Engineered MRI nanoprobes based on superparamagnetic iron oxide nanoparticles

This project aimed to engineer new T2 MRI contrast agents for cell labeling based on formulations containing monodisperse iron oxide magnetic nanoparticles (MNP) coated with natural and synthetic polymers. Monodisperse MNP capped with hydrophobic ligands were synthesized by a thermal decomposition method, and further stabilized in aqueous media with citric acid or meso-2,3-dimercaptosuccinic acid (DMSA) through a ligand exchange reaction. Hydrophilic MNP-DMSA, with optimal hydrodynamic size distribution, colloidal stability and magnetic properties, were used for further functionalization with different coating materials. A covalent coupling strategy was devised to bind the biopolymer gum Arabic (GA) onto MNPDMSA and produce an efficient contrast agent, which enhanced cellular uptake in human colorectal carcinoma cells (HCT116 cell line) compared to uncoated MNP-DMSA. A similar protocol was employed to coat MNP-DMSA with a novel biopolymer produced by a biotechnological process, the exopolysaccharide (EPS) Fucopol. Similar to MNP-DMSA-GA, MNP-DMSA-EPS improved cellular uptake in HCT116 cells compared to MNP-DMSA. However, MNP-DMSA-EPS were particularly efficient towards the neural stem/progenitor cell line ReNcell VM, for which a better iron dose-dependent MRI contrast enhancement was obtained at low iron concentrations and short incubation times. A combination of synthetic and biological coating materials was also explored in this project, to design a dynamic tumortargeting nanoprobe activated by the acidic pH of tumors. The pH-dependent affinity pair neutravidin/iminobiotin, was combined in a multilayer architecture with the synthetic polymers poy-L-lysine and poly(ethylene glycol) and yielded an efficient MRI nanoprobe with ability to distinguish cells cultured in acidic pH conditions form cells cultured in physiological pH conditions.

Bioproduction of succinic acid from renewable feedstocks by Actinobacillus succinogenes 130Z

Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)

Ursodeoxycholic acid does not interfere with in vivo Helicobacter pylori colonization

A low frequency of Helicobacter pylori in the gastric mucosa of patients with alkaline gastritis has been reported. At the same time, it can be noted that the growth of bacteria can be inhibited by bile acids. We studied 40 patients with chronic gastritis related to Helicobacter pylori in order to determine the effect of ursodeoxycholic acid on this infection. Diagnoses of the infection and the inflammatory process were obtained by histologic study of gastric biopsies collected during endoscopy. Two groups were studied: group I received ursodeoxycholic acid - 300 mg/day, and group II received the placebo, twice a day, both for 28 days. The colonization by Helicobacter pylori and the intensity of the mononuclear and polymorphonuclear inflammatory infiltrate were determined before (time 1) and after (time 2) treatment. Ursodeoxycholic acid had no effect on the Helicobacter pylori infection. A significant reduction in the intensity of the mononuclear inflammatory infiltrate of the gastric antrum mucosa was observed in patients from group I, when we compared not only times 1 and 2 but also groups I and II. However, this was not the case with the body mucosa. We concluded that ursodeoxycholic acid had no action on the colonization by Helicobacter pylori or on the polymorphonuclear inflammatory infiltrate, but it caused a significant reduction in the intensity of the mononuclear inflammatory infiltrate of the gastric antrum.

The effect of intravenous zoledronic acid on glucocorticoid-induced multiple vertebral fractures in juvenile systemic lupus erythematosus

Glucocorticoids are widely used in the treatment of lupus patients, and adverse effects, which include osteoporosis and associated fractures, are frequent. Treatment of osteoporosis of young patients should be effective and not harmful to bone growth and remodeling. Bisphosphonates are drugs that decrease the incidence of bone fractures, but their use in juvenile patients is still controversial because of their possible side effects on the growing skeleton. However, recently published studies showed that linear growth continued normally after treatment with these drugs, and there was no excessive suppression of bone remodeling or mineralization defects. Zoledronic acid is a new intravenous bisphosphonate that has been approved by the US FDA for use with hypercalcemia of malignancies and might be an effective treatment for postmenopausal osteoporosis. The authors report a case of a young girl with systemic lupus who developed multiple vertebral collapses due to glucocorticoid therapy, and zoledronic acid was used producing significant clinical and densitometric improvement.

