Establishing a cell biology platform: isolation and preservation of human blood products

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Experimental Gravimetric Adsorption Equilibrium of n-Alkanes and Alkenes, Carbon Dioxide and Nitrogen in MIL-53(Al) and Zeolite 5A

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Aqueous biphasic system based on cholinium ionic liquids: extraction of biologically active phenolic acids

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Due to their biological and pharmacological properties, they have been playing an important role in phytotherapy and consequently techniques for their separation and purification are in need. This thesis aims at exploring new sustainable separation processes based on ionic liquids (ILs) in the extraction of biologically active phenolic acids. For that purpose, three phenolic acids with similar chemical structures were selected: cinnamic acid, p-coumaric acid and caffeic acid. In the last years, it has been shown that ionic liquids-based aqueous biphasic systems (ABSs) are valid alternatives for the extraction, recovery and purification of biomolecules when compared to conventional ABS or extractions carried out with organic solvents. In particular, cholinium-based ILs represent a clear step towards a greener chemistry, while providing means for the implementation of efficient techniques for the separation and purification of biomolecules. In this work, ABSs were implemented using cholinium carboxylate ILs using either high charge density inorganic salt (K3PO4) or polyethylene glycol (PEG) to promote the phase separation of aqueous solutions containing three different phenolic acids. These systems allow for the evaluation of effect of chemical structure of the anion on the extraction efficiency. Only one imidazolium-based IL was used in order to establish the effect of the cation chemical structure. The selective extraction of one single acid was also researched. Overall, it was observed that phenolic acids display very complex behaviours in aqueous solutions, from dimerization to polymerization and also hetero-association are quite frequent phenomena, depending on the pH conditions. These phenomena greatly hinder the correct quantification of these acids in solution.

Molecular simulation of gas adsorption equilibria in nanoporous materials

This work is divided into two distinct parts. The first part consists of the study of the metal organic framework UiO-66Zr, where the aim was to determine the force field that best describes the adsorption equilibrium properties of two different gases, methane and carbon dioxide. The other part of the work focuses on the study of the single wall carbon nanotube topology for ethane adsorption; the aim was to simplify as much as possible the solid-fluid force field model to increase the computational efficiency of the Monte Carlo simulations. The choice of both adsorbents relies on their potential use in adsorption processes, such as the capture and storage of carbon dioxide, natural gas storage, separation of components of biogas, and olefin/paraffin separations. The adsorption studies on the two porous materials were performed by molecular simulation using the grand canonical Monte Carlo (μ,V,T) method, over the temperature range of 298-343 K and pressure range 0.06-70 bar. The calibration curves of pressure and density as a function of chemical potential and temperature for the three adsorbates under study, were obtained Monte Carlo simulation in the canonical ensemble (N,V,T); polynomial fit and interpolation of the obtained data allowed to determine the pressure and gas density at any chemical potential. The adsorption equilibria of methane and carbon dioxide in UiO-66Zr were simulated and compared with the experimental data obtained by Jasmina H. Cavka et al. The results show that the best force field for both gases is a chargeless united-atom force field based on the TraPPE model. Using this validated force field it was possible to estimate the isosteric heats of adsorption and the Henry constants. In the Grand-Canonical Monte Carlo simulations of carbon nanotubes, we conclude that the fastest type of run is obtained with a force field that approximates the nanotube as a smooth cylinder; this approximation gives execution times that are 1.6 times faster than the typical atomistic runs.

Valorization of olive pomace through combination of biocatalysis with supercritical fluid technology

