Genetica molecolare dell'autismo: studi di associazione ed analisi di geni candidati

Autism is a neurodevelpmental disorder characterized by impaired verbal communication, limited reciprocal social interaction, restricted interests and repetitive behaviours. Twin and family studies indicate a large genetic contribution to ASDs (Autism Spectrum Disorders). During my Ph.D. I have been involved in several projects in which I used different genetic approaches in order to identify susceptibility genes in autism on chromosomes 2, 7 and X: 1)High-density SNP association and CNV analysis of two Autism Susceptibility Loci. The International Molecular Genetic Study of Autism Consortium (IMGSAC) previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we evaluated the patterns of linkage disequilibrium (LD) and the distribution of haplotype blocks, utilising data from the HapMap project, across the two strongest peaks of linkage on chromosome 2 and 7. More than 3000 SNPs have been selected in each locus in all known genes, as well as SNPs in non-genic highly conserved sequences. All markers have been genotyped to perform a high-density association analysis and to explore copy number variation within these regions. The study sample consisted of 127 and 126 multiplex families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Association and CNV analysis implicated several new genes, including IMMP2L and DOCK4 on chromosome 7 and ZNF533 and NOSTRIN on the chromosome 2. Particularly, my contribution to this project focused on the characterization of the best candidate gene in each locus: On the AUTS5 locus I carried out a transcript study of ZNF533 in different human tissues to verify which isoforms and start exons were expressed. High transcript variability and a new exon, never described before, has been identified in this analysis. Furthermore, I selected 31 probands for the risk haplotype and performed a mutation screen of all known exons in order to identify novel coding variants associated to autism. On the AUTS1 locus a duplication was detected in one multiplex family that was transmitted from father to an affected son. This duplication interrupts two genes: IMMP2L and DOCK4 and warranted further analysis. Thus, I performed a screening of the cohort of IMGSAC collection (285 multiplex families), using a QMPSF assay (Quantitative Multiplex PCR of Short fluorescent Fragments) to analyse if CNVs in this genic region segregate with autism phenotype and compare their frequency with a sample of 475 UK controls. Evidence for a role of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family. 2)Analysis of X chromosome inactivation. Skewed X chromosome inactivation (XCI) is observed in females carrying gene mutations involved in several X-linked syndromes. We aimed to estimate the role of X-linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 164 affected girls. The study sample included families from different european consortia. I analysed the XCI inactivation pattern in a sample of italian mothers from singletons families with ASD and also a control groups (144 adult females and 40 young females). We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X-linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X-linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27-Xq28 region, with a peak Z score of 1.75 close to rs719489. In this region FMR1 and MECP2 have been associated in some cases with austim and therefore represent candidates for the disorder. I performed a mutation screen of MECP2 in 33 unrelated probands from IMGSAC and italian families, showing XCI skewness. Recently, Xq28 duplications including MECP2, have been identified in families with MR, with asymptomatic carrier females showing extreme (>85%) skewing of XCI. For these reason I used the sample of probands from X-skewed families to perform CNV analysis by Real-time quantitative PCR. No duplications have been found in our sample. I have also confirmed all data using as alternative method the MLPA assay (Multiplex Ligation dependent Probe Amplification). 3)ASMT as functional candidate gene for autism. Recently, a possible involvement of the acetylserotonin O-methyltransferase (ASMT) gene in susceptibility to ASDs has been reported: mutation screening of the ASMT gene in 250 individuals from the PARIS collection revealed several rare variants with a likely functional role; Moreover, significant association was reported for two SNPs (rs4446909 and rs5989681) located in one of the two alternative promoters of the gene. To further investigate these findings, I carried out a replication study using a sample of 263 affected individuals from the IMGSAC collection and 390 control individuals. Several rare mutations were identified, including the splice site mutation IVS5+2T>C and the L326F substitution previously reported by Melke et al (2007), but the same rare variants have been found also in control individuals in our study. Interestingly, a new R319X stop mutation was found in a single autism proband of Italian origin and is absent from the entire control sample. Furthermore, no replication has been found in our case-control study typing the SNPs on the ASMT promoter B.

Sviluppo di un algoritmo di calibrazione per analisi quantitative di immagini in microscopia a fluorescenza

