help in the objective characterization of ASD subjects and in the definition of a personalized therapeutic protocol for each child

Microscopy and Molecular Biology Techniques for the Study of Biocenosis Diversity in Semi-Confined Environments

This study is part of a wider conservation project of artistic and anthropological finds located in the Grotto of the Saints (Licodia Eubea, Alia, Sicily), and represents an opportunity for  investigating the micro-and macro biological systems colonizing this particular environment. It is well-known that the  bio-receptivity of surfaces is strongly related to its constituent materials and environmental parameters, whose effects promote the establishment of specific biotic communities. This is particularly true for caves, hypogea and semi-confined environments and, in particular for the Grotto of the Saints, where besides the presence of different nutrient sources, there are also high humidity values, percolating water and an aerobiological exchange with the surrounding countryside. Moreover, the weathering of this structure is enhanced by the canyon effect of the wind and the day-night temperature range. The identification and characterization of the biocenosis present in this environment was performed combining microscopy (optical, fluorescent, CLSM) and molecular biology analysis (DNA sequences). The aim was to identify the biological systems able to trigger the degradation processes, in order to plan their growth control and to prevent the colonization of the entire environment

How attention to faces and objects changes over time in toddlers with autism spectrum disorders: Preliminary evidence from an eye tracking study

Further understanding of the longitudinal changes in visual pattern of toddlers with autism spectrum disorders (ASDs) is needed. We examined twelve 19 to 33-month-old toddlers at their first diagnosis (mean age: 25.1 months) and after six months (mean age: 31.7 months) during two initiating joint attention (IJA) tasks using eye tracking. Results were compared with the performance of age-matched typically developing (TD) toddlers evaluated at a single time-point. Autistic toddlers showed longitudinal changes in the visual sensory processing of the IJA tasks, approaching TD performance with an improvement in the ability to disengage and to explore the global space. Findings suggest the use of eye tracking technology as an objective, non-intrusive, adjunctive tool to measure outcomes in toddlers with ASD

The Broad Autism (Endo)Phenotype: Neurostructural and Neurofunctional Correlates in Parents of Individuals with Autism Spectrum Disorders

Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with an early-onset and a strong genetic component in their pathogenesis. According to genetic and epidemiological data, ASD relatives present personality traits similar to, but not as severe as the defining features of ASD, which have been indicated as the "Broader Autism Phenotype" (BAP). BAP features seem to be more prevalent in first-degree relatives of individuals with ASD than in the general population. Characterizing brain profiles of relatives of autistic probands may help to understand ASD endophenotype. The aim of this review was to provide an up-to-date overview of research findings on the neurostructural and neurofunctional substrates in parents of individuals with ASD (pASD). The primary hypothesis was that, like for the behavioral profile, the pASD express an intermediate neurobiological pattern between ASD individuals and healthy controls. The 13 reviewed studies evaluated structural magnetic resonance imaging (MRI) brain volumes, chemical signals using magnetic resonance spectroscopy (MRS), task-related functional activation by functional magnetic resonance imaging (fMRI), electroencephalography (EEG), or magnetoencephalography (MEG) in pASD.The studies showed that pASD are generally different from healthy controls at a structural and functional level despite often not behaviorally impaired. More atypicalities in neural patterns of pASD seem to be associated with higher scores at BAP assessment. Some of the observed atypicalities are the same of the ASD probands. In addition, the pattern of neural correlates in pASD resembles that of adult individuals with ASD, or it is specific, possibly due to a compensatory mechanism. Future studies should ideally include a group of pASD and HC with their ASD and non-ASD probands respectively. They should subgrouping the pASD according to the BAP scores, considering gender as a possible confounding factor, and correlating these scores to underlying brain structure and function. These types of studies may help to understand the genetic mechanisms involved in the various clinical dimension of ASD

Gut to brain interaction in Autism Spectrum Disorders: A randomized controlled trial on the role of probiotics on clinical, biochemical and neurophysiological parameters

Background: A high prevalence of a variety of gastrointestinal (GI) symptoms is frequently reported in patients with Autism Spectrum Disorders (ASD). The GI disturbances in ASD might be linked to gut dysbiosis representing the observable phenotype of a "gut-brain axis" disruption. The exploitation of strategies which can restore normal gut microbiota and reduce the gut production and absorption of toxins, such as probiotics addition/supplementation in a diet, may represent a non-pharmacological option in the treatment of GI disturbances in ASD. The aim of this randomized controlled trial is to determine the effects of supplementation with a probiotic mixture (Vivomixx®) in ASD children not only on specific GI symptoms, but also on the core deficits of the disorder, on cognitive and language development, and on brain function and connectivity. An ancillary aim is to evaluate possible effects of probiotic supplementation on urinary concentrations of phthalates (chemical pollutants) which have been previously linked to ASD. Methods: A group of 100 preschoolers with ASD will be classified as belonging to a GI group or to a Non-GI (NGI) group on the basis of a symptom severity index specific to GI disorders. In order to obtain four arms, subjects belonging to the two groups (GI and NGI) will be blind randomized 1:1 to regular diet with probiotics or with placebo for 6 months. All participants will be assessed at baseline, after three months and after six months from baseline in order to evaluate the possible changes in: (1) GI symptoms; (2) autism symptoms severity; (3) affective and behavioral comorbid symptoms; (4) plasmatic, urinary and fecal biomarkers related to abnormal intestinal function; (5) neurophysiological patterns. Discussion: The effects of treatments with probiotics on children with ASD need to be evaluated through rigorous controlled trials. Examining the impact of probiotics not only on clinical but also on neurophysiological patterns, the current trial sets out to provide new insights into the gut-brain connection in ASD patients. Moreover, results could add information to the relationship between phthalates levels, clinical features and neurophysiological patterns in ASD. Trial registration: ClinicalTrials.gov Identifier: NCT02708901. Retrospectively registered: March 4, 2016

