Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.

Transport mechanism in ionic liquid membranes and the study of thiomersal biodegradation

The initial goal of this work was the development of a supported liquid membrane (SLM) bioreactor for the remediation of vaccine production effluents contaminated with a highly toxic organomercurial – thiomersal. Therefore, two main aspects were focused on: 1) the development of a stable supported liquid membrane – using room temperature ionic liquids (RTILs) – for the selective transport of thiomersal from the wastewater to a biological compartment, 2) study of the biodegradation kinetics of thiomersal to metallic mercury by a Pseudomonas putida strain. The first part of the work focused on the evaluation of the physicochemical properties of ionic liquids and on the SLMs’ operational stability. The results obtained showed that, although it is possible to obtain a SLM with a high stability, water possesses nonnegligible solubility in the RTILs studied. The formation of water clusters inside the hydrophobic ionic liquid was identified and found to regulate the transport of water and small ions. In practical terms, this meant that, although it was possible to transport thiomersal from the vaccine effluent to the biological compartment, complete isolation of the microbial culture could not be guaranteed and the membrane might ultimately be permeable to other species present in the aqueous vaccine wastewater. It was therefore decided not to operate the initially targeted integrated system but, instead, the biological system by itself. Additionally, attention was given to the development of a thorough understanding of the transport mechanisms involved in the solubilisation and transport of water through supported liquid membranes with RTILs as well as to the evaluation of the effect of water uptake by the SLM in the transport mechanisms of water-soluble solutes and its effect on SLM performance. The results obtained highlighted the determinant role played by water – solubilised inside the ionic liquids – on the transport mechanism. It became clear that the transport mechanism of water and water-soluble solutes through SLMs with [CnMIM][PF6] RTILs was regulated by the dynamics of water clusters inside the RTIL, rather than by molecular diffusion through the bulk of the ionic liquid. Although the stability tests vi performed showed that there were no significant losses of organic phase from the membrane pores, the formation of water clusters inside the ionic liquid, which constitute new, non-selective environments for solute transport, leads to a clear deterioration of SLM performance and selectivity. Nevertheless, electrical impedance spectroscopy characterisation of the SLMs showed that the formation of water clusters did not seem to have a detrimental effect on the SLMs’ electrical characteristics and highlighted the potential of using this type of membranes in electrochemical applications with low resistance requirements. The second part of the work studied the kinetics of thiomersal degradation by a pure culture of P. putida spi3 strain, in batch culture and using a synthe tic wastewater. A continuous ly stirred tank reactor fed with the synthetic wastewater was also operated and the bioreactor’s performance and robustness, when exposed to thiomersal shock loads, were evaluated. Finally, a bioreactor for the biological treatment of a real va ccine production effluent was set up and operated at different dilution rates. Thus it was possible to treat a real thiomersal-contaminated effluent, lowering the outlet mercury concentration to values below the European limit for mercury effluent discharges.

External and internal dynamics in the EU's counter-terrorism policy: a political and legal analysis

Tese de Doutoramento em Ciência Política e Relações Internacionais

Using mouse dynamics to assess stress during online exams

"Lecture notes in computer science series", ISSN 0302-9743, vol. 9121

Dynamics of HIV latency and reactivation in a primary CD4+ T cell model.

HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.

Setting boundaries: brain dynamics of modal and amodal illusory shape completion in humans.

Normal visual perception requires differentiating foreground from background objects. Differences in physical attributes sometimes determine this relationship. Often such differences must instead be inferred, as when two objects or their parts have the same luminance. Modal completion refers to such perceptual "filling-in" of object borders that are accompanied by concurrent brightness enhancement, in turn termed illusory contours (ICs). Amodal completion is filling-in without concurrent brightness enhancement. Presently there are controversies regarding whether both completion processes use a common neural mechanism and whether perceptual filling-in is a bottom-up, feedforward process initiating at the lowest levels of the cortical visual pathway or commences at higher-tier regions. We previously examined modal completion (Murray et al., 2002) and provided evidence that the earliest modal IC sensitivity occurs within higher-tier object recognition areas of the lateral occipital complex (LOC). We further proposed that previous observations of IC sensitivity in lower-tier regions likely reflect feedback modulation from the LOC. The present study tested these proposals, examining the commonality between modal and amodal completion mechanisms with high-density electrical mapping, spatiotemporal topographic analyses, and the local autoregressive average distributed linear inverse source estimation. A common initial mechanism for both types of completion processes (140 msec) that manifested as a modulation in response strength within higher-tier visual areas, including the LOC and parietal structures, is demonstrated, whereas differential mechanisms were evident only at a subsequent time period (240 msec), with amodal completion relying on continued strong responses in these structures.

A geometric mechanism of diffusion: Rigorous verification in a priori unstable Hamiltonian systems

In this paper we consider a representative a priori unstable Hamiltonian system with 2+1/2 degrees of freedom, to which we apply the geometric mechanism for diffusion introduced in the paper Delshams et al., Mem.Amer.Math. Soc. 2006, and generalized in Delshams and Huguet, Nonlinearity 2009, and provide explicit, concrete and easily verifiable conditions for the existence of diffusing orbits. The simplification of the hypotheses allows us to perform explicitly the computations along the proof, which contribute to present in an easily understandable way the geometric mechanism of diffusion. In particular, we fully describe the construction of the scattering map and the combination of two types of dynamics on a normally hyperbolic invariant manifold.

Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Actin filament dynamics impacts keratinocyte stem cell maintenance.

Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1.

