Phage display as a tool for development of novel therapeutics for breast cancer

Phage display technology is a powerful platform for the generation of highly specific human monoclonal antibodies (Abs) with potential use in clinical applications. Moreover, this technique has also proven to be a reliable approach in identifying and validating new cancer-related targets. For scientific or medical applications, different types of Ab libraries can be constructed. The use of Fab Immune libraries allows the production of high quality and affinity antigen-specific Abs. In this work, two immune human phage display IgG Fab libraries were generated from the Ab repertoire of 16 breast cancer patients, in order to obtain a tool for the development of new therapeutic Abs for breast cancer, a condition that has great impact worldwide. The generated libraries are estimated to contain more than 108 independent clones and a diversity over 90%. Libraries validation was pursued by selection against BSA, a foreign and highly immunogenic protein, and HER2, a well established cancer target. Preliminary results suggested that phage pools with affinity for these antigens were selected and enriched. Individual clones were isolated, however, it was not possible to obtain enough data to further characterize them. Selection against the DLL1 protein was also performed, once it is a known ligand of the Notch pathway, whose deregulation is associated to breast cancer, making it an interesting target for the generation of function-blocking Abs. Selection resulted in the isolation of a clone with low affinity and Fab expression levels. The validation process was not completed and further effort will have to be put in this task in the future. Although immune libraries concept implies limited applicability, the library reported here has a wide range of use possibilities, since it was not restrained to a single antigen but instead thought to be used against any breast cancer associated target, thus being a valuable tool.

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.

Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase

Protein Sci. 2009 Mar;18(3):619-28. doi: 10.1002/pro.69.

Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Biostratigraphie et paléoécologie des peuplements d'ostracodes dans le Domérien du Bassin Lusitanien, Portugal

The Domerian sections from the Lusitanian Basin of São Pedro de Muel, Rabaçal and Tomar have provided us with more than 1100 Ostracods belonging from 18 genus and about 48 species. The faunal diversity and density of the associations decrease in space (from Tomar to Rabaçal and São Pedro de Muel) and time, with favourable environments for the proliferation of Ostracods at the lower part of the sections (Stokesi subzone) and more hostile at the upper part (Ragazzonii subzone). The Monestieri and Nitescens horizons and the Subnodosus subzone are characterized by a typical assemblage of Ostracods. The palaeoecological Ostracod indexes reveal the fluctuations of the oxygenation, temperature, depth and hydrodynamism of the water, on the different sections and on the whole platform. They display a diachronous cooling in the Lower Domerian series. In the upper part of the Middle and in the Upper Domerian, the deeper, less oxygenated and cooler waters prevent the development of the Ostracod faunas.

Aspirations sociales: projects politiques et efficience socio-culturelle

Tese de doutoramento em Ciências da Educação

Les Plus Anciens Mastodontes Tetralophodontes (Langhien inférieur Vb), évolution et remarques sur la Tetralophodontie

This study deals with mastodont teeth found near Lisbon in Lower Langhian (lower Middle Miocene) fluviatile, feldspathic sands (Vb division). Conclusions are as follows: 1. Tetralophodont molars (even if at a still primitive stade of the tetralophodont condition) do exist at least since lower Langhian times, and not only since late Middle Miocene as was previously known. 2. Tri- and tetralophodont structures may (and indeed do) coexist in the same individual: such examples do not correspond to transitional forms, but instead to a mosaic of juxtaposed characters (however this does not mean there are no transitional forms in other instances). 3. So these structures coexisted in a population not yet genetically separated beyond fertile cross-breeding, i.e. beyond species' level. 4. Origin of the tetralophodont molar was due to some mutation (s). but without crossing species, limits and even more genus'ones. 5. At this times probably soon after the first appearance of tetralophodont mutants, animals with such characters were a small but significant minority among the population (17% if account is taken on D4's: only 2% after M2's). 6. There was not then any direct and clear correlation between number of lophs (transversal crests) and tooth size, even if the increase of such number goes along with length's increase. 7. Dimensions (length in special) in tetralophodont teeth tend to exceed those in «normal» trilophodont teeth, this being particularly clear in D4, even if there is no clear distinction: the situation is quite the same, maybe less marked, with the M2. 8. According to the preceding conclusions there are no reasons to segregate different taxa among such mastodont population on the grounds of the presence in D4, M1 and M2 of 3 or 4 crests (this character being regarded as diagnostic of the genus Tetralophodon). 9. On the contrary, if any natural (in biological sense) classification is disregarded and a morphological parataxonomy is adopted there should be considered both Gomphotherium angustidens and Tetralophodon sp.: however this is absolutely not our opinion.