Evaluation of antibacterial activity of caffeic acid encapsulated by -cyclodextrins

Context: Caffeic acid is described as antibacterial, but this bioactive molecule has some issues regarding solubility and stability to environmental stress. Thus, encapsulation devices are required. Objective: The aim of this work was to study the effect of the caffeic acid encapsulation by cyclodextrins on its antibacterial activity. Materials and methods: The interactions between the caffeic acid and three cyclodextrins (-cyclodextrin (CD), 2-hydroxypropyl--cyclodextrin (HPCD) and methyl--cyclodextrin were study. Results and discussion: The formation of an aqueous soluble inclusion complex was confirmed for CD and HPCD with a 1:1 stoichiometry. The CD/caffeic acid complex showed higher stability than HPCD/caffeic acid. Caffeic acid antibacterial activity was similar at pH 3 and pH 5 against the three bacteria (K. pneumoniae, S. epidermidis and S. aureus). Conclusions: The antibacterial activity of the inclusion complexes was described here for the first time and it was shown that the caffeic acid activity was remarkably enhanced by the cyclodextrins encapsulation.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Assessing the production of jet mix columns using alkali activated waste based on mechanical and financial performance and CO2 (eq) emissions

Recent research has proved the potential of alkaline activated fly-ash for soil stabilisation. However, such studies have not focused on the link between financial, mechanical and environmental aspects of this solution, but only on their absolute mechanical properties. The present paper characterises the mechanical behaviour of a large spectrum of activator-ash-soil combinations used to build jet mixing columns, analysing also the cost and CO2 (eq) emissions. The concern with these two vectors forced a decrease in the quantity of stabilising agent added to the soil, relatively to previous research, and the effects of such low quantities have not yet been published. However, the results clearly showed a significant improve in strength, still well above the average values expected when improving the stressstrain behaviour of a weak soil. Uniaxial compressive strength tests were used to assess the effects of the fly-ash percentage, the alkalieash ratio and the water content. The carbon calculator recently developed by the European Federation of Foundation Contractors and the Deep Foundations Institute was used to quantify the CO2 (eq) emissions associated with this technique. The financial cost was estimated based on the experience of a major Portuguese contractor. For comparison purposes, soil cement mixtures were also analysed, using similar conditions and tools used for the soil-ash analysis. Results showed that the cement and ash solutions are very similar in terms of overall performance, with some advantage of the former regarding financial cost, and a significant advantage of the latter regarding the CO2 (eq) emissions. This new grout, although it is in an embryonic stage, it has the potential for broader developments in the field.

Thermal characterization of polyhydroxyalkanoates and poly(lactic acid) blends obtained by injection molding

In this work we present the thermal characterization of the full scope of polyhydroxyalcanoate and poly(lactic acid) blends obtain by injection molding. Blends of polyhydroxyalcanoate and poly(lactic acid) (PHA/PLA) were prepared in different compositions ranging from 0–100% in steps of 10%. The blends were injection molded and then characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and wide angle X-ray diffraction (WAXD). The increment of PHA fraction increased the degree of crystallinity of the blend and the miscibility of the base polymers as verified by the Fox model. The WAXD analysis indicates that the presence of PHA hindered the PLA crystallization. The crystallization evolution trough PHA weight fraction (wf) shows a phase inversion around 50-60%. SEM analyses confirmed that the miscibility of PHA/PLA blends increased with the incorporation of PHA and became total for values of PHA higher that 50%.

Aminobenzocoumarinylmehtyl esters as photoactive precursors for the release of butyric acid

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2Hnaphtho[1,2-b] pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80:20 solutions as solvent. The data obtained showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. As well as the photolysis, the photophysics of the compounds were characterised by both steady state and time-resolved methods.

Microencapsulation of ellagic acid from pomegranate husk and karaya gum by spray drying

Objective: The aim of this study was to obtain and characterize microcapsules with Ellagic Acid (EA) from pomegranate as core material and Karaya Gum (KG) as wall material. Methods: EA was obtained from dry pomegranate peel powder via methanolysis and quantified by HPLC. Microcapsules were obtained preparing a dispersion containing KG and EA in phosphate buffer pH 8. The dispersion was processed in a spray dryer under specific conditions (inlet temperature at 150 °C, feed flow at 30% and aspirator at 100 %) for obtaining of microcapsules. Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were used for characterization. Results: Obtained material contains 98.03±2.82 mg EA/g of pomegranate peel. FTIR showed that there were changes in the molecular structure of microcapsules referred to raw materials. SEM confirmed that particles obtained had micron-size (1-5 µm). DSC analysis showed that raw materials had glass transition temperatures of 79.58 and 83.41 °C and for microcapsules the value was67.25 °C. Conclusion: Methanolysis is a viable technique for the obtaining of EA from the peel of pomegranate. KG shows good potential for be used as wall material for EA microencapsulation.

