Doctor of Philosophy

dissertationDespite the tremendous potential of short interfering RNA (siRNA) as a novel biopharmaceutical, its therapeutic utility has not been maximized mainly due to lack of proper in vivo delivery vehicle, off-target effects and several off-pathway protein interactions instigating immunostimulation. Judicious chemical modification of different parts of the siRNA was foreseen as a potential solution to the off-target gene silencing and the off-pathway protein binding. In this study, 8-alkoxyadenosines were explored as a nucleobase modification in the context of the siRNA-based RNA interference (RNAi). These nucleosides are unusual in that they have the potential to exist as an equilibrium mixture of the syn and anti conformers. When placed opposite to U in the complementary strand, 8- alkoxyadenosines can exist in normal anti conformation and form canonical Watson- Crick hydrogen bonding; interestingly, with G as the base-pairing partner, these unusual nucleosides can potentially flip into the syn conformation and form unorthodox Hoogsteen base-pairing. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions - two in the seed region, one at the cleavage junction and another nearer to the 3´-end of the guide strands. Thermal stabilities of the corresponding siRNA duplexes showed that U is still preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified iv positive control siRNAs, singly modified siRNAs have knocked down caspase 2 insert mRNA (generated from a recombinant plasmid) efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. Persistent placement of steric blockade in the minor groove affected the RNAi efficacy significantly; this observation supports the hypothesis and indicates the necessity of ‘switching' the bulky alkyloxy groups in the major groove, when modified siRNAs interact with the RISC assembly. SiRNAs modified at positions 6 and 10 of the guide strand were found to be effective against preventing interaction with the RNA-dependent protein kinase (PKR). In summary, 8-alkoxyadenosine-containing siRNAs prevented undesired off-pathway protein binding, without compromising the RNAi efficacy significantly

Etude du lien entre la fréquence et les puissances actives pour le dimensionnement d'un microréseau alternatif îloté avec sources d'énergie renouvelables

La qualité d’un réseau électrique se mesure notamment par sa résilience, à savoir sa capacité à continuer de fournir, de façon stable, une électricité de qualité malgré les incidents. Cette continuité d’alimentation est notamment assurée par le réglage de la fréquence, ce dernier faisant office de moyen de coopération entre les différentes sources du réseau. Les travaux de cette thèse étudient en profondeur les différentes façons d’assurer ce réglage de la fréquence sur des microréseaux alternatifs îlotés au regard des problématiques nouvelles : intégration croissante de sources d’énergie renouvelables intermittentes et baisse (voire disparition) de l’inertie inhérente aux groupes synchrones tournants. L’objectif principal est de proposer un ensemble de modèles analytiques permettant d’appréhender le comportement dynamique de la fréquence et des puissances actives transitant sur un microréseau, quelle que soit la topologie de ce dernier, afin de constituer une aide au dimensionnement. À cet objectif global s’ajoute un certain nombre de sous-objectifs qui structurent et guident l’ensemble des travaux :- Un objectif pédagogique : les couplages entre l’équilibre des puissances actives et la fréquence sur un réseau alternatif sont clairement explicités, que ces couplages relèvent de la structure et des paramètres physiques du microréseau ou qu’ils soient liés aux lois et paramètres de commande. La lecture des modèles doit ainsi permettre de comprendre analytiquement les différents liens de cause à effet entre les paramètres du système, la fréquence et les puissances,et les spécificités propres à chaque topologie de microréseau.- Un objectif de simplicité et de modularité : les modèles proposés doivent pouvoir être adaptés selon les cas particuliers de topologies, afin que tout lecteur puisse les réutiliser facilement en dehors du cadre de cette étude. Pour ce faire, ces travaux utilisent des modèles mathématiques sous forme de schémas blocs, intégrables sous Matlab.- Enfin, un objectif de modélisation « grand signal » : pour trancher avec l’approche petit signal de beaucoup de modélisations de microréseaux classiques, et pour permettre d’appréhender le comportement du microréseau sur une large plage de déséquilibre de puissanc

Time-dependent density functional theory calculation of van der Waals coefficient of sodium clusters

In this paper we employ all-electron \textit{ab-initio} time-dependent density functional theory based method to calculate the long range dipole-dipole dispersion coefficient (van der Waals coefficient) $C_{6}$ of sodium atom clusters containing even number of atoms ranging from 2 to 20 atoms. The dispersion coefficients are obtained via Casimir-Polder relation. The calculations are carried out with two different exchange-correlation potentials: (i) the asymptotically correct statistical average of orbital potential (SAOP) and (ii) Vosko-Wilk-Nusair representation of exchange-correlation potential within local density approximation. A comparison with the other theoretical results has been performed. We also present the results for the static polarizabilities of sodium clusters and also compare them with other theoretical and experimental results. These comparisons reveal that the SAOP results for C_{6} and static polarizability are quite accurate and very close to the experimental results. We examine the relationship between volume of the cluster and van der Waals coefficient and find that to a very high degree of correlation C_{6} scales as square of the volume. We also present the results for van der Waals coefficient corresponding to cluster-Ar atom and cluster-N_{2} molecule interactions.Comment: 22 pages including 6 figures. To be published in Journal of Chemical Physic

Ab initio calculations of the hydrogen bond

Recent x-ray Compton scattering experiments in ice have provided useful information about the quantum nature of the interaction between H$_2$O monomers. The hydrogen bond is characterized by a certain amount of charge transfer which could be determined in a Compton experiment. We use ab-initio simulations to investigate the hydrogen bond in H$_2$O structures by calculating the Compton profile and related quantities in three different systems, namely the water dimer, a cluster containing 12 water molecules and the ice crystal. We show how to extract estimates of the charge transfer from the Compton profiles.Comment: 16 pages, 7 figures, to appear in Phys. Rev.

