Bioconjugation and synthetic approach towards enantioenriched gem-difluoromethylene compounds through carbenium ions

Bioconjugation of peptides and asymmetric synthesis of gem-difluoromethylene compounds are areas of the modern organic chemistry for which mild and selective methods continue to be developed. This thesis reports new methodologies for these two areas based on the use of stabilized carbenium ions. The reaction that makes the bioconjugation of peptides possible takes place via the direct nucleophilic substitution of alcohols and is driven by the spontaneous formation of stabilized carbenium ions in water. By reacting with the thiol group of cysteine in very mild conditions and with a high selectivity, these carbenium ions allow the site-specific ligation of polypeptides containing cysteine and their covalent derivatization with functionalized probes. The ligation of the indole ring of tryptophan, an emerging target in bioconjugation, is also shown and takes place in the same conditions. The second area investigated is the challenging access to optically active gem-difluoromethylene compounds. We describe a methodology relying on the synthesis of enantioenriched 1,3-benzodithioles intermediates that are shown to be precursors of the corresponding gem-difluoromethylene analogues by oxidative desulfurization-fluorination. This synthesis takes advantage of the highly enantioselective organocatalytic α-alkylation of aldehydes with the benzodithiolylium ion and of the wide possibilities of synthetic transformations offered by the 1,3-benzodithiole group. This approach allows the asymmetric access to complex gem-difluoromethylene compounds through a late-stage fluorination step, thus avoiding the use of fluorinated building blocks.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.Laboratory Investigation advance online publication, 24 November 2014; doi:10.1038/labinvest.2014.141.

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

Truncated elongation factor G lacking the G domain promotes translocation of the 3' end but not of the anticodon domain of peptidyl-tRNA.

The mechanism by which elongation factor G (EF-G) catalyzes the translocation of tRNAs and mRNA on the ribosome is not known. The reaction requires GTP, which is hydrolyzed to GDP. Here we show that EF-G from Escherichia coli lacking the G domain still catalyzed partial translocation in that it promoted the transfer of the 3' end of peptidyl-tRNA to the P site on the 50S ribosomal subunit into a puromycin-reactive state in a slow-turnover reaction. In contrast, it did not bring about translocation on the 30S subunit, since (i) deacylated tRNA was not released from the P site and (ii) the A site remained blocked for aminoacyl-tRNA binding during and after partial translocation. The reaction probably represents the first EF-G-dependent step of translocation that follows the spontaneous formation of the A/P state that is not puromycin-reactive [Moazed, D. & Noller, H. F. (1989) Nature (London) 342, 142-148]. In the complete system--i.e., with intact EF-G and GTP--the 50S phase of translocation is rapidly followed by the 30S phase during which the tRNAs together with the mRNA are shifted on the small ribosomal subunit, and GTP is hydrolyzed. As to the mechanism of EF-G function, the results show that the G domain has an important role, presumably exerted through interactions with other domains of EF-G, in the promotion of translocation on the small ribosomal subunit. The G domain's intramolecular interactions are likely to be modulated by GTP binding and hydrolysis.

Is Regulatory Convergence Efficient? CES Germany & Europe Working Papers, No. 02.3, May 1992

One of the important themes in the new institutionalism is the convergence of market regulations in a world with three powerful clusters of countries (Western Europe, North America, and East Asia) on a small number of regimes, like disorganized capitalism, free market capitalism, and coordinated market capitalism. This paper examines the political-economic theory of regulatory convergence. It reconstructs and compares three welfarist approaches: the optimal regulatory regime (Tinbergen), the rule of constitutional law (Buchanan), and regulatory rivalry (Hayek). The paper concludes that most plausible results of convergence theory are completely opposite to the expressed political intentions of the theorists. Tinbergen's theory predicts neoliberalism, not social democracy. The theories of Buchanan and Hayek predict respectively a consensual or spontaneous formation of corporatist regulations, not the return of classical constitutionalism or liberalism. The paper summons new institutionalists to repair the weak scientific elements of convergence theory and to make a distinction between the ideological origins of this theory and its unintended ideological consequences.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Effect of rolling on dissipation in fault gouges

Sliding and rolling are two outstanding deformation modes in granular media. The first one induces frictional dissipation whereas the latter one involves deformation with negligible resistance. Using numerical simulations on two-dimensional shear cells, we investigate the effect of the grain rotation on the energy dissipation and the strength of granular materials under quasistatic shear deformation. Rolling and sliding are quantified in terms of the so-called Cosserat rotations. The observed spontaneous formation of vorticity cells and clusters of rotating bearings may provide an explanation for the long standing heat flow paradox of earthquake dynamics.

