Clinical and Molecular Characterization of Diastrophic Dysplasia in the Portuguese Population

SLC26A2-related dysplasias encompass a spectrum of diseases: from lethal achondrogenesis type 1B (ACG1B; MIM #600972) and atelosteogenesis type 2 (AO2; MIM #256050) to classical diastrophic dysplasia (cDTD; MIM #222600) and recessive multiple epiphyseal dysplasia (rMED; MIM #226900). This study aimed at characterizing clinically, radiologically and molecularly 14 patients affected by non-lethal SLC26A2-related dysplasias and at evaluating genotype-phenotype correlation. Phenotypically, eight patients were classified as cDTD, four patients as rMED and two patients had an intermediate phenotype (mild DTD - mDTD, previously 'DTD variant'). The Arg279Trp mutation was present in all patients, either in homozygosity (resulting in rMED) or in compound heterozygosity with the known severe alleles Arg178Ter or Asn425Asp (resulting in DTD) or with the mutation c.727-1G>C (causing mDTD). The 'Finnish mutation', c.-26+2T>C, and the p.Cys653Ser, both frequent mutations in non-Portuguese populations, were not identified in any of the patients of our cohort and are probably very rare in the Portuguese population. A targeted mutation analysis for p.Arg279Trp and p.Arg178Ter in the Portuguese population allows the identification of approximately 90% of the pathogenic alleles.

Food waste valorization through the production of polyhydroxyalkanoates by mixed microbial cultures

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

Development of chitosan-based microparticles for pulmonary drug delivery

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

Exopolysaccharide production by Enterobacter A47: optimization of cultivation conditions and study of polymer functional properties

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology

On the way towards the understanding of suberin degradation by Aspergillus nidulans

Dissertation presented to obtain the Ph.D degree in Biochemistry.

Smart windows with permanent memory effect

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

Constraint-based modelling of mixed microbial populations: Application to polyhydroxyalkanoates production

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

Design and characterization of Chitin- Glucan polymeric structures for wound dressing materials

The main objective of this work was the development of polymeric structures, gel and films, generated from the dissolution of the Chitin-Glucan Complex (CGC) in biocompatible ionic liquids for biomedical applications. Similar as chitin, CGC is only soluble in some special solvents which are toxic and corrosive. Due to this fact and the urgent development of biomedical applications, the need to use biocompatible ionic liquids to dissolve the CGC is indispensable. For the dissolution of CGC, the biocompatible ionic liquid used was Choline acetate. Two different CGC’s, KiOnutrime from KitoZyme and biologically produced CGC from Faculdade de Ciencias e Tecnologia (FCT) - Universidade Nova de Lisboa, were characterized in order to develop biocompatible wound dressing materials. The similar result is shown in term of the ratio of chitin:glucan, which is 1:1.72 for CGC-FCT and 1:1.69 for CGC-Commercial. For the analysis of metal element content, water and inorganic salts content and protein content, both polymers showed some discrepancies, where the content in CGC-FCT is always higher compared to the commercial one. The different characterization results between CGC-FCT and CGC-Commercial could be addressed to differences in the purification method, and the difference of its original strain yeast, whereas CGC-FCT is derived from P.pastoris and the commercial CGC is from A.niger. This work also investigated the effect of biopolymers, temperature dissolution, non-solvent composition on the characteristics of generated polymeric structure with biocompatible ionic liquid. The films were prepared by casting a polymer mixture, immersion in a non-solvent, followed by drying at ambient temperature. Three different non-solvents were tested in phase inversion method, i.e. water, methanol, and glycerol. The results indicate that the composition of non-solvent in the coagulation bath has great influence in generated polymeric structure. Water was found to be the best coagulant for producing a CGC polymeric film structure. The characterizations that have been done include the analysis of viscosity and viscoelasticity measurement, as well as sugar composition in the membrane and total sugar that was released during the phase inversion method. The rheology test showed that both polymer mixtures exhibit a non- Newtonian shear thinning behaviour. Where the viscosity and viscoelasticity test reveal that CGCFCT mixture has a typical behaviour of a viscous solution with entangled polymer chains and CGCCommercial mixture has true gel behaviour. The experimental results show us that the generated CGC solution from choline acetate could be used to develop both polymeric film structure and gel. The generated structures are thermally stable at 100° C, and are hydrophilic. The produced films have dense structure and mechanical stabilities against puncture up to 60 kPa.

Absence of experimental cross-protection induced by a Trypanosoma cruzi-like strain isolated from bats

This study evaluated the possibility of inoculation and reinoculation with a trypanosomatid isolated from bats that is morphologically, biologically and molecularly similar to Trypanosoma cruzi, to protect against infection by virulent strains. Non-isogenic mice were divided into 24 groups that received from zero to three inoculations of Trypanosoma cruzi-like strain RM1, in the presence or absence of Freund's adjuvant, and were challenged with the VIC or JG strains of Trypanosoma cruzi. Parasitemia and survival were monitored and animals were sacrificed for histopathological analysis. Animals immunized with Trypanosoma cruzi-like strain RM1 presented decreased parasitemia, independently of the number of inoculations or the presence of adjuvant. In spite of this reduction, these animals did not present any protection against histopathological lesions. Severe eosinophilic infiltrate was observed and was correlated with the number of inoculations of Trypanosoma cruzi-like strain RM1. These findings suggest that prior inoculation with this strain did not protect against infection but, rather, aggravated the tissue inflammatory process.