Questo progetto di tesi ha come obiettivo lo sviluppo di un algoritmo per la correzione e la calibrazione delle immagini in microscopia a fluorescenza e della sua implementazione come programma. Infatti, senza tale calibrazione le immagini di microscopia a fluorescenza sarebbero intrinsecamente affette da molteplici tipi di distorsioni ottiche. Questo limita fortemente la possibilità di effettuare analisi quantitative del livello di fluorescenza osservato. Il difetto sul quale ci siamo soffermati è la disomogeneità di campo, ossia una non uniforme fluorescenza causata dalla forma irregolare del fascio di eccitazione. Per conseguire l'obiettivo da noi proposto è necessaria l'acquisizione, in parallelo al campione in esame, di immagini di calibrazione contenenti sfere nanometriche a fluorescenza nota. A partire da queste, tramite procedure di image processing da noi implementate, abbiamo stimato la funzione di correzione della fluorescenza, localmente per ogni punto dell'immagine. Per la creazione di tale algoritmo abbiamo ipotizzato una possibile distribuzione dell'intensità dovuta alla non omogeneità del fascio ed abbiamo quindi stimato i parametri tramite un'apposita procedura di maximum likelihood. Tale stima è stata eseguita tenendo conto di possibili effetti dovuti alla luminosità di background, alla sovrapposizione di più nanosfere e ad effetti di bordo nel corso dell'elaborazione. Questa procedura è stata ripetuta su quattro diverse immagini di calibrazione, per valutarne la consistenza e la validità. Inoltre, per poter verificare che il software di elaborazione abbia le desiderate proprietà di linearità tra segnale misurato ed intensità nota, ci siamo serviti di un'ulteriore immagine di calibrazione contenente una mistura di sfere nanometriche con intensità variabili su due ordini di grandezza. Il risultato di questo lavoro di tesi verrà incluso in un programma per la calibrazione delle immagini in fluorescenza acquisite al laboratorio di biofisica del Dipartimento di Fisica ed Astronomia di Bologna.

Applicazioni di interferometria radar da satellite per la generazione di mappe di spostamento nell'analisi di fenomeni dinamici

Scopo di questa tesi è la produzione di una serie di mappe di deformazione del suolo o della copertura superficiale del terreno mediante l'utilizzo di una tecnica di monitoraggio territoriale da satellite denominata Interferometria SAR. Tale tecnica si colloca nell'ambito del Telerilevamento a sensori attivi. Le mappe di spostamento sono state prodotte per quattro casi di studio, due riguardanti eventi sismici (terremoto del 2009 a L'Aquila in Italia e terremoto del 2003 a Bam in Iran) e due relativi al movimento di deriva conseguente allo scioglimento dei ghiacciai alpini (ghiacciaio dell'Aletsch in Svizzera) e delle calotte polari (ghiacciaio David-Drygalski in Antartide).

Il modello di Fister-Panetta per la crescita di cellule tumorali : analisi qualitativa e aspetti didattici

Si analizza il modello di Fister-Panetta per la crescita di cellule tumorali nella fase avascolare del tumore e si propone un percorso didattico di introduzione alla modellistica matematica in una quinta liceo. Si riporta poi una riflessione sulle motivazioni per cui l'introduzione degli aspetti modellistici e applicativi della matematica è importante anche nella scuola superiore.

Impiego della spettrometria di massa nell'analisi di fumonisine in alimenti e matrici di origine animale

Mycotoxins are heterogeneous chemical compounds characterized by a low molecular weight and synthesized by the secondary metabolism of different molds. Fumonisins are water-soluble mycotoxins produced by Fusarium species spoiling corn and derived produc ts. These mycotoxins can be a health hazard when consuming contaminated cereals, but they can reach humans also indirectly through the consumption of food products derived from animals fed with contaminated feed. Fumonisins have been associated with several animal and human diseases: they are suspected risk factors for esophageal and liver cancers, neural tube defects and cardiovascular problems. Improved methods are needed to accurately assess fumonisins concentrations in food of vegetable and animal origin, in order to prevent acute and chronic human exposure. The aim of the present work was to evaluate the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis from foods and matrices of animal origin. Different methods for the identification and quantification of fumonisins and related products have been developed and validated to determine fumonisin B1 in milk, fumonisin B1, fumonisin B2 and their complete hydrolyzed products (HFB1 and HFB2) in pig liver and fumonisins B1 and B2 in complete and complementary dry dog food. The experimental procedures have been carefully studied, considering matrices features, number and type of molecules to detect. Therefore, several extraction, clean up and separation techniques were tested in order to obtain the better conditions of sample processing. The fit for purpose sample preparation, matched with high mass spectrometry sensibility and specificity, have allowed to achieve good results in any tested animal matrices. Hence, the developed methods were validated and have shown a high accuracy, sensibility and precision, fulfilling performance requirements of Decision 2002/657/EC and of European Project Standard, Measuring and Testing (SMT). In any developed method, the analytes were identified and quantified even at very low concentrations : the limits of quantification resulted lower than other similar works, performed with different detectors. These methods were applied to some commercial samples and to some samples collected for research projects in the Department of Veterinary Public Health and Animal Pathology (DVPHAP) of University of Bologna. Although the disclosed data must be considered completely preliminary and without statistical significance, they emphasize the presence of mycotoxins in animal products. The outcomes obtained from the processed samples (bovine milk, pig liver and dry dog food) suggest the efficacy of these methods also on other food matrices, confirming the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis. Moreover the results underline the need to set up a large scale monitoring in order to evaluate the presence of fumonisins in food of animal origin for human consumption.