Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.

Ptolemaiida, a new order of Mammalia--with description of the cranium of Ptolemaia grangeri.

All records of the exotic mammalian family Ptolemaiidae are known from 182 m of section in the lower to middle parts of the upper Eocene and lower Oligocene Jebel Qatrani Formation, Fayum Depression, Egypt. Previous tentative assignments of ptolemaiid affinity have suggested that these animals are allied with the primitive suborder Pantolesta (currently placed in the order Cimolesta). Though perhaps ultimately derived from an unknown member of that group, the likelihood that ptolemaiids constitute a distinct group is considered, and analysis of all known materials of Ptolemaia, Qarunavus, and Cleopatrodon demonstrates that these genera belong in their own order, the Ptolemaiida, described here. The morphologically unique dentition and only known ptolemaiid cranium, that of Ptolemaia grangeri, is described. Although Qarunavus and Cleopatrodon show some similarities in primitive characters to European merialine Paroxyclaenidae (suborder Pantolesta), their affinities clearly lie with Ptolemaia and the Ptolemaiida.

Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.

Dalla catalisi all’inibizione della crescita da contatto: le molteplici funzioni di O-acetilserina sulfidrilasi batterica

CysK, uno degli isoenzimi di O-acetilserina sulfidrilasi (OASS) presenti in piante e batteri, è un enzima studiato da molto tempo ed il suo ruolo fisiologico nella sintesi della cisteina è stato ben definito. Recentemente sono state scoperte altre funzioni apparentemente non collegate alla sua funzione enzimatica (moonlighting). Una di queste è l’attivazione di una tossina ad attività tRNAsica, CdiA-CT, coinvolta nel sistema di inibizione della crescita da contatto (CDI) di ceppi patogeni di E. coli. In questo progetto abbiamo studiato il ruolo di CysK nel sistema CDI e la formazione di complessi con due differenti partner proteici: CdiA-CT e CysE (serina acetiltransferasi, l’enzima che catalizza la reazione precedente nella biosintesi della cisteina). I due complessi hanno le stesse caratteristiche spettrofluorimetriche e affinità molto simili, ma la cinetica di raggiungimento dell’equilibrio per il complesso tossina:CysK è più lenta che per il complesso CysE:CysK (cisteina sintasi). In entrambi i casi la formazione veloce di un complesso d’incontro è seguita da un riarrangiamento conformazionale che porta alla formazione di un complesso ad alta affinità. L’efficienza di formazione del complesso cisteina sintasi è circa 200 volte maggiore rispetto al complesso CysK:tossina. Una differenza importante, oltre alla cinetica di formazione dei complessi, è la stechiometria di legame. Infatti mentre CysE riesce a legare solo uno dei due siti attivi del dimero di CysK, nel complesso con CdiA-CT entrambi i siti attivi dell’enzima risultano essere occupati. Le cellule isogeniche esprimono un peptide inibitore della tossina (CdiI), e sono quindi resistenti all’azione tRNAsica. Tuttavia, siccome CdiI non altera la formazione del complesso CdiA-CT:CysK, CdiA-CT può esercitare comunque un ruolo nel metabolismo della cisteina e quindi nella fitness dei batteri isogenici, attraverso il legame e l'inibizione di CysK e la competizione con CysE. La via biosintetica della cisteina, un precursore di molecole riducenti, risulta essere molto importante per i batteri soprattutto in condizioni avverse come all’interno dei macrofagi nelle infezioni persistenti. Perciò questa via metabolica è di interesse per lo sviluppo di nuovi antibiotici, e in particolare le due isoforme dell’OASS negli enterobatteri, CysK e CysM, sono potenziali target per lo sviluppo di nuove molecole ad azione antibatterica. Partendo dall’analisi delle modalità di interazione con CysK del suo partner ed inibitore fisiologico, CysE, si è studiato dapprima l’interazione di pentapeptidi che mimassero la regione C-terminale di quest'ultimo, e in base ai dati ottenuti sono stati sviluppati piccoli ligandi sintetici. La struttura generale di questi composti è costituita da un gruppo acido ed un gruppo lipofilo, separati da un linker ciclopropanico che mantiene questi due gruppi in conformazione trans, ottimale per l’interazione col sito attivo dell’enzima. Sulla base di queste considerazioni, di docking in silico e di dati sperimentali ottenuti con la tecnica dell’STD-NMR e con saggi di binding spettrofluorimetrici, si è potuta realizzare una analisi di relazione struttura-attività che ha portato via via all’ottimizzazione dei ligandi. Il composto più affine che è stato finora ottenuto ha una costante di dissociazione nel range del nanomolare per entrambe le isoforme, ed è un ottimo punto di partenza per lo sviluppo di nuovi farmaci.