Double-walled PLLA/PLGA particles for the sustained release of Meloxicam: preparation, characterisation and contolled release studies

There were two main objectives in this thesis investigation, first, the production, characterisation, in vitro degradation and release studies of double walled microspheres for drug release control. The second one, and the most challenging, was the production of double walled nanospheres, also for drug control delivery. The spheres were produced using two polymers, the Poly(L-lactide)Acid, PLLA, and the Poly(L-lactide-co-glycolic)Acid, PLGA.Afterwards, a model drug, Meloxicam, which is an antiinflammatory drug, was encapsulated into the particles. Micro and nanospheres were produced by the solvent extraction/evaporation method, where perfect spherical particles were obtained. By varying the polymers PLLA/PLGA mass ratio, different core and shell composition, as well as several shell and core thickness were observed. In the particles with a PLLA/PLGA mass ratio 1:1, the shell is composed by PLLA and the core by PLGA. It was also verified that the Meloxicam has a tendency to be distributed in the PLGA layer. Micro and nanoparticles were characterised in morphology, size, polymer cristalinity properties and drug distribution. Particles degradation studies was performed, where the particles in a PVA solution of pH 7,4 where placed in an incubator, during approximately 40 days, at 120rpm, and 37ºC, simulating, as much as possible, the human body environment. From these studies, the conclusion was that particles containing a PLGA shell and a PLLA core degrade more rapidly, due to the fact that PLLA is more hydrophobic than the PLGA. Concerning the drug release controlled results, done also for 40 and 50 days, they showed that the microspheres containing a shell of PLLA release more slowly than when the shell is composed of PLGA. This result was predictable, since the drug is solubilised in the PLGA polymer and so, in that case, the PLLA shell works like a barrier between the drug and the outer medium. Another positive aspect presented by this study is the lower initial burst effect, obtained when using double walled particles, which is one of the advantages of the same. In a second part of this investigation, the production of the nanospheres was the main goal, since it was not yet accomplished by other authors or investigators. After several studies, referring to the speed, time and type of agitation, as well as, the concentration and volume of the first aqueous solution of poly-vinyl-alcohol (PVA) during the process of solvent extraction/evaporation it was possible to obtain double walled nanospheres.(...)

Fiber functionalization for biomedical applications

Deep-eutectic solvents (DES) are considered novel renewable and biodegradable solvents, with a cheap and easy synthesis, without waste production. Later it was discovered a new subclass of DES that even can be biocompatible, since their synthesis uses primary metabolites such as amino acids, organic acids and sugars, from organisms. This subclass was named natural deep-eutectic solvents (NADES). Due to their properties it was tried to study the interaction between these solvents and biopolymers, in order to produce functionalized fibers for biomedical applications. In this way, fibers were produced by using the electrospinning technique. However, it was first necessary to study some physical properties of NADES, as well as the influence of water in their properties. It has been concluded that the water has a high influence on NADES properties, which can be seen on the results obtained from the rheology and viscosity studies. The fluid dynamics had changed, as well as the viscosity. Afterwards, it was tested the viability of using a starch blend. First it was tested the dissolution of these biopolymers into NADES, in order to study the viability of their application in electrospinning. However the results obtained were not satisfactory, since the starch polymers studied did not presented any dissolution in any NADES, or even in organic solvents. In this way it was changed the approach, and it was used other biocompatible polymers. Poly(ethylene oxide), poly(vinyl alcohol) and gelatin were the others biopolymers tested for the electrospinning, with NADES. All polymers show good results, since it was possible to obtain fibers. However for gelatin it was used only eutectic mixtures, containing active pharmaceutical ingredients (API’s), instead of NADES. For this case it was used mandelic acid (antimicrobial properties), choline chloride, ibuprofen (anti-inflammatory properties) and menthol (analgesic properties). The polymers and the produced fibers were characterized by scanning electron microscope (SEM), Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With the help of these techniques it was possible to conclude that it was possible to encapsulate NADES within the fibers. Rheology it was also study for poly(ethylene oxide) and poly(vinyl alcohol), in a way to understand the influence of polymer concentration, on the electrospinning technique. For the gelatin, among the characterization techniques, it was also performed cytotoxicity and drug release studies. The gelatin membranes did not show any toxicity for the cells, since their viability was maintained. Regarding the controlled release profile experiment no conclusion could be drawn from the experiments, due to the rapid and complete dissolution of the gelatin in the buffer solution. However it was possible to quantify the mixture of choline chloride with mandelic acid, allowing thus to complete, and confirm, the information already obtained for the others characterization technique.