High-Risk Siblings without Autism: Insights from a Clinical and Eye-Tracking Study

Joint attention (JA)—the human ability to coordinate our attention with that of other people—is impaired in the early stage of Autism Spectrum Disorder (ASD). However, little is known about the JA skills in the younger siblings of children with ASD who do not develop ASD at 36 months of age [high-risk (HR)-noASD]. In order to advance our understanding of this topic, a prospective multicenter observational study was conducted with three groups of toddlers (age range: 18–33 months): 17 with ASD, 19 with HR-noASD and 16 with typical development (TD). All subjects underwent a comprehensive clinical assessment and an eye-tracking experiment with pre-recorded stimuli in which the visual patterns during two tasks eliciting initiating joint attention (IJA) were measured. Specifically, fixations, transitions and alternating gaze were analyzed. Clinical evaluation revealed that HR-noASD subjects had lower non-verbal cognitive skills than TD children, while similar levels of restricted and repetitive behaviors and better social communication skills were detected in comparison with ASD children. Eye-tracking paradigms indicated that HR-noASD toddlers had visual patterns resembling TD in terms of target-object-to-face gaze alternations, while their looking behaviors were similar to ASD toddlers regarding not-target-object-to-face gaze alternations. This study indicated that high-risk, unaffected siblings displayed a shared profile of IJA-eye-tracking measures with both ASD patients and TD controls, providing new insights into the characterization of social attention in this group of toddlers

An Integrated Approach for the Monitoring of Brain and Autonomic Response of Children with Autism Spectrum Disorders during Treatment by Wearable Technologies

Autism Spectrum Disorders (ASD) are associated with physiological abnormalities, which are likely to contribute to the core symptoms of the condition. Wearable technologies can provide data in a semi-naturalistic setting, overcoming the limitations given by the constrained situations in which physiological signals are usually acquired. In this study an integrated system based on wearable technologies for the acquisition and analysis of neurophysiological and autonomic parameters during treatment is proposed and an application on five children with ASD is presented. Signals were acquired during a therapeutic session based on an imitation protocol in ASD children. Data were analyzed with the aim of extracting quantitative EEG (QEEG) features from EEG signals as well as heart rate and heart rate variability (HRV) from ECG. The system allowed evidencing changes in neurophysiological and autonomic response from the state of disengagement to the state of engagement of the children, evidencing a cognitive involvement in the children in the tasks proposed. The high grade of acceptability of the monitoring platform is promising for further development and implementation of the tool. In particular if the results of this feasibility study would be confirmed in a larger sample of subjects, the system proposed could be adopted in more naturalistic paradigms that allow real world stimuli to be incorporated into EEG/psychophysiological studies for the monitoring of the effect of the treatment and for the implementation of more individualized therapeutic programs

Disentangling the initiation from the response in joint attention: An eye-tracking study in toddlers with autism spectrum disorders

Joint attention (JA), whose deficit is an early risk marker for autism spectrum disorder (ASD), has two dimensions: (1) responding to JA and (2) initiating JA. Eye-tracking technology has largely been used to investigate responding JA, but rarely to study initiating JA especially in young children with ASD. The aim of this study was to describe the differences in the visual patterns of toddlers with ASD and those with typical development (TD) during both responding JA and initiating JA tasks. Eye-tracking technology was used to monitor the gaze of 17 children with ASD and 15 age-matched children with TD during the presentation of short video sequences involving one responding JA and two initiating JA tasks (initiating JA-1 and initiating JA-2). Gaze accuracy, transitions and fixations were analyzed. No differences were found in the responding JA task between children with ASD and those with TD, whereas, in the initiating JA tasks, different patterns of fixation and transitions were shown between the groups. These results suggest that children with ASD and those with TD show different visual patterns when they are expected to initiate joint attention but not when they respond to joint attention. We hypothesized that differences in transitions and fixations are linked to ASD impairments in visual disengagement from face, in global scanning of the scene and in the ability to anticipate object's action