The additive effects in matrix dispersed liquid crystals

Dissertation presented at Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa to obtain the degree of Master in Chemical and Biochemical Engineering

Influence of the molecular weight in mechanical properties and degradation kinetics of chitosan inverted colloidal crystals

Tissue engineering arises from the need to regenerate organs and tissues, requiring the development of scaffolds, which can provide an optimum environment for tissue growth. In this work, chitosan with different molecular weights was used to develop biodegradable 3D inverted colloidal crystals (ICC) structures for bone regeneration, exhibiting uniform pore size and interconnected network. Moreover, in vitro tests were conducted by studying the influence of the molecular weight in the degradation kinetics and mechanical properties. The production of ICC included four major stages: fabrication of microspheres; assembly into a cohesive structure, polymeric solution infiltration and microsphere removal. Chitosan’s degree of deacetylation was determined by infrared spectroscopy and molecular weight was obtained via capillary viscometry. In order to understand the effect of the molecular weight in ICC structures, the mass loss and mechanical properties were analyzed after degradation with lysozyme. Structure morphology observation before and after degradation was performed by scanning electron microscopy. Cellular adhesion and proliferation tests were carried out to evaluate ICC in vitro response. Overall, medium molecular weight ICC revealed the best balance in terms of mechanical properties, degradation rate, morphology and biological behaviour.

Superoxide reductase from the syphilis spirochete Treponema pallidum

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P21 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A ° , = 106.9 ) and diffracted beyond 1.60 A ° resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A ° , = 104.9 ) and diffracted beyond 1.55 A ° . A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator ( = 1.542 A ° ) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P21 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

In-situ X-ray diffraction studies during growth of Ni-Ti shape memory alloy films and their complementary ex-situ characterization

Shape Memory Alloy (SMA) Ni-Ti films have attracted much interest as functional and smart materials due to their unique properties. However, there are still important issues unresolved like formation of film texture and its control as well as substrate effects. Thus, the main challenge is not only the control of the microstructure, including stoichiometry and precipitates, but also the identification and control of the preferential orientation since it is a crucial factor in determining the shape memory behaviour. The aim of this PhD thesis is to study the optimisation of the deposition conditions of films of Ni-Ti in order to obtain the material fully crystallized at the end of the deposition, and to establish a clear relationship between the substrates and texture development. In order to achieve this objective, a two-magnetron sputter deposition chamber has been used allowing to heat and to apply a bias voltage to the substrate. It can be mounted into the six-circle diffractometer of the Rossendorf Beamline (ROBL) at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, enabling an in-situ characterization by X-ray diffraction(XRD) of the films during their growth and annealing. The in-situ studies enable us to identify the different steps of the structural evolution during deposition with a set of parameters as well as to evaluate the effect of changing parameters on the structural characteristics of the deposited film. Besides the in-situ studies, other complementary ex-situ characterization techniques such as XRD at a laboratory source, Rutherford backscattering spectroscopy(RBS), Auger electron spectroscopy (AES), cross-sectional transmission electron microscopy (X-TEM), scanning electron microscopy (SEM), and electrical resistivity (ER) measurements during temperature cycling have been used for a fine structural characterization. In this study, mainly naturally and thermally oxidized Si(100) substrates, TiN buffer layers with different thicknesses (i.e. the TiN topmost layer crystallographic orientation is thickness dependent) and MgO(100) single crystals were used as substrates. The chosen experimental procedure led to a controlled composition and preferential orientation of the films. The type of substrate plays an important role for the texture of the sputtered Ni-Ti films and according to the ER results, the distinct crystallographic orientations of the Ni-Ti films influence their phase transformation characteristics.

Structure to function studies in UDP-glucose dehydrogenases and nitroreductases

The thesis is divided into two parts corresponding to structural studies on two different proteins. The first part concerns the study of two UDP-glucose dehydrogenases (UGDs) from Sphingomonas elodea ATCC 31461 and Burkholderia cepacia IST 408, both involved in exopolysaccharide production. Their relevance arises because some of these bacterial exopolysaccharides are valuable as established biotechnological products, the former case, whilst others are highly problematic, when used by pathogens in biofilm formation over biological surfaces, as the latter case, namely in the human lungs. The goal of these studies is to increase our knowledge regarding UGDs structural properties, which can potentiate either the design of activity enhancers to respond to the increased demand of useful biofilms, or the design of inhibitors of biofilm production, in order to fight invading pathogens present in several infections. The thesis reports the production and crystallisation of both proteins, the determination of initial phases by single-wavelength anomalous dispersion (SAD) in S. elodea crystals using a seleno-methionine isoform, and phasing of B. cepacia crystals by molecular replacement (MR) using the S. elodea model, as well as the refinement, structural analysis and comparison between the several UGDs structures available during this work.(...)

Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-alpha-arabinanase from Bacillus subtilis.

Acta Crystallographica F64 (2008) 636-638

Historically accurate reconstructions and characterisation of chrome yellow pigments

Dissertation presented at the Faculty of Sciences and Technology of the New University of Lisbon in fulfillment of the requirements for the Master degree in Conservation Science

Crystallographic studies on molybdopterin-dependent enzymes

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from desulfovibrio gigas

Acta Crystallographica Section F Structural Biology and Crystallization Communications Volume 65, Part 8

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jul 1;64(Pt 7):593-5

Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis

Acta Cryst. (2007). F63, 516–519

Using gold nanoparticles in protein crystallography: studies in crystal growth and derivatization

Dissertation for the Master Degree in Structural and Functional Biochemistry

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

J Biol Inorg Chem (2011) 16:51–61 DOI 10.1007/s00775-010-0700-8

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448r

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

J Biol Inorg Chem (2007) 12:353–366 DOI 10.1007/s00775-006-0191-9