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.

Effet ajouté et participation des pères à un programme d'entraînement aux habiletés parentales

Selon l'American Psychiatric Association (APA, 2004), les troubles du comportement perturbateur (TCP) se divisent en trois diagnostics : le trouble d'attention avec ou sans hyperactivité (TDA/H), le trouble d'opposition avec ou sans provocation (TOP) et le trouble des conduites (TC). Les programmes d'entraînement aux habiletés parentales (PEHP) sont reconnus comme étant des pratiques probantes dans le traitement de ces troubles (Hartman, Stage et Webster-Stratton, 2003; Kazdin, 2001; Nock & Kazdin, 2001; Webster-Stratton, 2007; Weersing & Weisz, 2000). Toutefois, les pères s'impliquent généralement moins que les mères dans ce type d'intervention alors que leur implication dans la vie de l'enfant est associée à de nombreux bénéfices (Coplin & Houts, 1991; Lamb, 2004). Les objectifs de cette étude sont de vérifier si la participation des pères au programme est similaire à celle des mères et de vérifier quel est l'effet ajouté de leur participation à l'efficacité d'un PEHP sur l'enfant, les parents et la famille. Au total, 33 familles ont participé au programme et leur présence est justifiée par le TDA/H de leur enfant. Le groupe "sans père" (SP) est formé de 9 mères qui sont venues seules aux rencontres et le groupe "avec pères" (AP) est formé de 24 pères et mères venus ensemble. Ils ont participé au programme Ces années incroyables qui inclut 16 rencontres de groupe de deux heures chacune. L'évaluation est faite avant le programme (pré-test, Tl), à la fin (post-test, T2) et 6 mois plus tard (suivi, T3) et inclut des mesures du comportement de l'enfant, des pratiques éducatives parentales, du stress parental, du sentiment d'autoefficacité parental et du fonctionnement familial. Les résultats démontrent que les pères accompagnés sont moins assidus que les mères accompagnées, mais ne se distinguent pas des mères qui sont venues seules. Leur engagement dans les rencontres est toutefois similaire à celui des mères. Les analyses de variance à mesures répétées démontrent une amélioration plus immédiate des comportements extériorisés chez l'enfant dont le père était présent au programme. Suite au programme (post-test), sa présence n'a pas d'effet sur le stress parental, mais six mois après la fin de ce dernier (suivi), les mères qui y étaient seules se sentent plus stressée [i.e. stressées] que celles qui étaient accompagnées du père. Toutefois, très peu d'effets ont été décelés sur les pratiques éducatives parentales et le fonctionnement familial. La discussion analyse ces résultats et y fait suivre des retombées cliniques.

Spatial and temporal variation of commercially important bivalve species in the Algarve coast, Portugal

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Notable differences in gene expression between injured turbot (Scophthalmus maximus) and brill (Scophthalmus rhombus) skin

Dissertação de mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Péptidos para a detecção de agregados de alfa-sinucleína

Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2013

Partial volume correction using bimodal contrast agents in PET-MRI

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014

Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015

Development of a new cellular model for the visualization and study of GFAP oligomerization in living cells