Notes sur la géologie et la paléontologie du Miocène de Lisbonne. XXI Primeira caracterização de dinoflagelados dos níveis mais baixos da série miocénica de Lisboa

This is the first report on Dinoflagellata from lowermost Miocene beds in Portugal (Tagus basin, Lisbon region, exposure at Benfica). Some general data about Dinoflagellata are presented. Descriptions are provided for some forms: Gonyaulacysta tenuitabulata, Spiniferites ramosus, Achomosphaera sp., Hystrichokolpoma rigaudae, Homotriblium cf. pallidum, Cordosphaeridium sp, and Lingulodinium machaerophorum. Lithostratigraphical study of an important section along Circular highway at Benfica has shown that there is a hitherto unknown sedimentary cycle in Lisbon's lowermost Miocene (upper Oligocene?). Pollen and Ostracoda point out to an Lower Aquitanian or even Upper Chattian age for the first Neogene marine transgression in Portugal, previously considered as Upper Aquitanian or Lower Burdigalian.

Notes sur la géologie et la paleontology du Miocène de Lisbonne. XXII - Une Espèce nouvelle Pokornyella Lusitanica (Ostrocoda), dans le Miocène Inférieur du bassin du Tage

A new species, Pokornyella lusitanica (Ostracoda), from the Lower Miocene (Aquitanian) of the Lisbon area, is described. Some palaeoecological data are presented.

Notes sur la géologie et la paleontology du Miocène de Lisbonne

This note deals with the stratigraphical and paleontological study of the Palença section on the southern bank of the river Tagus, Portugal, and specially with its coccolithophorids. Three main lithostratigraphical units may be recognized: the lowest one does correspond to the upper part of COTTER's division II, the intermediate one to divisions III and IV-a, the third corresponding to pratically the whole division IV-b, However other and higher levels are also represented. Higher beds are also represented in the same sections; they are less well exposed and were not studied in detail. Caracterisation of biozones on the basis of Coccoliths so far found at Palença section is difficultsince MARTINI's zones have been defined mainly by forms of Discoaster and other genera that are wanting. However we can recognize that the richest assemblage (from beds 17-18, the uppermost layers of blue clays IV-a) may correspond to NN4. This is not in opposition to the results of the study of planctonic foraminifera, that are characteristic of BLOW's N7. Coccoliths from lower beds do not allow at present any valid comparisons.

Contributions à la Paléontologie du Miocène moyen continental du Bassin du Tage. III - Mammifères - Póvoa de Santarém, Pêro Filho et Chões (Secorio). Conclusions générales

This study on middle Miocene mammalian faunas from Tagus'basin deals particularly with some small mammals hitherto undescribed from Portugal, including a new Glirid (Paraglirulus scalabicensis nov. sp.); it allows an accurate datation by biostratigraphical standards, Megacricetodon crusafonti, Fahlbuschia darocensis, Cricetodon jotae being characteristic of mammalian MN6 unit, thus their age is nearly that of Sansan and Manchones (however the presence of Peridyromys hamadryas and Lagopsis verus do suggest, amidst this biozone, a somewhat later age than Sansan's); it contributes with indirect correlation data with marine formations, as underlying oyster-bearing beds most probably are in correspondance to the apogee of the same transgression that deposed near Lisbon ”schlier" facies from VI-a division (Serravalian, Blow's zones 10-13, Globorotalia meyeri zone); the diversity of mammalian assemblages is surely related to an environement with varied biotopes, whose characterisation becomes easier if account is taken of the preceding papers on mollusks (G. Truc) and Cyprinid fishes (J. Gaudant), and also according to some unpublished paleobotanical data (J. Pais). A table with a synthesis of all paleontological data so far known is presented.

