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Sviluppo di metodi per l'identificazione e l'analisi di sequenze geniche fetali nella circolazione materna per una diagnosi prenatale non invasiva
Get PDFDuring last decade, molecular biology evolution has lead to a deep knowledge of human genome allowing the in-utero diagnosis of a large number of genetic diseases. Now adays performing a prenatal diagnosis of chromosomal abnormalities and of genetic diseases, requires genetic material from the fetus and this is possible only by invasive procedures such as amniocentesis and chorionic villus sampling. The risk of miscarriage of these technics is between 0.5% and 1% and considering these limits, new alternative and non-invasive screening methods, based on ultrasound and/or biochemical parameters of maternal blood, were developed. The disavantages of these methods are the probabilistic aspect of the results.
There was an increasing interest in a new approach that could keep to zero the risk of miscarriage, with an extension of fetal genetic materials screening to a large number of women at the beginning of pregnancy, leading a decreased physic and psychological impact.
In 1997 it was demonstrated the presence of cell-free fetal DNA (cffDNA) in the maternal blood and this has opened up new possibilities for a non-invasive prenatal diagnosis (NIPD).
The cell-free fetal DNA originates from apoptotic trophoblasts on placenta’s surface and comprises around 3-6% of total cell-free DNA in maternal circulation. The cffDNA consists of short DNA fragments (<200bp). Fetal DNA can be detected from 32 days of gestation (though only reliably from 7 weeks) and it’s concentration increases with gestational age (21% per week during the first three months) with a sharp peak during last 8 weeks of pregnancy. The cffDNA is completely removed after delivery.
The main problem related to the application of this NIPD is the high maternal background, so researchers have initially focused their attention on the detection of sequences that are absent in the maternal genome, such as DNA sequences from chromosome Y of male fetuses, the determination of fetal RhD genotype in women RhD-negative, and the detection of paternal mutations in maternal plasma.
Although, the Real Time qPCR amplification of fetal sequences is the widespread method for cffDNA study, there are still a lot of studies focused on the development different protocols for detection of minimal differences between maternal and fetal DNA.
The aim of this PhD thesis is the study and setup of different methods to identify fetal genomic sequences in maternal circulation.
The research project was divided in three steps: the first one was focused on the validation of Real-Time qPCR protocol, applied to the detection and quantification of total free DNA and of cffDNA in pregnancies with a male fetus; the second was focused on the develop of a reliable protocol for the identification of fetal genomic sequences useful to recognize cffDNA in pregnancies with a female fetus, by the mini-sequencing technics; finally, the last step was focused on the validation of an analytical protocol to verify the presence/absence of paternal mutations in maternal plasma using nested PCR allele-specific.
The results demonstrated here for each technic, show a high sensibility and resolution capacity, together with an easy and rapid handling. Furthermore, the combination of the two technics (Real-Time qPCR and mini-sequencing reaction) was essential in order to exclude the presence of a paternal mutation in the fetal genome in every case where a mutation wasn’t identified. The ability to exclude the presence of the paternal mutation in the fetus by analysis of maternal plasma avoid the utilization of invasive prenatal tests and, at the same time, reassures the couple early in the course of the pregnancy about the recurrence of the disease, thus avoiding significant anxiety.Negli ultimi anni l'evoluzione della biologia molecolare ha permesso una conoscenza sempre maggiore del genoma umano e consente ormai la diagnosi in utero di un numero crescente di patologie genetiche. Al momento attuale la diagnostica prenatale di anomalie cromosomiche o di malattie genetiche richiede il prelievo di cellule fetali ottenibili con metodiche invasive, l'amniocentesi e la villocentesi. Queste tecniche comportano un rischio di provocare un aborto compreso fra 0.5% e 1%. Di fronte a questi limiti delle metodiche invasive sono stati messi a punto alcuni metodi di screening alternativi non invasivi, basati su parametri ecografici fetali e/o biochimici su sangue materno, che però hanno il limite di fornire risposte solo di tipo probabilistico.
L'introduzione di un test realmente alternativo di diagnosi prenatale non invasiva che permetta di eliminare il rischio di aborto legato all’amniocentesi e alla villocentesi è fortemente attesa. Infatti un simile test permetterebbe una estensione dell'analisi del materiale genetico fetale a molte più donne all'inizio della gravidanza e diminuirebbe l'impatto fisico e psicologico di questo tipo di indagine.
Nel 1997 uno studio ha dimostrato la presenza di DNA libero fetale (cell-free fetal DNA, cffDNA) nel plasma materno e ha evidenziato la sua potenzialità per una diagnosi prenatale non invasiva (NIPD). Durante i 15 anni successivi, si è sviluppata la ricerca di base sulle caratteristiche del DNA libero fetale e molti ricercatori hanno studiato la biologia e il significato clinico del trasporto di cffDNA nel circolo materno.
