Structural and functional analysis of centromeric chromatin

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.

Characterization of circulating steroids and endocannabinoids levels in obese subjects by liquid chromatography tandem mass spectrometry

Nowadays obesity can be defined as a global epidemic. The precise identification of circulating biomarkers involved in this pathology could be essential to early diagnose potential co-morbidities and to better address the development of future therapeutic strategies. Published evidences show that circulating steroid hormones and endocannabinoids might have a role in the physiopathology of obesity; however, a precise and reliable quantification of these molecules is still lacking. In the first part of the present thesis, we developed a sensitive, specific and accurate quantification method for nine steroid hormones using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system. This method has been used first for a comparative study with immunoassays, currently used in the clinical practice to quantify these molecules and then to redefine circulating reference intervals in healthy subjects. Furthermore, we measured circulating steroid hormones in three groups of subjects: normo-weight, over-weight and obese, defining different steroid hormones profiles depending on the obesity state. The role of circulating endocannabinoids in humans is still unclear, however there are several evidences concerning their involvement in obesity. In the second part of the thesis, we determined changes of circulating endocannabinoids in obese patients after a weight loss induced by bariatric surgery, currently the most effective long-term treatment for obesity, using LC/MS-MS. We measured basal and dynamic endocannabinoids plasma levels in 12 patients with severe obesity before, one month after and six months after the Roux-en-Y gastric bypass intervention, currently one of the most performed types of bariatric surgery. All together the findings illustrated in this thesis project will help better define the role of steroid hormones and endocannabinoids in the framework of obesity in humans and the role that each type of molecule might have in its pathophysiology.