Ações encobertas

The global and increasingly technological society requires the States to adopt security measures that can maintain the balance between the freedom, on the one hand, and the security and the respect for fundamental rights of a democratic state, on the other. A State can only achieve this aim if it has an effective judicial system and in particular a criminal procedure adequate to the new criminogenic realities. In this context, the national legislator has adopted, following other international legal systems, special means of obtaining proof more stringent of rights. Within those special means are included the covert actions, that, being a means to use sparingly, is a key element to fight against violent and highly organized crime. Therefore, the undercover agent, voluntary by nature, develops a set of activities that enables the investigation to use other means of taking evidence and/or probationary diligences itself, with the purpose of providing sufficient proof to the case file. In this milieu, given the high risks involved during the investigation, as well as after its completion, the undercover agent can act upon fictitious identity. This measure can be maintained during the evidentiary phase of the trial. Similarly, given the latent threat that the undercover agent suffers by its inclusion in criminal organizations, as well as the need for his inclusion in future covert actions it is crucial that his participation as a witness in the trial is properly shielded. Thus, when the undercover agent provides, exceptionally, statements in the trial, he shall do so always through videoconference with voice and image distortion. This measure can guarantee the anonymity of the undercover agent and concomitantly, that the adversarial principle and the right of the accused to a fair trial is not prejudiced since, in those circumstances, the diligence will be supervised in its entirety (in the audience and with the undercover agent) by a judge.

Design of bio-inspired ionic liquids for protein stabilisation

Ionic Liquids (ILs) consist in organic salts that are liquid at/or near room temperature. Since ILs are entirely composed of ions, the formation of ion pairs is expected to be one essential feature for describing solvation in ILs. In recent years, protein - ionic liquid (P-IL) interactions have been the subject of intensive studies mainly because of their capability to promote folding/unfolding of proteins. However, the ion pairs and their lifetimes in ILs in P-IL thematic is dismissed, since the action of ILs is therefore the result of a subtle equilibrium between anion-cation interaction, ion-solvent and ion-protein interaction. The work developed in this thesis innovates in this thematic, once the design of ILs for protein stabilisation was bio-inspired in the high concentration of organic charged metabolites found in cell milieu. Although this perception is overlooked, those combined concentrations have been estimated to be ~300 mM among the macromolecules at concentrations exceeding 300 g/L (macromolecular crowding) and transient ion-pair can naturally occur with a potential specific biological role. Hence the main objective of this work is to develop new bio-ILs with a detectable ion-pair and understand its effects on protein structure and stability, under crowding environment, using advanced NMR techniques and calorimetric techniques. The choline-glutamate ([Ch][Glu]) IL was synthesized and characterized. The ion-pair was detected in water solutions using mainly the selective NOE NMR technique. Through the same technique, it was possible to detect a similar ion-pair promotion under synthetic and natural crowding environments. Using NMR spectroscopy (protein diffusion, HSQC experiments, and hydrogen-deuterium exchange) and differential scanning calorimetry (DSC), the model protein GB1 (production and purification in isotopic enrichment media) it was studied in the presence of [Ch][Glu] under macromolecular crowding conditions (PEG, BSA, lysozyme). Under dilute condition, it is possible to assert that the [Ch][Glu] induces a preferential hydration by weak and non-specific interactions, which leads to a significant stabilisation. On the other hand, under crowding environment, the [Ch][Glu] ion pair is promoted, destabilising the protein by favourable weak hydrophobic interactions , which disrupt the hydration layer of the protein. However, this capability can mitigates the effect of protein crowders. Overall, this work explored the ion-pair existence and its consequences on proteins in conditions similar to cell milieu. In this way, the charged metabolites found in cell can be understood as key for protein stabilisation.