Technologies for Seduction: The “Espacio Doméstico” VideoArt Center in Blanca [EDOM]

Sustainability, understood in its beginnings as a common horizon for multiple practices and fields of study, has gradually given way to the development of increasingly sophisticated tools, with distinct dominant meanings established for each discipline. Within the field of material technologies for architectural production, sustainability seems to have found its most fertile ground in topics such as recycling, the use of "bio" materials, or energetic efficiency. However, to improve the understanding of the impact of technology on our ways of living, it appears increasingly necessary to move from the deterministic logic of sustainability into the relational domain of ecology, where the use and deployment of technologies can be observed through the multiplicity of its effects and the diversity of actors involved. In this paper we will address the case of the rehabilitation of several traditional houses located in the Murcian town of Blanca to host the “Espacio Doméstico” VideoArt Center (EDOM). In this action the selection and implementation of technologies have been aimed at impacting on diverse aspects including local communities, digital manufacturing, recycling, and policies regarding the rehabilitation of heritage buildings. While the initial approach was to address housing recovery as a heterogeneous accumulation of stories, technologies or material deployments of the domestic, our intervention strategies ascribed to the different technologies the role of mediating with existing elements through the incorporation of the very different visions of sustainability. Thus, we displayed artifacts produced by digitally manufactured methacrylate assembled on IKEA structures, fluorescent power lines supported by insulators on the wall, fluorescent tattoos on walls and ceilings that guide and extend the configuration of existing flooring, esparto furniture and fabrics produced by the esparto women workers’ and village women’s associations, re-appropriations of old furniture through the implementation of new media technologies, etc. If we can see seduction as the process of converting affinities and disagreements into affirmative communication, then the EDOM proposal can be seen as an active seduction process between technologies and users who approach this kind of cultural artifacts. Through these permanently active processes, art technologies will refer the viewer to complex sensory experiences, where a combination of parody, memory and sound pushes the user to the limit of mere comprehension of works of art. This more relational approach to the issue of heritage rehabilitation, technology or art institutions is offered as an area of controversy and debate on the scope of political ecology and its potential impact on the architect’s professional practice.

Ethnogenesis and Craniofacial Change in Japan from the Perspective of Nonmetric Traits

To examine population affinities in light of the ‘dual structure model’, frequencies of 21 nonmetric cranial traits were analyzed in 17 prehistoric to recent samples from Japan and five from continental northeast Asia. Eight bivariate plots, each representing a different bone or region of the skull, as well as cluster analysis of 21-trait mean measures of divergence using multidimensional scaling and additive tree techniques, revealed good discrimination between the Jomon-Ainu indigenous lineage and that of the immigrants who arrived from continental Asia after 300 BC. In Hokkaido, in agreement with historical records, Ainu villages of Hidaka province were least, and those close to the Japan Sea coast were most, hybridized with Wajin. In the central islands, clines were identified among Wajin skeletal samples whereby those from Kyushu most resembled continental northeast Asians, while those from the northernmost prefectures of Tohoku apparently retained the strongest indigenous heritage. In the more southerly prefectures of Tohoku, stronger traces of Jomon ancestry prevailed in the cohort born during the latest Edo period than in the one born after 1870. Thus, it seems that increased inter-regional mobility and gene flow following the Meiji Restoration initiated the most recent episode in the long process of demic diffusion that has helped to shape craniofacial change in Japan.

Inference of binding affinity from neuronal receptors in humans

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2016

Palaeoenvironmental and palaeoceanographic changes during the lower to middle Jurassic (Toarcian-Aalenian, ~183-171 Ma). : new evidences from calcareous nannofossils of the Lusitanian Basin

Tese de doutoramento (co-tutela), Geologia (Paleontologia e Estratigrafia), Faculdade de Ciências da Universidade de Lisboa, Université Claude Bernard Lyon 1, 2016

