Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp strain NCIMB 12038

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear nonheme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 Angstrom, alpha = beta = gamma = 90degrees, and diffract to 2.3 Angstrom resolution.

Numerical study of the effects of reinforcement/matrix interphase on stress-strain behavior of YAl2 particle reinforced MgLiAl composites

For the potential influence produced by the reinforcement/matrix interphase in particle reinforced metal matrix composites (PMMCs), a unit cell model with transition interphase was proposed. Uniaxial tensile loading was simulated and the stress/strain behavior was predicted. The results show that a transition interphase with both appropriate strength and thickness could affect the failure mode, reduce the stress concentration, and enhance the maximum strain value of the composite.

Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies.

The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Epitaxial strain and electric boundary condition effects on the structural and ferroelectric properties of BiFeO3 films

The influence of both compressive and tensile epitaxial strain along with the electrical boundary conditions on the ferroelastic and ferroelectric domain patterns of bismuth ferrite films was studied. BiFeO3 films were grown on SrTiO3(001), DyScO3(110), GdScO3(110), and SmScO3(110) substrates to investigate the effect of room temperature in-plane strain ranging from -1.4% to +0.75%. Piezoresponse force microscopy, transmission electron microscopy, x-ray diffraction measurements, and ferroelectric polarization measurements were performed to study the properties of the films. We show that BiFeO3 films with and without SrRuO3 bottom electrode have different growth mechanisms and that in both cases reduction of the domain variants is possible. Without SrRuO3, stripe domains with reduced variants are formed on all rare earth scandate substrates because of their monoclinic symmetry. In addition, tensile strained films exhibit a rotation of the unit cell with increasing film thickness. On the other side, the presence of SrRuO3 promotes step flow growth of BiFeO3. In case of vicinal SrTiO3 and DyScO3 substrates with high quality SrRuO3 bottom electrode and a low miscut angle of approximate to 0.15 degrees we observed suppression of the formation of certain domain variants. The quite large in-plane misfit of SrRuO3 with GdScO3 and SmScO3 prevents the growth of high quality SrRuO3 films and subsequent domain variants reduction in BiFeO3 on these substrates, when SrRuO3 is used as a bottom electrode.

Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187

We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M. A. Valvano, and J. H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of beta-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.

A Burkholderia cenocepacia MurJ (MviN) homolog is essential for cell wall peptidoglycan synthesis and bacterial viability

The cell wall peptidoglycan (PG) of Burkholderia cenocepacia, an opportunistic pathogen, has not yet been characterized. However, the B. cenocepacia genome contains homologs of genes encoding PG biosynthetic functions in other bacteria. PG biosynthesis involves the formation of the undecaprenyl-pyrophosphate-linked N-acetyl glucosamine-N-acetyl muramic acid-pentapeptide, known as lipid II, which is built on the cytosolic face of the cell membrane. Lipid II is then translocated across the membrane and its glycopeptide moiety becomes incorporated into the growing cell wall mesh; this translocation step is critical to PG synthesis. We have investigated candidate flippase homologs of the MurJ family in B. cenocepacia. Our results show that BCAL2764, herein referred to as murJBc, is indispensable for viability. Viable B. cenocepacia could only be obtained through a conditional mutagenesis strategy by placing murJBc under the control of a rhamnose-inducible promoter. Under rhamnose depletion, the conditional strain stopped growing and individual cells displayed morphological abnormalities consistent with a defect in PG synthesis. Bacterial cells unable to express MurJBc underwent cell lysis, while partial MurJBc depletion sensitized the mutant to the action of β-lactam antibiotics. Depletion of MurJBc caused accumulation of PG precursors consistent with the notion that this protein plays a role in lipid II flipping to the periplasmic compartment. Reciprocal complementation experiments of conditional murJ mutants in B. cenocepacia and Escherichia coli with plasmids expressing MurJ from each strain indicated that MurJBc and MurJEc are functional homologs. Together, our results are consistent with the notion that MurJBc is a PG lipid II flippase in B. cenocepacia.

Influence of strain and polycrystalline ordering on magnetic properties of high moment rare earth metals and alloys

Despite being the most suitable candidates for solenoid pole pieces in state-of-the-art superconductor- based electromagnets, the intrinsic magnetic properties of heavy rare earth metals and their alloys have gained comparatively little attention. With the potential of integration in micro- and nanoscale devices, thin films of Gd, Dy, Tb, DyGd and DyTb were plasma-sputtered and investigated for their in-plane magnetic properties, with an emphasis on magnetisation vs. temperature profiles. Based on crystal structure analysis of the polycrystalline rare earth films, which consist of a low magnetic moment FCC layer at the seed interface topped with a higher moment HCP layer, an experimental protocol is introduced which allows the direct magnetic analysis of the individual layers. In line with the general trend of heavy lanthanides, the saturation magnetisation was found to drop with increasing unit cell size. In-situ annealed rare earth films exceeded the saturation magnetisation of a high-moment Fe65Co35 reference film in the cryogenic temperature regime, proving their potential for pole piece applications; however as-deposited rare earth films were found completely unsuitable. In agreement with theoretical predictions, sufficiently strained crystal phases of Tb and Dy did not exhibit an incommensurate magnetic order, unlike their single-crystal counterparts which have a helical phase. DyGd and DyTb alloys followed the trends of the elemental rare earth metals in terms of crystal structure and magnetic properties. Inter-rare-earth alloys hence present a desirable blend of saturation magnetisation and operating temperature.

