Phage display as a tool for generation of bio-therapeutic agents for breast cancer

Notch is a conserved signalling pathway, which plays a crucial role in a multiple cellular processes such as stem cell self-renewal, cell division, proliferation and apoptosis. In mammalian, four Notch receptors and five ligands are described, where interaction is achieved through their extracellular domains, leading to a transcription activation of different target genes. Increased expression of Notch ligands has been detected in several types of cancer, including breast cancer suggesting that these proteins represent possible therapeutic targets. The goal of this work was to generate quality protein targets and, by phage display technology, select function-blocking antibodies specific for Notch ligands. Phage display is a powerful technique that allows the generation of highly specific antibodies to be used for therapeutics, and it has also proved to be a reliable approach in identifying and validating new cancer-related targets. Also, we aimed at solving the tri-dimensional structure of the Notch ligands alone and in complex with selected antibodies. In this work, the initial phase focused on the optimization of the expression and purification of a human Delta-like 1 ligand mutant construct (hDLL1-DE3), by refolding from E. coli inclusion bodies. To confirm the biological activity of the produced recombinant protein cellular functional studies were performed, revealing that treatment with hDLL1-DE3 protein led to a modulation of Notch target genes. In a second stage of this study, Antibody fragments (Fabs) specific for hDLL1-DE3 were generated by phage display, using the produced protein as target, in which one good Fab candidate was selected to determine the best expression conditions. In parallel, multiple crystallization conditions were tested with hDLL1-DE3, but so far none led to positive results.

O Display da morte na ficção de José Saramago

Tese de Doutoramento em Ciências da Literatura (área de especialização em Literatura Portuguesa).

User expectations for media sharing practices in open display networks

Open Display Networks have the potential to allow many content creators to publish their media to an open-ended set of screen displays. However, this raises the issue of how to match that content to the right displays. In this study, we aim to understand how the perceived utility of particular media sharing scenarios is affected by three independent variables, more specifically: (a) the locativeness of the content being shared; (b) how personal that content is and (c) the scope in which it is being shared. To assess these effects, we composed a set of 24 media sharing scenarios embedded with different treatments of our three independent variables. We then asked 100 participants to express their perception of the relevance of those scenarios. The results suggest a clear preference for scenarios where content is both local and directly related to the person that is publishing it. This is in stark contrast to the types of content that are commonly found in public displays, and confirms the opportunity that open displays networks may represent a new media for self-expression. This novel understanding may inform the design of new publication paradigms that will enable people to share media across the display networks.

The display gamut available to simulate colors perceived by anomalous trichromats

The aim of this work was to investigate the effect on a display gamut of varying the optical density and the position of the maximum sensitivity of the cones spectra of anomalous trichromatic observers. The anomalous cone spectral sensitivities were estimated for a set of varying optical density and maximum sensitivity spectra conditions and used to compute the display color gamut. The computed display gamut simulated for normal observers the chro- matic diversity perceived by anomalous observers. It was found that even small variations on the optical density and on the position of the maximum sensitivity spectra have an impact on the simulations of the display gamut for anomalous observers. It was also found that simulations for deuteroanomalous observers are the ones with greater impact if the estimation of the corresponding color display gamut is not carefully adjusted for the observer.

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Television before TV : a transnational history of an experimental medium on display, 1928 - 1939

