UN APPROCCIO METAGENOMICO PER STUDIARE COMUNITÀ NATURALI DI PROTISTI A VITA LIBERA

Protists are a polyphyletic group including all the eukaryotic unicellular microorganisms. They are the leading source of biodiversity on our planet and play an essential role in maintaining global processes, which in turn regulate the functioning of the biosphere. Nevertheless, we know little about the enormous biodiversity of these microorganisms. Protists are also a natural reservoir of bioactive substances of ecological-environmental and industrial hygieneinterest. Indeed, some protists are able to produce molecules with a toxicological potential that can be transmitted through the food chain up to its final levels (man), while other substances may constitute the active ingredient of pharmaceutical products and agro-food products. The main tools currently applied in the study of free-living protists from different environments, require as a prerequisite the cultivation of these microorganisms. Moreover, conventional cultures can not restore the environmental conditions, resulting in the failure of their multiplication. Thus, these methods are not always able to provide comprehensive information on biodiversity and biotechnological potential of these microorganisms in environmental samples. However, modern molecular techniques allow quick access to microbial diversity and facilitate the discovery of new groups of microorganisms, otherwise difficult to be identified. In order to contribute to the knowledge of the biodiversity of free-living protists, in my work, I used a metagenomic approach to study natural communities of these microorganisms. Metagenomics is a new approach to afor the scientific research, originally initially used for microbial communities’ studies of microbial communities of prokaryotic naturees. It is based on the use of modern genomic techniques in studying microbial communities directly from environmental samples, avoiding the problem of isolation and culture in laboratory. It is, in practice, a technology that , firstly, provides for the simultaneous analysis of the genome of all the microorganisms present in particular environmental samples,; secondly, allowings the search for new species and, finally,or contributes to create a profile of known species. To this endFor this purpose, I have identified some natural aquatic habitats, both marine and freshwater, from where I took appropriate samples. After a preliminary concentration of the samples by centrifugationFirst of all, I increased the protists concentration through centrifugation, in order to separate the sediment from the liquid phase, . Secondly, a preliminary subsequent filtration of the latter phase through filters of different mesh sizes was performed in order to exclude microorganisms larger than those of protists bigger microorganisms, such as small crustaceans and other metazoans. Subsequently, the samples were subjected to techniques of DNA extraction, of DNA based on specifically developed protocols. The obtained DNA samples were then subjected to gene amplification by the PCR technique.; the As genetic marker useful for the species identification, selected was the nuclear ribosomal gene for ribosomal RNA (rRNA) 18S gene, unanimously considered a good species-specific marker, was selected. Hence, the obtained PCR products were cloned and recombinant clones subjected to sequencing. Finally, the obtained sequences were compared with those homologous available in the homologous genomic Ggenbank databases . The obtained sequences were alsoand subjected to phylogenetic analysis. The final obtained results have allowed to identify, on a molecular basis, several taxa of protists and to outline a sufficiently representative framework of the community of protists of the sampled sites. In order to obtain a support for the taxonomic identifications made with the genetic approach, environmental samples were subjected also to morphological analysis, carried out by observation under the an optical microscope, acquisition of photographic images relating to presentof protists present in the samples and their subsequent identification on a morphologicy basis