Development of Monoolein-based lipofection vectors for therapeutic siRNA delivery

Tese de Doutoramento em Biologia Molecular e Ambiental - Especialidade em Biologia Celular e Saúde

Cationic Liposomes as antigenic delivery systems: development of an immunoprotective strategy against Candida albicans

Tese de Doutoramento Biologia Molecular e Ambiental - Especialidade em Biologia Celular e Saúde

Enzymatic phosphorylation of hair keratin enhances fast adsorption of cationic moieties

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by 31P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 minutes for cationic moiety like Methylene Blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance.

Otimização do perfil farmacocinético e farmacodinâmico de fármacos anticancerígenos através da sua veiculação em nanossistemas lipídicos

Dissertação de mestrado em Biofísica e Bionanossistemas

Lipids under stress - a lipidomic approach for the study of mood disorders

The emerging field of lipidomics has identified lipids as key players in disease physiology. Their physicochemical diversity allows precise control of cell structure and signaling events through modulation of membrane prop- erties and trafficking of proteins. As such, lipids are important regulators of brain function and have been implicated in neurodegenerative and mood disorders. Importantly, environmental chronic stress has been associated with anxiety and depression and its exposure in rodents has been extensively used as a model to study these diseases. With the accessibility to modern mass- spectrometry lipidomic platforms, it is now possible to snapshot the extensively interconnected lipid network. Here, we review the fundamentals of lipid biology and outline a framework for the interpretation of lipidomic studies as a new approach to study brain pathophysiology. Thus, lipid profiling provides an exciting avenue for the identification of disease signatures with important implications for diagnosis and treatment of mood disorders.

Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...)

Biotechnological valorization of waste cooking oils: lipase and microbial lipids production by Yarrowia lipolytica

[Excerpt] Waste cooking oils (WCO) generated from vegetable oils used at high temperatures in food frying, cause environmental problems and must be reutilized. New strategies to valorize these wastes are attracting a great scientific interest due to the important advantages offered from an economic and environmental point of view. A microbial platform can be established to convert low-value hydrophobic substrates, such as waste cooking oils, to microbial lipids (single cell oil, SCO) and other value-added bioproducts, such as lipase. (...)

Energy production from lipids by novel anaerobes

[Excerpt] Anaerobic microbial diversity encloses a very high potential that can be used for biotechnological applications. This potential is still largely unexplored, since the majority of the microorganisms in Nature are unknown or poorly characterized. This work is focused on the study of novel anaerobic microorganisms that participate in the metabolism of lipids, long chain fatty acids (LCFA) and glycerol, with the main goal of producing valuable energy-rich organic compounds. For that, conventional anaerobic culturing procedures were combined with continuous bioreactors operation and allied to microbial ecology approaches. Two main examples of the work performed will be presented. (...)

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Phenanthroimidazoles as fluorimetric and colourimetric chemosensors in aqueous solution

2,4,5-Triaryl-imidazoles are versatile compounds with application in medicine, due to their biological activity, and materials sciences, for their interesting optical properties. These properties can be tuned by careful selection of substituents at positions 2, 4 and 5: replacement of the aryl group by an heterocyclic group results in larger π-conjugated systems with improved optical properties for application in nonlinear optics, OLEDs, DNA intercalators, and chemosensors. Moreover, it is expected that introducing more conjugation and rigidity into the resulting system will further improve its properties. The development of chromo/fluorescent probes that are capable of detecting ions with high sensitivity and selectivity in aqueous media is currently a topic of strong interest and the design of heteroditopic receptors that contain two or more different binding sites for the simultaneous complexation of cationic and anionic guests is a emerging field of supramolecular chemistry. In this communication, we report the synthesis of new phenanthroimidazoles substituted at position 2 with arylthienyl or arylfuryl moieties possessing substituents of different electronic character, in order to tune the chromo/fluoro response in the presence of relevant anions and metal cations. Their photophysical properties and chemosensory ability were studied in acetonitrile and mixtures of acetonitrile and water, and selective detection of cyanide was achieved in aqueous mixtures for some of the derivatives.

Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Assessment of liposome disruption to quantify drug delivery in vitro

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

Hydrocarbonoclastic bacteria: from bioremediation to bioenergy feedstock

Tese de Doutoramento em Engenharia Química e Biológica.

Development of folate-targeted liposomes for rheumatoid arthritis therapy

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).