Il DNA libero fetale origina dall’apoptosi dei trofoblasti costantemente rimpiazzati sulla superficie della placenta e comprende circa il 3-6% del DNA libero totale nella circolazione materna. E’ stato inoltre osservato che è costituito da corti frammenti di DNA (<200bp) piuttosto che interi cromosomi e che la sua concentrazione aumenta gradualmente durante la gravidanza: è rilevabile nella circolazione materna già a 32 giorni circa di gestazione (in quantità affidabile solo dopo le 7 settimane) e aumenta del 21% ogni settimana nel primo trimestre, con un forte picco durante le ultime 8 settimane di gravidanza per poi venire rapidamente rimosso dalla circolazione materna subito dopo il parto.
L’utilizzo del DNA fetale libero come mezzo per la diagnosi prenatale non invasiva di malattie genetiche fino ad ora è fortemente limitato dall’elevato background di DNA materno pertanto le prime strategie messe a punto per l’identificazione del DNA libero fetale si sono basate sul rilevamento di sequenze assenti nel genoma materno, come le sequenze Y-specifiche, la determinazione del genotipo RhD fetale in donne RhD-negative e l’identificazione di sequenze fetali con mutazioni ereditate dal padre.
L’amplificazione tramite Real Time qPCR di sequenze unicamente fetali nel plasma di donne gravide rappresenta ad oggi il metodo di elezione per lo studio del DNA libero fetale, ma numerosi sforzi sono stati realizzati per la messa a punto di sistemi sempre più sensibili ed universali per rilevare le più piccole differenze tra il DNA fetale libero e quello materno, al fine di consentire un utilizzo su larga scala del cffDNA per la diagnosi prenatale non invasiva.
Lo scopo di questa ricerca è stato quindi, la messa a punto di protocolli validi per l’identificazione e l’analisi di sequenze geniche fetali nella circolazione materna.
Il progetto è stato quindi suddiviso in tre fasi: la prima fase ha comportato la messa a punto di opportuni protocolli di Real-Time qPCR per il rilevamento e la quantificazione sia del DNA libero totale estratto sia del cffDNA estratto in gravidanze con feto di sesso maschile; la seconda fase del progetto si è occupata di testare e sviluppare un protocollo affidabile per l’identificazione di sequenze geniche fetali utili al riconoscimento di cffDNA nelle gravidanze con feto di sesso femminile, mediante l’utilizzo della tecnica di mini-sequencing; l’ultima fase del progetto si è concentrata infine sulla messa a punto di un protocollo di verifica della presenza/assenza di mutazioni paterne in plasma materno mediante nested PCR allele-specifica.
L’analisi dei risultati ha dimostrato, per ogni tecnica sviluppata, elevate sensibilità e capacità di risoluzione accoppiate a velocità e facilità di esecuzione, confermandone quindi l’applicabilità per una diagnosi prenatale non invasiva. Inoltre l’utilizzo in combinazione delle due tecniche (Real-Time qPCR e mini-sequencing) per la conferma della presenza del cffDNA nel campione in analisi, si è rilevata indispensabile per poter escludere che il feto abbia ereditato la mutazione paterna, in tutti i casi in cui non viene identificata la mutazione. L’esclusione della mutazione paterna nel feto attraverso l’analisi su plasma materno permetterebbe quindi di evitare l’utilizzo di procedure invasive per il prelievo di cellule fetali e avere questa informazione alla 9-10 settimana di gravidanza consentirebbe di rassicurare la coppia prima di qualsiasi altro test diagnostico di routine
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
CATECHOL-O-METHYLTRANSFERASE GENOTYPE MODIFIES EXECUTIVE FUNCTIONING AND PREFRONTAL FUNCTIONAL CONNECTIVITY IN WOMEN WITH ANOREXIA NERVOSA.
No full textBackground: Anorexia nervosa (AN) is characterised by high levels of perseveration and inflexibility which interfere with successful treatments. Dopamine signalling seems to play a key role in modulating the prefrontal cortex, since both dopamine deficiency and excess negatively influences the efficiency of cognitive functions. The present study explores the effect of a functional polymorphism (Val158Met) in the catechol-O-methyltransferase gene on the set-shifting abilities and prefrontal functional connectivity of patients with AN.
Methods: All subjects of the study (166 subjects with DSM-IV lifetime AN and 140 healthy women) performed the Wisconsin Card Sorting Task, and a subsample (33 AN patients and 30 controls) underwent resting-state functional magnetic resonance imaging scanning.
Results: Both underweight and weight-recovered AN patients showed high levels of perseveration, but only in the underweight group did the Val158Met polymorphism affect cognitive performance, showing the U-shaped curve characteristic of increased dopamine signalling in the prefrontal cortex. Underweight AN patients who are Met homozygotes had significantly higher levels of perseveration and increased prefrontal functional connectivity, indicating abnormal regional cortical processing.
Limitations: Although our data show that grey matter reduction in starving AN did not explain our findings, the cross-sectional design of the present study did not allow to distinguish between the effects of starvation and those of low estrogen levels.
Conclusions: Starvation affects dopamine release in the prefrontal cortex of AN patients with different effects on executive functioning and prefrontal functional connectivity according to catechol-O-methyltransferase genotype. This observation has several therapeutic implications that need to be addressed by future studies
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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