Analisi del profilo tossicologico e cancerogeno di contaminanti ambientali e identificazione dei rischi per la salute umana correlati all'esposizione

L’attenta analisi della letteratura scientifica su argomenti riguardanti i contaminanti ambientali oggetto di studio (i policlorodifenili) ha permesso di raccogliere dati utili riguardanti le proprietà di queste molecole, la loro diffusione e la loro pericolosità. Oggetto della ricerca è stato lo studio in vitro del potenziale citotossico e trasformante dei PCB, utilizzando come riferimento una miscela commerciale di PCB, l’Aroclor 1260, e di un MIX di 18 congeneri ricostituito in laboratorio. Il lavoro è proseguito con la valutazione degli effetti di queste miscele e di due congeneri singoli (PCB 118 e PCB 153) su linee cellulari diverse in test di vitalità a breve termine. L’utilizzo di test specifici ha poi permesso la valutazione di un possibile potenziale estrogenico. Una volta ottenuto un quadro generale sui possibili effetti delle miscele grazie ai risultati dei test funzionali, è stata valutata la modulazione, da parte delle molecole e/o di miscele delle stesse, dell’espressione di geni coinvolti nella risposta ad estrogeni o a composti diossino simili, andando ad effettuare un’analisi di tipo molecolare con Real-Time PCR (RT-PCR) e analizzando nello specifico marcatori di pathway dell’Aryl Hydrocarbon Receptor (AhR) o dell’Estrogen Receptor (ER). In ultima analisi al fine di verificare l’applicabilità di biomarkers di espressione a situazioni di contaminazioni reali, ci si è focalizzati su campioni estratti da matrici ambientali, ed in particolare linee cellulari di interesse sono state esposte a estratti di sedimenti provenienti da siti inquinati. L’approccio scelto è stato di tipo molecolare, con lo scopo di individuare pathway da valutare in un secondo momento in test funzionali specifici. L’attività di ricerca si è avvalsa della tecnica del DNA-microarray per valutare la modulazione dell’espressione genica in risposta all’esposizione a contaminanti ambientali. In questo modo è possibile definire i profili di espressione genica che sottendono a risposte biologiche complesse nell’intento di individuare biomarcatori in grado di predire il rischio per l’uomo, e di consentire la stima di una relazione diretta tra esposizione ed effetti possibili.

Mechanisms of cell senescence: p21 and DNA damage response in aging and longevity

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

Genomic characterization of the italian wolf (Canis lupus): the genes involved in black coat colour determination and application of microarray technique for snps detection.

This study provides a comprehensive genetic overview on the endangered Italian wolf population. In particular, it focuses on two research lines. On one hand, we focalised on melanism in wolf in order to isolate a mutation related with black coat colour in canids. With several reported black individuals (an exception at European level), the Italian wolf population constituted a challenging research field posing many unanswered questions. As found in North American wolf, we reported that melanism in the Italian population is caused by a different melanocortin pathway component, the K locus, in which a beta-defensin protein acts as an alternative ligand for the Mc1r. This research project was conducted in collaboration with Prof. Gregory Barsh, Department of Genetics and Paediatrics, Stanford University. On the other hand, we performed analysis on a high number of SNPs thanks to a customized Canine microarray useful to integrate or substitute the STR markers for genotyping individuals and detecting wolf-dog hybrids. Thanks to DNA microchip technology, we obtained an impressive amount of genetic data which provides a solid base for future functional genomic studies. This study was undertaken in collaboration with Prof. Robert K. Wayne, Department of Ecology and Evolutionary Biology, University of California, Los Angeles (UCLA).