Photoactivation of butyric acid from 6-aminobenzocoumarin cages

A new benzocoumarin bearing an amino group is proposed as a photocleavable protecting group for carboxylic acids. The novel heterocycle, 6-amino-4-chloromethyl-2-oxo-2H-naphtho[1,2-b]pyran was used in the preparation of ester conjugates of butyric acid, and of the corresponding mono- and di-methylated or ethylated derivatives. The photolability of the ester conjugates was studied by irradiation at selected wavelengths in methanol/HEPES buffer (80:20) solutions, and the release of butyric acid was followed with HPLC/UV and 1H NMR monitoring. Release of the carboxylic acid was faster for the monoalkylated derivatives (approximately within 20 min), at the longer wavelengths of irradiation (350 and 419 nm). The photophysics of the heterocyclic conjugates was also evaluated by both steady state and time-resolved methods.

Cost-efficient one-part alkali-activated mortars with low global warming potential for floor heating systems applications

Increasing building energy efficiency is one the most cost-effective ways to reduce emissions. The use of thermal insulation materials mitigates heat loss in buildings, therefore minimising heat energy needs. In recent years, several papers were published on the subject of foam alkali-activated cements with enhanced thermal conductivity. However, on those papers cost analysis was strangely avoided. This paper presents experimental results on one-part alkali-activated cements. It also includes global warming potential assessment and cost analysis. Foam one-part alkali-activated cements cost simulations considering two carbon dioxide social costs scenarios are also included. The results show that one-part alkali-activated cements mixtures based on 26%OPC + 58.3%FA + 8%CS + 7.7%CH and 3.5% hydrogen peroxide constitute a promising cost-efficient (67 euro/m3), thermal insulation solution for floor heating systems. This mixture presents a low global warming potential of 443 KgCO2eq/m3. The results confirm that in both carbon dioxide social cost scenarios the mixture 26 OPC + 58.3 FA + 8 CS + 7.7 CH with 3.5% hydrogen peroxide foaming agent is still the most cost efficient.

Cheese whey: a cost-effective alternative for hyaluronic acid production by Streptococcus zooepidemicus

This study focuses on the optimization of cheese whey formulated media for the production of hyaluronic acid HA by Streptococcus zooepidemicus. Culture media containing whey (W; 2.1 g/L) or whey hydrolysate (WH; 2.4 g/L) gave the highest HA productions. Both W and WH produced high yields on protein consumed, suggesting cheese whey is a good nitrogen source for S. zooepidemicus production of HA. Polysaccharide concentrations of 4.0 g/L and 3.2 g/L were produced in W and WH in a further scale-up to 5 L bioreactors, confirming the suitability of the low-cost nitrogen source. Cheese whey culture media provided high molecular weight (> 3000 kDa) HA products. This study revealed replacing the commercial peptone by the low-cost alternative could reduce HA production costs by up to a 70%compared to synthetic media.

Development of hyaluronic acid, dextrin and extracellular matrix hydrogels for cell expansion

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Effect of acid or alkaline catalyst and of different capping agents on the optical properties of CdS nanoparticles incorporated within a diureasil hybrid matrix

CdS nanoparticles (NPs) were synthesized using colloidal methods and incorporated within a diureasil hybrid matrix. The surface capping of the CdS NPs by 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-aminopropyltrimethoxysilane (APTMS) organic ligands during the incorporation of the NPs within the hybrid matrix has been investigated. The matrix is based on poly(ethylene oxide)/poly(propylene oxide) chains grafted to a siliceous skeleton through urea bonds and was produced by sol–gel process. Both alkaline and acidic catalysis of the sol–gel reaction were used to evaluate the effect of each organic ligand on the optical properties of the CdS NPs. The hybrid materials were characterized by absorption, steady-state and time-resolved photoluminescence spectroscopy and High Resolution Transmission Electron Microscopy (HR-TEM). The preservation of the optical properties of the CdS NPs within the diureasil hybrids was dependent on the experimental conditions used. Both organic ligands (APTMS and MPTMS) demonstrated to be crucial in avoiding the increase of size distribution and clustering of the NPs within the hybrid matrix. The use of organic ligands was also shown to influence the level of interaction between the hybrid host and the CdS NPs. The CdS NPs showed large Stokes shifts and long average lifetimes, both in colloidal solution and in the xerogels, due to the origin of the PL emission in surface states. The CdS NPs capped with MPTMS have lower PL lifetimes compared to the other xerogel samples but still larger than the CdS NPs in the original colloidal solution. An increase in PL lifetimes of the NPs after their incorporation within the hybrid matrix is related to interaction between the NPs and the hybrid host matrix.