Molecular mapping of microbial assemblages in aqueous ecosystems of Epirus and study of potential use as antineoplastic factors (Doctoral thesis)

Τούκα, Αναστασία

Ancient ecosystems are hotspots of biodiversity and their longevity and geographical isolation make them important agents for evolutionary and climate history research. Many ancient lakes are found in Greece. One of them is lake Pamvotis which we studied. Lake Pamvotis is a shallow, eutrophic lake and to our knowledge this is the first study to reveal bacterial and archaeal diversity in lake Pamvotis. Subject of this study was also the isolation and culture of bacterial cells from lake Pamvotis. Methanol and aquatic extracts from those cultures would be evaluated for their potential cytotoxic and antineoplastic effects in cancer cell lines.We used water and sediment samples from lake Pamvotis from two stations and each season. Firstly, we determined the physicochemical parameters and then in order to reveal the diversity of bacteria and archaea we used extraction DNA protocol, PCR protocols, DGGE, cloning, sequencing, phylogenetic analysis tools and next generation sequencing. To quantify bacteria and archaea in lake Pamvotis samples, we used real-time PCR and we used BIOLOG method to study the bacterial biochemical profile. We also performed statistical analysis to investigate possible relationships among bacterial and archaeal abundances and the physicochemical variables. In case of bacterial and cyanobacterial cultures, we used R2A and BG11 medium, respectively. Methanol and aquatic extracts from isolated bacteria and cyanobacteria were tested in cancer cell lines A549, HepG2, MCF7 and MRC7 (control) for cytotoxic/antineoplastic activity. Concerning lake Pamvotis sediments, we identified many bacterial (50.88%) and archaeal (20.65%) sequences corresponding to potentially new species and most of the bacterial (84.21%) and archaeal (95.65%) sequences were found not to have cultivated counterparts (compared to GenBank entries). Proteobacteria, Cyanobacteria and methanogenic archaea were in abundance in the sediments of the lake and archaea represent 11.13%-23.88% of the total prokaryotic cells. Of the determined physicochemical variables, Ni,Cr,Ca,pH,TKN,As,TC were found to affect strongly the prokaryotic abundances. Upon phylogenetic analysis, a new class of Cyanobacteria was identified and named “Lake Pamvotis cluster” (LPC). In the water samples of lake Pamvotis we also identified bacterial sequences corresponding to potentially new species. Archaea represent 0.14%-9.77% of the total prokaryotic cells and we noticed that cyanobacteria related to Synechococcus sp. might be responsible for a bloom in spring, different than Microcystis sp. bloom in summer.Finally, the extracts from bacterial and cyanobacterial cultures which we used for toxicity tests, revealed a potential bioactive compound. The cyanobacterial methanol extract had cytotoxic effects (apoptosis) on MCF7 cells and on control fibroblasts (MRC5 cells). This extract was derived from the cyanobacterium related to Pseudanabaena/Limnothrix sp. and the bioactive compound was not belonging to known cyanotoxins.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής
Subject classification: Βακτήρια
Keywords: Μοριακή χαρτογράφηση,Λίμνη Παμβώτιδα,Βακτήρια,Αρχαία,Κυανοβακτήρια,Αποδιατακτική ηλεκτροφόρηση σε πήκτωμα διαβάθμισης,Ποσοτική αλυσιδωτή αντίδραση πολυμεράσης,Αλληλούχιση επόμενης γενιάς,Βακτηριακά εκχυλίσματα,Τοξίνες,Κυτταροτοξικότητα,Molecular mapping,Lake Pamvotis,Bacteria,Archaea,Cyanobacteria,DGGE,qPCR,Next generation sequencing,Bacterial extracts,Toxins,Cytotoxicity
Appears in Collections:Διδακτορικές Διατριβές

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