Dès le milieu des années 1920 et le développement des premiers prototypes fonctionnels, la télévision se fait objet d'exposition. La nouvelle technologie est montrée à des foires industrielles et dans des grands magasins, à des expositions universelles et nationales : exposée, la télévision devient un mass media avant même qu'elle ne diffuse des émissions régulières. En étudiant les présentations publiques de la télévision en Allemagne, en Grande-Bretagne et aux Etats-Unis entre 1928 - date de l'ouverture des premières expositions annuelles dans les trois pays - et 1939 - moment de l'interruption de celles-ci suite à l'éclatement de la Deuxième Guerre mondiale - Television before TV montre que les lieux où le médium rencontre son premier public constituent les mêmes espaces qui, rétrospectivement, permettent de comprendre son avènement. Cette recherche propose ainsi de nouvelles pistes pour l'histoire des médias en interrogeant la définition de la télévision en tant que médium du privé. Elle rappelle l'importance historique de l'espace public pour la « vision à distance » et, dépassant la perspective nationale, reconstitue les différences et similarités techniques, médiatiques et institutionnelles de la télévision allemande, américaine et britannique dans l'entre-deux-guerres. Développée dans un contexte qui est simultanément nationaliste et nourri d'échanges et de compétitions internationales, la télévision sert de comparatif dans les rivalités entre pays, mais stimule également la circulation de savoir-faire et de personnes. Sa forte valeur symbolique comme emblème du progrès scientifique et, dans le cas allemand, comme preuve de la modernité du régime national-socialiste se traduit dans les salles d'expositions par des scénographies souvent innovatrices, dont les photographies et descriptions se propagent au-delà de la presse locale. Au centre d'un réseau discursif et représentationnel, les expositions jouent alors un rôle essentiel pour la construction sociale, politique et culturelle de la télévision. Remplaçant l'analyse de textes audiovisuels par l'étude de la machine exposée, ma recherche reformule la question bazinienne de qu'est-ce que la télévision ? pour demander où le médium se situe-t-il ? Cette prémisse méthodologique permet de contourner une évaluation qualitative de la technologie qui déplorerait ses multiples imperfections, et éclaire d'un nouveau jour des dispositifs télévisuels qui, même sans programme, intègrent un univers voué à la culture de consommation et du loisir.

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells.

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 degrees C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents.

IPH Response to Dept of Justice and Equality Review of the Code of Practice on the Sale and Display of Alcohol in Mixed Trading Premises

IPH responded to the Department of Justice, Equality and Defence review of the voluntary Code of Practice for the display and sale of alcohol in supermarkets, convenience stores and similar mixed trading outlets. The voluntary Code was introduced in 2008 as an alternative to the statutory rules for structural separation of alcohol products in mixed trading outlets which are set out in section 9 of the Intoxicating Liquor Act 2008. Interested bodies and individuals were invited to submit comments on the Compliance Report for 2011 and on the effectiveness of the voluntary approach to structural separation by 20th December 2011. The Minister said he intended to also seek the views of the Minister for Health and the Joint Oireachtas Committee on Justice, Defence and Equality before reaching any decision on whether to bring the statutory rules in the 2008 Act into operation.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)

Calotropis procera R. Br. (Asclepiadaceae) is a well-known medicinal plant with leaves, roots, and bark being exploited by popular medicine to fight many human and animal diseases. This work deals with the fractionation of the crude latex produced by the green parts of the plant and aims to evaluate its toxic effects upon egg hatching and larval development of Aedes aegypti. The whole latex was shown to cause 100% mortality of 3rd instars within 5 min. It was fractionated into water-soluble dialyzable (DF) and non-dialyzable (NDF) rubber-free materials. Both fractions were partially effective to prevent egg hatching and most of individuals growing under experimental conditions died before reaching 2nd instars or stayed in 1st instars. Besides, the fractions were very toxic to 3rd instars causing 100% mortality within 24 h. When both fractions were submitted to heat-treatment the toxic effects were diminished considerably suggesting low thermostability of the toxic compounds. Polyacrylamide gel electrophoresis of both fractions and their newly fractionated peaks obtained through ion exchange chromatography or desalting attested the presence of proteins in both materials. When submitted to protease digestion prior to larvicidal assays NDF lost most of its toxicity but DF was still strongly active. It may be possible that the highly toxic effects of the whole latex from C. procera upon egg hatching and larvae development should be at least in part due to its protein content found in NDF. However the toxicity seems also to involve non protein molecules present in DF.

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Group A rotavirus and norovirus display sharply distinct seasonal profiles in Belém, northern Brazil

Several viruses have been associated with acute gastroenteritis (AGE), and group A rotavirus (RVA) and nor-ovirus (NoV) are the most prevalent. This study aimed to assess their prevalence among children hospitalised for diarrhoea during a three-year surveillance study. From May 2008-April 2011, overall positivity rates of 21.6% (628/2904) and 35.4% (171/483) were observed for RVA and NoV, respectively. The seasonality observed indicated distinct patterns when both viruses were compared. This finding may explain why hospitalisation for AGE remains constant throughout the year. Continuous AGE monitoring is needed to better assess the patterns of infection.