Effect of a methyl group on the spontaneous resolution of a square-pyramidal coordination compound: crystal packing and conglomerate formation

Reaction of [Cu(pic)2]·2H2O (where pic stands for 2-picolinato) with 2-({[2-(dimethylamino)ethyl]amino}methyl)phenol (HL1) produces the square-pyramidal complex [CuL1(pic)] (1), which crystallizes as a conglomerate (namely a mixture of optically pure crystals) in the Sohncke space group P212121. The use of the methylated ligand at the benzylic position, i.e. (±)-2-(1-{[2-(dimethylamino)ethyl]amino}ethyl)phenol (HL2), yields the analogous five-coordinate complex [CuL2(pic)] (2) that crystallizes as a true racemate (namely the crystals contain both enantiomers) in the centrosymmetric space group P21/c. Density functional theory (DFT) calculations indicate that the presence of the methyl group indeed leads to a distinct crystallization behaviour, not only by intramolecular steric effects, but also because its involvement in non-covalent C–H···π and hydrophobic intermolecular contacts appears to be an important factor contributing to the crystal-lattice (stabilizing) energy of 2

Formation of haemozoin/β-haematin under physiological conditions is not spontaneous

Malaria parasite detoxifies free haem, released as a result of haemoglobin digestion, by converting it into an stable, crystalline, black brown pigment known as 'malaria pigment' or 'haemozoin'. Earlier studies have demonstrated the involvement of a parasite-specific enzyme 'haem polymerase' in the formation of haemozoin. However, recently it has been proposed that the polymerization of haem may be a spontaneous process that could take place by incubation of haematin with carboxylic acids (pH 4.2-5.0) even without presence of any parasitic or biological component (FEBS Letters, 352, 54-57 (1994). Here we report that no spontaneous haem polymerization occurs at physiological conditions and the product described in the study mentioned above is not haemozoin/beta-haematin (haem polymer) as characterized by us on the basis of solubility characteristics and thin layer chromatography. The infra-red spectroscopic analysis of the product formed though exhibits the bands corresponding to formation of iron-carboxylate bond, similar to that in haemozoin/beta-haematin, but was identified as haem-acid adduct. Thus polymerization of haem may not occur spontaneously under the reaction conditions corresponding to food vacuoles of the malarial parasite, the physiological site of haemozoin formation.

Investigation of spontaneous microemulsion formation in supercritical carbon dioxide using high-pressure NMR

NMR spectroscopy and relaxometry were used to investigate microemulsion formation in supercritical CO2. The droplets were stabilised by the salt of a perfluorinated polyether. Spontaneous microemulsion formation was observed over a period of 5 h in the absence of applied sheer. Time-resolved relaxation times of the surfactant tail showed a stepwise increase in mobility of the tail over this period. Conversely, the translational mobility of water confined within the droplet decreased over the same interval. This data is consistent with the gradual decrease in droplet size as time progressed. Indeed, NMR self-diffusion coefficients were used to show that droplets with a radius of approximately 5 nm were formed at equilibrium.

Scenes, quarters and clusters : new experiments in the formation and governance of creative places in China

This thesis examines the formation and governance patterns of the social and spatial concentration of creative people and creative business in cities. It develops a typology for creative places, adding the terms 'scene' and 'quarter' to 'clusters', to fill in the literature gap of partial emphasis on the 'creative clusters' model as an organising mechanism for regional and urban policy. In this framework, a cluster is the gathering of firms with a core focus on economic benefits; a quarter is the urban milieu usually driven by a growth coalition consisting of local government, real estate agents and residential communities; and a scene is the spontaneous assembly of artists, performers and fans with distinct cultural forms. The framework is applied to China, specifically to Hangzhou – a second-tier city in central eastern China that is ambitious to become a 'national cultural and creative industries centre'. The thesis selects three cases – Ideal & Silian 166 Creative Industries Park, White-horse Lake Eco-creative City and LOFT49 Creative Industries Park – to represent scene, quarter and cluster respectively. Drawing on in-depth interviews with initiators, managers and creative professionals of these places, together with extensive documentary analysis, the thesis investigates the composition of actors, characteristics of the locality and the diversity of activities. The findings illustrate that, in China, planning and government intervention is the key to the governance of creative space; spontaneous development processes exist, but these need a more tolerant environment, a greater diversity of cultural forms and more time to develop. Moreover, the main business development model is still real estate based: this model needs to incorporate more mature business models and an enhanced IP protection system. The thesis makes a contribution to literature on economic and cultural geography, urban planning and creative industries theory. It advocates greater attention to self-management, collaborative governance mechanisms and business strategies for scenes, quarters and clusters. As intersections exist in the terms discussed, a mixed toolkit of the three models is required to advance the creative city discourse in China.

Scenes, quarters and clusters: The formation and governance of creative places in urban China

This paper presents a PhD program examining the formation and governance patterns of the social and spatial concentration of creative people and creative businesses in cities. It develops a typology for creative places, adding the terms ‘scene’ and ‘quarter’ to ‘clusters’, to fill in the literature gap of partial emphasis on the ‘creative clusters’ model as an organising mechanism for regional and urban policy. The framework is then applied to China, specifically to Hangzhou, a second-tier city in central eastern China that is ambitious to become a ‘national cultural and creative industries centre’. Drawing on in-depth interviews with initiators, managers and creative professionals from three cases selected respectively for scene, quarter and cluster, together with extensive documentary analysis, the paper investigates the composition of actors, characteristics of the locality and the diversity of activities of the three places. The findings demonstrate a convergence of the three terms. Furthermore, in China, planning and government intervention is the key to the governance of creative places; spontaneous development processes exist, but these need a more tolerant environment, a greater diversity of cultural forms and more time to develop. Moreover, the main business development model is still real estate based: this model needs to incorporate more mature business models and an enhanced IP protection system. Finally, the business strategies need to be combined with a self-management model for the creative class, and a collaborative governance mechanism with other stakeholders such as government, real estate developers and education providers.