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

Old conflicts, new rivals? Airline competition in the European market

Christoph Franz of Lufthansa recently identified Ryanair, easyJet, Air Berlin and Emirates as the company’s main competitors – gone are the days when it could benchmark itself against BA or Air France-KLM! This paper probes behind the headlines to assess the extent to which different airlines are in competition, using evidence from the UK and mainland European markets. The issue of route versus network competition is addressed. Many regulators have put an emphasis on the former whereas the latter, although less obvious, can be more relevant. For example, BA and American will cease to compete between London and Dallas Fort Worth if their alliance obtains anti-trust immunity but 80% of the passengers on this route are connecting at one or both ends and hence arguably belong to different markets (e.g. London-San Francisco, Zurich-Dallas, Edinburgh-New Orleans) which may be highly contested. The remaining 20% of local traffic is actually insufficient to support a single point to point service in its own right. Estimates are made of the seat capacity major airlines are offering to the local market as distinct from feeding other routes. On a sector such as Manchester–Amsterdam, 60% of KLM’s passengers are transferring at Schiphol as against only 1% of bmibaby’s. Thus although KLM operates 5 flights and 630 seats per day against bmibaby’s 2 flights and 298 seats, in the point to point market bmibaby offers more seats than KLM. The growth of the Low Cost Carriers (LCCs) means that competition increasingly needs to be viewed on city pair markets (e.g. London-Rome) rather than airport pair markets (e.g. Heathrow-Fiumicino). As the stronger LCCs drive out weaker rivals and mainline carriers retrench to their major hubs, some markets now have fewer direct options than existed prior to the low cost boom. Timings and frequencies are considered, in particular the extent to which services are a true alternative especially for business travellers. LCCs typically offer lower frequencies and more unsociable timings (e.g. late evening arrivals at remote airports) as they are more focused on providing the cheapest service rather than the most convenient schedule. Interesting findings on ‘monopoly’ services are presented (including alliances) - certain airlines have many more of these than others. Lufthansa has a significant number of sectors to itself whereas at the other extreme British Airways has direct competition on almost every route in its network. Ryanair and flybe have a higher proportion of monopoly routes than easyJet or Air Berlin. In the domestic US market it has become apparent since deregulation that better financial returns can come from dominating a large number of smaller markets rather than being heavily exposed in the major markets - which are hotly fought over. Regional niches that appear too thin for Ryanair to serve (with its all 189 seat 737-800 fleet) are identified. Fare comparisons in contrasting markets provide some insights to marketing and pricing strategies. Data sources used include OAG (schedules and capacity), AEA (traditional European airlines traffic by region), the UK CAA (airport, airline and route traffic plus survey information of passenger types) and ICAO (international route traffic and capacity by carrier). It is concluded that airlines often have different competitors depending on the context but in surprisingly many cases there are actually few or no direct substitutes. The competitive process set in train by deregulation of European air services in the 1990s is leading back to one of natural monopolies and oblique alternatives. It is the names of the main participants that have changed however!

pH Limitations in environmental geochemical monitoring

Senior thesis written for Oceanography 445

Reading Maud’s remains: Tennyson, geological processes, and palaeontological reconstructions

As Tennyson's “little Hamlet ,” Maud (1855) posits a speaker who, like Hamlet, confronts the ignominious fate of dead remains. Maud's speaker contemplates such remains as bone, hair, shell, and he experiences his world as one composed of hard inorganic matter, such things as rocks, gems, flint, stone, coal, and gold. While Maud's imagery of “stones, and hard substances” has been read as signifying the speaker's desire “unnaturally to harden himself into insensibility” (Killham 231, 235), I argue that these substances benefit from being read in the context of Tennyson's wider understanding of geological processes. Along with highlighting these materials, the text's imagery focuses on processes of fossilisation, while Maud's characters appear to be in the grip of an insidious petrification. Despite the preoccupation with geological materials and processes, the poem has received little critical attention in these terms. Dennis R. Dean, for example, whose Tennyson and Geology (1985) is still the most rigorous study of the sources of Tennyson's knowledge of geology, does not detect a geological register in the poem, arguing that by the time Tennyson began to write Maud, he was “relatively at ease with the geological world” (Dean 21). I argue, however, that Maud reveals that Tennyson was anything but “at ease” with geology. While In Memoriam (1851) wrestles with religious doubt that is both initiated, and, to some extent, alleviated by geological theories, it finally affirms the transcendence of spirit over matter. Maud, conversely, gravitates towards the ground, concerning itself with the corporal remains of life and with the agents of change that operate on all matter. Influenced by his reading of geology, and particularly Charles Lyell's provocative writings on the embedding and fossilisation of organic material in strata in his Principles of Geology (1830–33) volume 2, Tennyson's poem probes the taphonomic processes that result in the incorporation of dead remains and even living flesh into the geological system.

Media ‘Globalization’ as Survival Strategy for Authoritarian Regimes In the Arab Middle East

Sakr challenges the notion that transnational media technologies have forced states in the Arab Middle East to cede ever more control to non-state players since the 1990s. Taking account of a long history of foreign political engineering in Arab countries, she probes the realities of Arab broadcasting privatization, intra-regional harmonization of government communication policies, and external financial support for media freedom and reform, to show how Arab governments were large successfully in harnessing forces implicated in media globalization in a way that entrenched authoritarian elements of the status quo. The findings validate an alternative to globalization theory that places a dual focus on the agency of national ruling elites and the international structures that underpin the power of those elites today, as in the past.

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.