Cheirogaster sp. (O. Testudines, Fam. Testudinidae, Geochelone sl.) du Paléogène de Naia, Tondela et l’âge du gisement

A land tortoise from a new locality at Naia, Tondela, is described. It is to be reported either to an advanced form of the genus Hadrianus or to an archaic representative of Cheirogaster; it may be included in the comprehensive genus Geochelone s.l., excluding however Ergilemys and its descendants. There is a strong possibility in favour of Cheirogaster. Testudo must also be excluded. It is not possible to classify this specimen at species'level. Our specimen does agree best with Upper Eocene Testudinidae and with some Lower Oligocene ones. Its age is certainly not Upper Oligocene or later, nor Lower and Middle Eocene. This datation is not opposed to the age of the fossiliferous clays of Naia as supposed by correlation with another locality - Côja, about 30 km to the South - which yielded an assemblage of mammals whose Ludian (Upper Bartonian s.l.) age seems well established. Naia and Côja's fossil-bearing clays must be nearly synchronous; their origin is well in place among the phenomena related to the surrection of iberian Central Chain during paroxysmal phase of pyrenean orogenesis.

Faciès, biostratigraphie et paléogéographie du Jurassique portugais

The facies distribution along the Jurassic stages in an already well established stratigraphic frame is defined for the three portuguese basins: North of Tagus, Santiago de Cacém and Algarve. The deposits are organized in two sedimentary cycles. The first one from the Liassic to Calovian shows, in the Tagus Basin, a transgression from NW which did not surpass the Meseta present limits. The iniatilly brackish deposits only changed to marine by the end of Lotharingian. The sedimentation, mainly marly during the Liassic became more calcareous since the Aalenian. During the Dogger the basin differentiated into platform deposits towards East and South and open sea zone towards West. This zone underwent a progressive reduction and, during the Callovian, two small basins were individualized: Cabo Mondego basin in the North and Serra de El-Rei-Montejunto in the South. It is from the latter that the second sedimentary cycle (Middle Oxfordian-Portlandian) developed with open sea deposits along the Sintra–Torres Vedras axis surrounded by platform and litoral brackish formations. During the first sedimentary cycle only litoral platform deposits are known in Santiago de Cacém and Algarve basins. During the second sedimentary cycle temporary sea open deposits are known in Santiago de Cacém and Central Algarve.

Éssai de synthèse paléogéographique et paléobiostratigraphique du bassin occidental portugais au cours du crétacé supérieur

The Upper-Cretaceous transgressive serie is described by the authors, on the whole Occidental Portuguese Basin: it begins in the Lisbon region in the Albian and finishes in the Beira litoral province in the lower Turonian, while the sedimentation zones migrate Northward. The lithologic composition is given for each stage and sub-stage, taking into cgnsideration, in particular in the Upper Cenomanian, the lateral variations from one region to another. The paleogeography becomes clear by the study of the sedimentation environments (6 fig.). In conclusion, the authors propose a correlation between the whole serie and the accepted zonation of the Northwestern Europe. Tables show the repartition of the main macrofauna and microfauna.

La marge continentale sud-portugaise: évolution structurale et sédimentaire

The structural and sedimentary evolution of the portuguese continental margin South of Setúbal Canyon is outlined from the study of many seismic reflection profiles and rocks samples. During Triassic and Early Liassic time, a distension affects the Algarve margin that belongs to the Mesogean area. Off Baixo Alentejo rifting phases at Late Jurassic and Early Cretaceous times induced opening or widening of the adjacent part of the Atlantic ocean. Alpine orogeny is inferred to explain the Eocene and Miocene deformation of the margin specially along the main NE-SW fractures.