A supercritical carbon dioxide (scCO2) based oil extraction method was implemented on olive pomace (alperujo), and an oil yield of 25,5 +/- 0,8% (goil/gdry residue) was obtained. By Soxhlet extraction with hexane, an oil extraction yield of 28,9 +/- 0,8 % was obtained, which corresponds to an efficiency of 88,4 +/- 4,8 % for the supercritical method. The scCO2 extraction process was optimized for operating conditions of 50 MPa and 348,15 K, for which an oil loading of 32,60 g oil/kg CO2 was calculated. As a proof of concept, olive pomace was used as feedstock for biodiesel production, in a process combining the use of lipase as a catalyst with the use of scCO2 as a solvent, and integrating the steps of oil extraction, oil to biodiesel transesterification and subsequent separation of the latter. In the conducted experiments, FAME (fatty acid methyl ester) purities of 90% were obtained, with the following operating parameters: an oil:methanol molar ratio of 1:24; a residence time of 7,33 and 11,6 mins; a pressure of 40 MPa; a temperature of 313,15 K; and Lipozyme (Mucor miehei; Sigma-Aldritch) as an enzyme. However, oscillations of FAME purity were registered throughout the experiments, which could possibly be due to methanol accumulation in the enzymatic reactor. Finally, the phenolic content of olive pomace, and the effect of the drying process – oven or freeze-drying – and the extraction methods – hydro-alcoholic method and supercritical method – on the phenolic content were analysed. It was verified that the oven-drying process on the olive pomace preserved 90,1 +/- 3,6 % of the total phenolic content. About 62,3 +/- 5,53% of the oven-dried pomace phenolic content was extracted using scCO2 at 60 MPa and 323,15 K. Seven individual phenols – hydroxytyrosol, tyrosol, oleuropein, quercetin, caffeic acid, ferulic acid and p-coumaric acid – were identified and quantified by HPLC.

The determinants of net interest margins & net interest spreads in the Russian & Japanese Commercial Banking sectors

This study presents an empirical investigation of the determinants of net interest margins and spreads in the Russian and Japanese banking sectors with a particular focus on commercial banks. Net interest mar-gins and spreads serve as indicators of financial intermediation efficiency. This paper employed a bank-level unbalanced panel dataset prolonging from 2005 to 2014. My main empirical results show that bank characteristics explain the most of the variation in not only net interest margins but also in spreads. Capi-talization, liquidity risk, inflation, economic growth, private and government debt are important determi-nants of margin in Russia. In Japan to the contrary loan and deposit market concentration along with bank size do predominate. Common significant variables in both countries are the substitution effect, cost effi-ciency and profitability. Turning to net interest spreads, micro- and macro-specific variables are the main significant drivers in Russia. I reach the conclusion that there are no significant determinants of net interest spreads in Japan within the original selection of variables, but operating efficiency and deposits to total funding seem to prevail. In both countries, there are solid differences in the net interest margins as well as spreads once the pre- and the post-crisis periods are considered.

Photochemical synthesis and functional transformations of bicyclic vinyl aziridines

Aziridines, a class of organic compounds containing a three membered heterocycle with a nitrogen atom, are extremely valuable molecules in organic and medicinal chemistry. They are frequently used as versatile precursors in the synthesis of natural products, and many biologically active molecules possess the aziridine moiety. The reactivity of aziridines has been studied, for example, in ring-opening reactions with thiols. However, not much interest seems to be given to reactions of aziridines in aqueous media, despite the numberless advantages of using water as solvent in organic chemistry. The nucleophilic ring-opening reaction of aziridines in aqueous media was here explored. Following the Kaplan aziridine synthetic methodology, in which pyridinium salts undergo a photochemical transformation to give bicyclic vinyl aziridines, new aziridines were synthetized. Their nucleophilic ring-opening reaction in water under physiological conditions was investigated and a range of sulphur, nitrogen, carbon and oxygen nucleophiles tested. Thiols, anilines and azide proved to be good nucleophiles to react with the aziridines, giving the ring-opening product in moderate to good yields. The best results were obtained with thiols, more specifically with cysteine-derived nucleophiles. Preliminary results show that these bicyclic vinyl aziridines can modify calcitonin, a peptide containing two cysteine amino acids residues, grating them the potential to be used in bioconjugation as ligands to cysteine-containing proteins, or even as enzyme inhibitors of, for example, cysteine proteases. Additionally, exploratory investigations suggest that the separation of both enantiomers of the bicyclic vinyl aziridine can be performed by taking advantage of an enzymatic methodology for the resolution of racemic secondary alcohols. Both enantiomers would be highly valuable as precursors in the synthesis of enantiomerically pure molecules, as no other method is currently reported for their separation.