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.

Optical and mechanical analysis on a biological cell in optical tweezers

La réponse mécanique d’une cellule à une force externe permet d’inférer sa structure et fonction. Les pinces optiques s’avèrent une approche particulièrement attrayante pour la manipulation et caractérisation biophysique sophistiquée des cellules de façon non invasive. Cette thèse explore l’utilisation de trois types de pinces optiques couramment utilisées : 1) statiques (static), 2) à exposition partagée (time-sharing) et 3) oscillantes (oscillating). L’utilisation d’un code basé sur la méthode des éléments finis en trois dimensions (3DFEM) nous permet de modéliser ces trois types de piégeage optique afin d’extraire les propriétés mécaniques cellulaires à partir des expériences. La combinaison des pinces optiques avec la mécanique des cellules requiert des compétences interdisciplinaires. Une revue des approches expérimentales sur le piégeage optique et les tests unicellulaires est présentée. Les bases théoriques liant l’interaction entre la force radiative optique et la réponse mécanique de la cellule aussi. Pour la première fois, une simulation adaptée (3DFEM) incluant la diffusion lumineuse et la distribution du stress radiatif permet de prédire la déformation d’une cellule biconcave –analogue aux globules rouges—dans un piège statique double (static dual-trap). À l’équilibre, on observe que la déformation finale est donnée par l’espacement entre les deux faisceaux lasers: la cellule peut être étirée ou même comprimée. L’exposition partagée (time-sharing) est la technique qui permet de maintenir plusieurs sites de piégeage simultanément à partir du même faisceau laser. Notre analyse quantitative montre que, même oscillantes, la force optique et la déformation sont omniprésentes dans la cellule : la déformation viscoélastique et la dissipation de l’énergie sont analysées. Une autre cellule-type, la tige cubique, est étudiée : cela nous permet d’élucider de nouvelles propriétés sur la symétrie de la réponse mécanique. Enfin, l’analyse de la déformation résolue en temps dans un piége statique ou à exposition partagée montre que la déformation dépend simultanément de la viscoélasticité, la force externe et sa forme tridimensionnelle. La technique à force oscillante (oscillating tweezers) montre toutefois un décalage temporel, entre la force et la déformation, indépendant de la forme 3D; cette approche donnerait directement accès au tenseur viscoélastique complexe de la cellule.

Enhanced bio-decolourisation of acid orange 7 by Shewanella oneidensis through co-metabolism in a microbial fuel cell

The decolourisation of acid orange 7 (AO7) (C.I.15510) through co-metabolism in a microbial fuel cell by Shewanella oneidensis strain 14063 was investigated with respect to the kinetics of decolourisation, extent of degradation and toxicity of biotransformation products. Rapid decolourisation of AO7 (>98% within 30 h) was achieved at all tested dye concentrations with concomitant power production. The aromatic amine degradation products were recalcitrant under tested conditions. The first-order kinetic constant of decolourisation (k) decreased from 0.709 ± 0.05 h−1 to 0.05 ± 0.01 h−1 (co-substrate – pyruvate) when the dye concentration was raised from 35 mg l−1 to 350 mg l−1. The use of unrefined co-substrates such as rapeseed cake, corn-steep liquor and molasses also indicated comparable or better AO7 decolourisation kinetic constant values. The fully decolourised solutions indicated increased toxicity as the initial AO7 concentration was increased. This work highlights the possibility of using microbial fuel cells to achieve high kinetic rates of AO7 decolourisation through co-metabolism with concomitant electricity production and could potentially be utilised as the initial step of a two stage anaerobic/aerobic process for azo dye biotreatment.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

The mechanism of the regulation of energy distribution between photosystems 1 and 2 in the cyanobacterium Synechococcus sp. strain PCC 7002 /

Cyanobacteria are able to regulate the distribution of absorbed light energy between photo systems 1 and 2 in response to light conditions. The mechanism of this regulation (the state transition) was investigated in the marine cyanobacterium Synechococcus sp. strain PCC 7002. Three cell types were used: the wild type, psaL mutant (deletion of a photo system 1 subunit thought to be involved in photo system 1 trimerization) and the apcD mutant (a deletion of a phycobilisome subunit thought to be responsible for energy transfer to photo system 1). Evidence from 77K fluorescence emission spectroscopy, room temperature fluorescence and absorption cross-section measurements were used to determine a model of energy distribution from the phycobilisome and chlorophyll antennas in state 1 and state 2. The data confirm that in state 1 the phycobilisome is primarily attached to PS2. In state 2, a portion of the phycobilisome absorbed light energy is redistributed to photo system 1. This energy is directly transferred to photo system 1 by one of the phycobilisome terminal emitters, the product of the apcD gene, rather than via the photo system 2 chlorophyll antenna by spillover (energy transfer between the photo system 2 and photo system 1 chlorophyll antenna). The data also show that energy absorbed by the photo system 2 chlorophyll antenna is redistributed to photo system 1 in state 2. This could occur in one of two ways; by spillover or in a way analogous to higher plants where a segment of the chlorophyll antenna is dissociated from photo system 2 and becomes part of the photo system 1 antenna. The presence of energy transfer between neighbouring photo system 2 antennae was determined at both the phycobilisome and chlorophyll level, in states 1 and 2. Increases in antenna absorption cross-section with increasing reaction center closure showed that there is energy transfer (connectivity) between photosystem 2 antennas. No significant difference was shown in the amount of connectivity under these four conditions.