Biological effects of acrylic engineered particulate-systems

Polymeric particulate-systems are of great relevance due to their possible biomedical applications, among them as carriers for the nano- or microencapsulation of drugs. However, due to their unique specific properties, namely small size range, toxicity issues must be discarded before allowing its use on health-related applications. Several polymers, as poly(methyl methacrylate) (PMMA), have proved to be suitable for the preparation of particulate-systems. However, a major drawback of its use refers to incomplete drug release from particles matrix. Recent strategies to improve PMMA release properties mention the inclusion of other acrylic polymers as Eudragit (EUD) on particles formulation. Though PMMA and EUD are accepted by the FDA as biocompatible, their safety on particle composition lacks sufficient toxicological data. The main objective of this thesis was to evaluate the biological effects of engineered acrylic particulate-systems. Preparation, physicochemical characterization and in vitro toxicity evaluation were assessed on PMMA and PMMA-EUD (50:50) particles. The emulsification-solvent evaporation methodology allowed the preparation of particles with spherical and smooth surfaces within the micrometer range (±500 nm), opposing surface charges and different levels of hydrophobicity. It was observed that particles physicochemical properties (size and charge) were influenced by biological media composition, such as serum concentration, ionic strength or pH. In what concerns to the in vitro toxicological studies, particle cellular uptake was observed on different cell lines (macrophages, osteoblasts and fibroblasts). Cytotoxicity effects were only found after 72 h of cells exposure to the particles, while no oxidative damage was observed neither on osteoblasts nor fibroblasts. Also, no genotoxicity was found in fibroblast using the comet assay to assess DNA damage. This observation should be further confirmed with other validated genotoxicity assays (e.g. Micronucleus Assay). The present study suggests that the evaluated acrylic particles are biocompatible, showing promising biological properties for potential use as carriers in drug-delivery systems.

Cellulose nanorods in liquid crystalline elastomers for improved actuators

Nanotechnology plays a central role in ‘tailoring’ materials’ properties and thus improving its performances for a wide range of applications. Coupling nature nano-objects with nanotechnology results in materials with enhanced functionalities. The main objective of this master thesis was the synthesis of nanocrystalline cellulose (NCCs) and its further incorporation in a cellulosic matrix, in order to produce a stimuli-responsive material to moisture. The induced behaviour (bending/unbending) of the samples was deeply investigated, in order to determine relationships between structure/properties. Using microcrystalline cellulose as a starting material, acid hydrolysis was performed and the NCC was obtained. Anisotropic aqueous solutions of HPC and NCC were prepared and films with thicknesses ranging from 22μm to 61μm were achieved, by using a shear casting technique. Microscopic and spectroscopic techniques as well as mechanical and rheological essays were used to characterize the transparent and flexible films produced. Upon the application of a stimulus (moisture), the bending/unbending response times were measured. The use of NCC allowed obtaining films with response times in the order of 6 seconds for the bending and 5 seconds for the unbending, improving the results previously reported. These promising results open new horizons for building up improved soft steam engines.

Synthesis of Polymeric Nanoparticles for biomedical delivery applications

Polymeric nanoparticles (PNPs) have attracted considerable interest over the last few years due to their unique properties and behaviors provided by their small size. Such materials could be used in a wide range of applications such as diagnostics and drug delivery. Advantages of PNPs include controlled release, protection of drug molecules and its specific targeting, with concomitant increasing of the therapeutic index. In this work, novel sucrose and cholic acid based PNPs were prepared from different polymers, namely polyethylene glycol (PEG), poly(D,L-lactic-co-glycolic acid) (PLGA) and PLGA-co-PEG copolymer. In these PNP carriers, cholic acid will act as a drug incorporation site and the carbohydrate as targeting moiety. The uptake of nanoparticles into cells usually involves endocytotic processes, which depend primarily on their size and surface characteristics. These properties can be tuned by the nanoparticle preparation method. Therefore, the nanoprecipitation and the emulsion-solvent evaporation method were applied to prepare the PNPs. The influence of various parameters, such as concentration of the starting solution, evaporation method and solvent properties on the nanoparticle size, size distribution and morphology were studied. The PNPs were characterized by using atomic force microscopy (AFM), scanning electron microscopy (SEM) and dynamic light scattering (DLS) to assess their size distribution and morphology. The PNPs obtained by nanoprecipitation ranged in size between 90 nm and 130 nm with a very low polydispersity index (PDI < 0.3). On the other hand, the PNPs produced by the emulsion-solvent evaporation method revealed particle sizes around 300 nm with a high PDI value. More detailed information was found in AFM and SEM images, which demonstrated that all these PNPs were regularly spherical. ζ-potential measurements were satisfactory and evidenced the importance of sucrose moiety on the polymeric system, which was responsible for the obtained negative surface charge, providing colloidal stability. The results of this study show that sucrose and cholic acid based polymeric conjugates can be successfully used to prepare PNPs with tunable physicochemical characteristics. In addition, it provides novel information about the materials used and the methods applied. It is hoped that this work will be useful for the development of novel carbohydrate based nanoparticles for biomedical applications, specifically for targeted drug delivery.