Implementation, elimination of weakly dominated strategies and evolutionary dynamics

This paper is concerned with the realism of mechanisms that implementsocial choice functions in the traditional sense. Will agents actually playthe equilibrium assumed by the analysis? As an example, we study theconvergence and stability properties of Sj\"ostr\"om's (1994) mechanism, onthe assumption that boundedly rational players find their way to equilibriumusing monotonic learning dynamics and also with fictitious play. Thismechanism implements most social choice functions in economic environmentsusing as a solution concept the iterated elimination of weakly dominatedstrategies (only one round of deletion of weakly dominated strategies isneeded). There are, however, many sets of Nash equilibria whose payoffs maybe very different from those desired by the social choice function. Withmonotonic dynamics we show that many equilibria in all the sets ofequilibria we describe are the limit points of trajectories that havecompletely mixed initial conditions. The initial conditions that lead tothese equilibria need not be very close to the limiting point. Furthermore,even if the dynamics converge to the ``right'' set of equilibria, it stillcan converge to quite a poor outcome in welfare terms. With fictitious play,if the agents have completely mixed prior beliefs, beliefs and play convergeto the outcome the planner wants to implement.

Speculative dynamics in the term structure of interest rates

When long maturity bonds are traded frequently and traders have non-nestedinformation sets, speculative behavior in the sense of Harrison and Kreps (1978) arises.Using a term structure model displaying such speculative behavior, this paper proposesa conceptually and observationally distinct new mechanism generating time varying predictableexcess returns. It is demonstrated that (i) dispersion of expectations about futureshort rates is sufficient for individual traders to systematically predict excess returns and(ii) the new term structure dynamics driven by speculative trade is orthogonal to publicinformation in real time, but (iii) can nevertheless be quantified using only publicly availableyield data. The model is estimated using monthly data on US short to medium termTreasuries from 1964 to 2007 and it provides a good fit of the data. Speculative dynamicsare found to be quantitatively important, potentially accounting for a substantial fractionof the variation of bond yields and appears to be more important at long maturities.

Adaptive dynamics and the implementation problem with complete information

This paper studies the equilibrating process of several implementationmechanisms using naive adaptive dynamics. We show that the dynamics convergeand are stable, for the canonical mechanism of implementation in Nash equilibrium.In this way we cast some doubt on the criticism of ``complexity'' commonlyused against this mechanism. For mechanisms that use more refined equilibrium concepts,the dynamics converge but are not stable. Some papers in the literatureon implementation with refined equilibrium concepts have claimed that themechanisms they propose are ``simple'' and implement ``everything'' (incontrast with the canonical mechanism). The fact that some of these ``simple''mechanisms have unstable equilibria suggests that these statements shouldbe interpreted with some caution.

International Standardisation and Local Participatory Dynamics : The INTERNORM Project

This paper presents a pilot project (INTERNORM) funded by the University of Lausanne (2010 - 2013) to support the involvement of civil society organisations (CSO) in international standard setting bodies such as the ISO. It analyses how a distinct participatory mechanism can influence the institutional environment of technical diplomacy in which standards are shaped. The project is an attempt to respond to the democratic deficit attested in the field of international standardisation, formally open to civil society participation, but still largely dominated by expert knowledge and market players. Many international standards have direct implications on society as a whole, but CSOs (consumers and environmental associations, trade unions) are largely under-represented in negotiation arenas. The paper draws upon international relations literature on new institutional forms in global governance and studies of participation in science and technology. It argues that there are significant limitations to the rise of civil society participation in such global governance mechanisms. The INTERNORM project has been designed as a platform of knowledge exchange between CSO and academic experts, with earmarked funding and official membership to a national standardisation body. But INTERNORM cannot substitute for a long- established lack of resources in time, money and expertise of CSOs. Despite high entry costs into technical diplomacy, participation thus appears as less a matter of upstream engagement, or of procedure only, than of dedicated means to shift the geometry of actors and the framing of socio-technical change.

Influence of ionization state on the activation of temocapril by hCES1: a molecular-dynamics study.

Temocapril is a prodrug whose hydrolysis by carboxylesterase 1 (CES1) yields the active ACE inhibitor temocaprilat. This molecular-dynamics (MD) study uses a resolved structure of the human CES1 (hCES1) to investigate some mechanistic details of temocapril hydrolysis. The ionization constants of temocapril (pK1 and pK3) and temocaprilat (pK1, pK2, and pK3) were determined experimentally and computationally using commercial algorithms. The constants so obtained were in good agreement and revealed that temocapril exists mainly in three ionic forms (a cation, a zwitterion, and an anion), whereas temocaprilat exists in four major ionic forms (a cation, a zwitterion, an anion, and a dianion). All these ionic forms were used as ligands in 5-ns MS simulations. While the cationic and zwitterionic forms of temocapril were involved in an ion-pair bond with Glu255 suggestive of an inhibitor behavior, the anionic form remained in a productive interaction with the catalytic center. As for temocaprilat, its cation appeared trapped by Glu255, while its zwitterion and anion made a slow departure from the catalytic site and a partial egress from the protein. Only its dianion was effectively removed from the catalytic site and attracted to the protein surface by Lys residues. A detailed mechanism of product egress emerges from the simulations.

Comparison between molecular dynamics abd Monte Carlo simulations of an ordering process in a binary alloy

Ordering in a binary alloy is studied by means of a molecular-dynamics (MD) algorithm which allows to reach the domain growth regime. Results are compared with Monte Carlo simulations using a realistic vacancy-atom (MC-VA) mechanism. At low temperatures fast growth with a dynamical exponent x>1/2 is found for MD and MC-VA. The study of a nonequilibrium ordering process with the two methods shows the importance of the nonhomogeneity of the excitations in the system for determining its macroscopic kinetics.