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dc.contributor.advisorLuisi, Pier Luigi-
dc.contributor.authorCarrara, Paolo-
dc.description.abstractOne of the open questions in origin of life is about the formation of primitive cells from separated molecular components. In recent years, this subject has been approached in the “Minimal Cell” project, namely the laboratory study on cell-like compartments with the minimal and sufficient number of components that may allow cellular life. The theoretical framework of this research is the theory of autopoiesis, that elegantly defines what minimal life is in terms of self-bounded biochemical network capable of reconstructing itself (boundary included) thanks to its own reactions. Current conceptual and experimental studies are focused on the study of cell-models created from lipid compartments, i.e., lipid vesicles (or liposomes). Until now, great attention has been devoted to consider the emergence of cellular life as an event that is bases on individual compartments, e.g., a single cell within a population that coentrap simple solutes that later internally develop a minimal metabolism, or complex solutes that are already part of an external metabolism. This view can be however not realistic, due to the low probability of such events. An alternative view explicitly considers the interaction between compartments as a way of stepwise increase of metabolic molecular complexity thanks to fusion or solute exchange processes. The aim of this work is to put forward this new view, developing experimental strategies for its study and possibly revealing key aspects of this novel approach. No previous experimental reports on the subject can be found in the literature. This can be done by achieving two main goals. Firstly, define and develop cell-model systems, and then propose a novel view about the possible important steps in primitive pre-cellular evolution, from the viewpoint of cell communities. Giant vesicles (GVs) are used as cell model, because of their large size (1–100 um) which allows direct observation by light and fluorescent microscopy.GVs have been produced with emulsion inversion method. This was selected because of its versatility and for the high solute entrapment that characterize it. However, this method was just discovered when this PhD work started and essentially no standard protocol was available. We have therefore, as a first step, outlined an experimental protocol that allows the highly reproducible production of GVs at high yield (10000 GVs/ul). It was possible to obtain GVs with an internal environment different from that outside, and entrap a wide range of water-soluble solutes as calcein (a small soluble molecule), FITC and RITC-dextran (sugars), allophycocyanin (APC), phycoerythrin (PE) (proteins) in their aqueous core. Preliminary results have extended this method to the entrapment of nucleic acids and enzymes. In order to create a model of primitive colonies we exploited the electrostatic attraction, a very basic and long-range physical force that had to be present also in ancient time. As model cationic peptide we employed poly(arginine) (PLA) to trigger the aggregation of negatively charged GVs (POPC:oleate 1:1). Immediately after addition of PLA, GVs move towards each other, stick together and form GVs colonies of various size. The process of the formation of colonies has been characterized by changing the variables affecting its occurrence. The first step was to test the threshold concentration of PLA. The second step was to verify whether the formation of colonies depended on the number of GVs initially present in the slide well. Thanks to these experiments we are able to reproduce the process of vesicle aggregation into colonies in a reproducible and controlled way. Regardless of the amount of PLA added or the number of GVs initially present in the slide well, it was always possible to detect 1-5% GVs that derive, without any doubts, from the fusion of two or more GVs. This first important result confirms that the colony formation brings about an increase of complexity, giving an advantage to the colony with respect to individual GVs. Additional advantages of the GVs colonies with respect to individual (free) GVs lies in their resistance to flow, and against osmotic stress. Moreover, GVs colonies can grow by incorporating new GVs.In conclusion, this work has opened a new vision with regard to phenomena at the base of the origin of life. It is the first time that from a vision of a single compartment it is passed to that of a community. A colony of individuals that work together is able to have selective advantages in respect to the individual one and thus evolve and achieve a greater level of complexity. We think that this model of primordial cell community is able to simulate a more likely the reality of cellular life.it_IT
dc.publisherUniversità degli studi Roma Treit_IT
dc.titleConstructing a minimal cellit_IT
dc.typeDoctoral Thesisit_IT
dc.subject.miurSettori Disciplinari MIUR::Scienze biologiche::BIOLOGIA MOLECOLAREit_IT
dc.subject.isicruiCategorie ISI-CRUI::Scienze biologiche::Molecular Biology & Geneticsit_IT
dc.subject.anagraferoma3Scienze biologicheit_IT
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Appears in Collections:X_Dipartimento di Biologia
T - Tesi di dottorato
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