Biotransformation/separation routes to obtain pure enantiomers

The objective of the work presented in this thesis was the development of an innovative approach for the separation of enantiomers of secondary alcohols, combining the use of an ionic liquid (IL) - both as solvent for conducting enzymatic kinetic resolution and as acylating agent - with the use of carbon dioxide (CO2) as solvent for extraction. Menthol was selected for testing this reaction/separation approach due to the increasing demand for this substance, which is widely used in the pharmaceutical, cosmetics and food industries. With a view to using an ionic ester as acylating agent, whose conversion led to the release of ethanol, and due to the need to remove this alcohol so as to drive reaction equilibrium forward, a phase equilibrium study was conducted for the ehtanol/(±)-menthol/CO2 system, at pressures between 8 and 10 MPa and temperatures between 40 and 50 oC. It was found that CO2 is more selective towards ethanol, especially at the lowest pressure and highest temperature tested, leading to separation factors in the range 1.6-7.6. The pressure-temperature-composition data obtained were correlated with the Peng-Robinson equation of state and the Mathias-Klotz-Prausnitz mixing rule. The model fit the experimental results well, with an average absolute deviation (AAD) of 3.7 %. The resolution of racemic menthol was studied using two lipases, namely lipase from Candida rugosa (CRL) and immobilized lipase B from Candida antarctica (CALB), and two ionic acylating esters. No reaction was detected in either case. (R,S)-1-phenylethanol was used next, and it was found that with CRL low, nonselective, conversion of the alcohol took place, whereas CALB led to an enantiomeric excess (ee) of the substrate of 95%, at 30% conversion. Other acylating agents were tested for the resolution of (±)-menthol, namely vinyl esters and acid anhydrides, using several lipases and varying other parameters that affect conversion and enantioselectivity, such as substrate concentration, solvent and temperature. One such acylating agent was propionic anhydride. It was thus performed a phase equilibrium study on the propionic anhydride/CO2 system, at temperatures between 35 and 50 oC. This study revealed that, at 35 oC and pressures from 7 MPa, the system is monophasic for all compositions. The enzymatic catalysis studies carried out with propionic anhydride revealed that the extent of noncatalyzed reaction was high, with a negative effect on enantioselectivity. These studies showed also that it was possible to reduce considerably the impact of the noncatalyzed reaction relative to the reaction catalyzed by CRL by lowering temperature to 4 oC. Vinyl decanoate was shown to lead to the best results at conditions amenable to a process combining the use of supercritical CO2 as agent for post-reaction separation. The use of vinyl decanoate in a number of IL solvents, namely [bmim][PF6], [bmim][BF4], [hmim][PF6], [omim][PF6], and [bmim][Tf2N], led to an enantiomeric excess of product (eep) values of over 96%, at about 50% conversion, using CRL. In n-hexane and supercritical CO2, reaction progressed more slowly.(...)

Assessment of NDVI, land surface temperature and precipitation anomalies for drought monitoring in Bayankhongor province, Mongolia

During the last decade Mongolia’s region was characterized by a rapid increase of both severity and frequency of drought events, leading to pasture reduction. Drought monitoring and assessment plays an important role in the region’s early warning systems as a way to mitigate the negative impacts in social, economic and environmental sectors. Nowadays it is possible to access information related to the hydrologic cycle through remote sensing, which provides a continuous monitoring of variables over very large areas where the weather stations are sparse. The present thesis aimed to explore the possibility of using NDVI as a potential drought indicator by studying anomaly patterns and correlations with other two climate variables, LST and precipitation. The study covered the growing season (March to September) of a fifteen year period, between 2000 and 2014, for Bayankhongor province in southwest Mongolia. The datasets used were MODIS NDVI, LST and TRMM Precipitation, which processing and analysis was supported by QGIS software and Python programming language. Monthly anomaly correlations between NDVI-LST and NDVI-Precipitation were generated as well as temporal correlations for the growing season for known drought years (2001, 2002 and 2009). The results show that the three variables follow a seasonal pattern expected for a northern hemisphere region, with occurrence of the rainy season in the summer months. The values of both NDVI and precipitation are remarkably low while LST values are high, which is explained by the region’s climate and ecosystems. The NDVI average, generally, reached higher values with high precipitation values and low LST values. The year of 2001 was the driest year of the time-series, while 2003 was the wet year with healthier vegetation. Monthly correlations registered weak results with low significance, with exception of NDVI-LST and NDVI-Precipitation correlations for June, July and August of 2002. The temporal correlations for the growing season also revealed weak results. The overall relationship between the variables anomalies showed weak correlation results with low significance, which suggests that an accurate answer for predicting drought using the relation between NDVI, LST and Precipitation cannot be given. Additional research should take place in order to achieve more conclusive results. However the NDVI anomaly images show that NDVI is a suitable drought index for Bayankhongor province.