Sedimentology of surface samples from the Weddell Sea, Antarctica

Mineralogical and granulometric properties of glacial-marine surface sediments of the Weddell Sea and adjoining areas were studied in order to decipher spatial variations of provenance and transport paths of terrigenous detritus from Antarctic sources. The silt fraction shows marked spatial differences in quartz contents. In the sand fractions heavy-mineral assemblages display low mineralogical maturity and are dominated by garnet, green hornblende, and various types of clinopyroxene. Cluster analysis yields distinct heavy-mineral assemblages, which can be attributed to specific source rocks of the Antarctic hinterland. The configuration of modern mineralogical provinces in the near-shore regions reflects the geological variety of the adjacent hinterland. In the distal parts of the study area, sand-sized heavy minerals are good tracers of ice-rafting. Granulometric characteristics and the distribution of heavy-mineral provinces reflect maxima of relative and absolute accumulation of ice-rafted detritus in accordance with major iceberg drift tracks in the course of the Weddell Gyre. Fine-grained and coarse-grained sediment fractions may have different origins. In the central Weddell Sea, coarse ice-rafted detritus basically derives from East Antarctic sources, while the fine-fraction is discharged from weak permanent bottom currents and/or episodic turbidity currents and shows affinities to southern Weddell Sea sources. Winnowing of quartz-rich sediments through intense bottom water formation in the southern Weddell Sea provides muddy suspensions enriched in quartz. The influence of quartz-rich suspensions moving within the Weddell Gyre contour current can be traced as far as the continental slope in the northwestern Weddell Sea. In general, the focusing of mud by currents significantly exceeds the relative and absolute contribution of ice-rafted detritus beyond the shelves of the study area.

Dinocyst datum events of several holes in the Norwegian-Greenland Sea

The presence of abundant age-diagnostic dinoflagellate cysts in Ocean Drilling Program (ODP) Hole 913B (Leg 151), Deep Sea Drilling Project Hole 338 (Leg 38) and ODP Hole 643A (Leg 104) has enabled the development of a new biostratigraphy for the Eocene-Oligocene interval in the Norwegian-Greenland Sea. This development is important because the calcareous microfossils usually used for biostratigraphy in this age interval are generally absent in high latitude sediments as a result of dissolution. In parallel with this biostratigraphic analysis, we developed a magnetic reversal stratigraphy for these Norwegian-Greenland Sea sequences. This has allowed independent age determination and has enabled the dinocyst biostratigraphy to be firmly tied into the global geomagnetic polarity timescale (GPTS). The relatively high resolution of this study has enabled identification of dinoflagellate cyst assemblages that have affinities with those from the North Sea and the North Atlantic, which allows regional correlation. Correlation of each site with the GPTS has also allowed comparison of the stratigraphic record preserved in each drill-hole. Hole 913B is the most complete and is the best-preserved record of the Eocene and Oligocene in the Northern Hemisphere high latitudes, and can serve as a reference section for palaeoenvironmental reconstructions of this age interval.

Sea-surface temperature reconstructions for the Quaternary western Atlantic

Paleotemperature estimates calculated by the SIMMAX Modern Analog Technique are presented for two gravity cores from the Rio Grande Rise, one from the Brazil Slope, and one from the Ceara Rise. The estimates are based on comparisons between modern and fossil planktonic foraminiferal assemblages and were carried out on samples from Quaternary sediments. Estimated warm-season temperatures from the Rio Grande Rise (at approx. 30° S) range from around 19°C to 24°C, with some coincidence of warm peaks with interglacial stages. The temperature estimates (also warm-season) from the more tropical Brazil Slope (at approx. 8° S) and Ceara Rise (at approx. 4° N) cores are more stable, remaining between 26°C and 28°C throughout most of their lengths. This fairly stable situation in the tropical western Atlantic is interrupted in oxygen isotope stage 6 by a significant drop of 2-3°C in both of these cores. Temperature estimates from the uppermost samples in all cores compare very well to the modern-day measured values. Affinities of some foraminiferal species for warmer or cooler surface temperatures are identified within the temperature range of the examined samples based on their abundance values. Especially notable among the warmer species are, Globorotalia menardii, Globigerinita glutinata, Globigerinoides ruber, and Globigerinoides sacculifer. Species indicative of cooler surface temperatures include Globorotalia inflata, Globigerina bulloides, Neogloboquadrina pachyderma, and Globigerina falconensis. A cluster analysis was carried out to assist in understanding the degree of variation which occurs in the foraminiferal assemblages, and how temperature differences influence the faunal compositions of the samples. It is demonstrated that fairly similar samples may have unexpectedly different estimated temperatures due to small differences in key species and, conversely, quite different assemblages can result in similar or identical temperature estimates which confirms that other parameters than just temperature affect faunal content.