Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis.

Microbiology 154 (2008) 2719-2729

Bioactive beads for local sensing of proteases in 3D engineered tissues

Dissertação para obtenção do Grau de Doutor em Bioengenharia (MIT)

Old cellulose for new multifunctional networks

Dissertação para obtenção do Grau de Doutor em Ciência e Engenharia de Materiais

Assessment of a novel Cu (II) complex as a potential anticancer agent

Widely used in cancer treatment, chemotherapy still faces hindering challenges, ranging from severe induced toxicity to drug resistance acquisition. As means to overcome these setbacks, newly synthetized compounds have recently come into play with the basis of improved pharmacokinetic/pharmacodynamic properties. With this mind-set, this project aimed towards the antiproliferative potential characterization of a group of metallic compounds. Additionally the incorporation of the compounds within a nanoformulation and within new combination strategies with commercial chemotherapeutic drugs was also envisaged. Cell viability assays presented copper (II) compound (K4) as the most promising, presenting an IC50 of 6.10 μM and 19.09 μM for HCT116 and A549 cell line respectively. Exposure in fibroblasts revealed a 9.18 μM IC50. Hoechst staining assays further revealed the compound’s predisposition to induce chromatin condensation and nuclear fragmentation in HCT116 upon exposure to K4 which was later demonstrated by flow cytometry and annexin V-FITC/propidium iodide double staining analysis (under 50 % cell death induction). The compound further revealed the ability to interact with major macromolecules such as DNA (Kb = 2.17x105 M-1), inducing structural brakes and retardation, and further affecting cell cycle progression revealing delay in S-phase. Moreover BSA interactions were also visible however not conclusive. Proteome profiling revealed overexpression of proteins involved in metabolic activity and underexpression of proteins involved in apoptosis thus corroborating Hoechst and apoptosis flow cytometry data. K4 nanoformulation suffered from several hindrances and was ill succeeded in part due to K4’s poor solubility in aqueous buffers. Other approaches were considered in this regard. Combined chemotherapy assays revealed high cytotoxicity for afatinib and lapatinib strategies. Lapatinib and K4 proteome profiling further revealed high apoptosis rates, high metabolic activity and activation of redundant proteins as part of compensatory mechanisms.

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.