Calculation of valence electron momentum densities using the projector augmented-wave method

We present valence electron Compton profiles calculated within the density-functional theory using the all-electron full-potential projector augmented-wave method (PAW). Our results for covalent (Si), metallic (Li, Al) and hydrogen-bonded ((H_2O)_2) systems agree well with experiments and computational results obtained with other band-structure and basis set schemes. The PAW basis set describes the high-momentum Fourier components of the valence wave functions accurately when compared with other basis set schemes and previous all-electron calculations.Comment: Submitted to Journal of Physics and Chemistry of Solids on September 17 2004. Revised version submitted on December 13 200

Network equilibration and first-principles liquid water.

Motivated by the very low diffusivity recently found in ab initio simulations of liquid water, we have studied its dependence with temperature, system size, and duration of the simulations. We use ab initio molecular dynamics (AIMD), following the Born-Oppenheimer forces obtained from density-functional theory (DFT). The linear-scaling capability of our method allows the consideration of larger system sizes (up to 128 molecules in this study), even if the main emphasis of this work is in the time scale. We obtain diffusivities that are substantially lower than the experimental values, in agreement with recent findings using similar methods. A fairly good agreement with D(T) experiments is obtained if the simulation temperature is scaled down by approximately 20%. It is still an open question whether the deviation is due to the limited accuracy of present density functionals or to quantum fluctuations, but neither technical approximations (basis set, localization for linear scaling) nor the system size (down to 32 molecules) deteriorate the DFT description in an appreciable way. We find that the need for long equilibration times is consequence of the slow process of rearranging the H-bond network (at least 20 ps at AIMDs room temperature). The diffusivity is observed to be very directly linked to network imperfection. This link does not appear an artifact of the simulations, but a genuine property of liquid water

SCN1A variants from bench to bedside-improved clinical prediction from functional characterization

Variants in the SCN1A gene are associated with a wide range of disorders including genetic epilepsy with febrile seizures plus (GEFS+), familial hemiplegic migraine (FHM), and the severe childhood epilepsy Dravet syndrome (DS). Predicting disease outcomes based on variant type remains challenging. Despite thousands of SCN1A variants being reported, only a minority has been functionally assessed. We review the functional SCN1A work performed to date, critically appraise electrophysiological measurements, compare this to in silico predictions, and relate our findings to the clinical phenotype. Our results show, regardless of the underlying phenotype, that conventional in silico software correctly predicted benign from pathogenic variants in nearly 90%, however was unable to differentiate within the disease spectrum (DS vs. GEFS+ vs. FHM). In contrast, patch‐clamp data from mammalian expression systems revealed functional differences among missense variants allowing discrimination between disease severities. Those presenting with milder phenotypes retained a degree of channel function measured as residual whole‐cell current, whereas those without any whole‐cell current were often associated with DS (p = .024). These findings demonstrate that electrophysiological data from mammalian expression systems can serve as useful disease biomarker when evaluating SCN1A variants, particularly in view of new and emerging treatment options in DS

A new approach to local hardness

The applicability of the local hardness as defined by the derivative of the chemical potential with respect to the electron density is undermined by an essential ambiguity arising from this definition. Further, the local quantity defined in this way does not integrate to the (global) hardness - in contrast with the local softness, which integrates to the softness. It has also been shown recently that with the conventional formulae, the largest values of local hardness do not necessarily correspond to the hardest regions of a molecule. Here, in an attempt to fix these drawbacks, we propose a new approach to define and evaluate the local hardness. We define a local chemical potential, utilizing the fact that the chemical potential emerges as the additive constant term in the number-conserving functional derivative of the energy density functional. Then, differentiation of this local chemical potential with respect to the number of electrons leads to a local hardness that integrates to the hardness, and possesses a favourable property; namely, within any given electron system, it is in a local inverse relation with the Fukui function, which is known to be a proper indicator of local softness in the case of soft systems. Numerical tests for a few selected molecules and a detailed analysis, comparing the new definition of local hardness with the previous ones, show promising results.Comment: 30 pages (including 6 figures, 1 table

Work functions, ionization potentials, and in-between: Scaling relations based on the image charge model

We revisit a model in which the ionization energy of a metal particle is associated with the work done by the image charge force in moving the electron from infinity to a small cut-off distance just outside the surface. We show that this model can be compactly, and productively, employed to study the size dependence of electron removal energies over the range encompassing bulk surfaces, finite clusters, and individual atoms. It accounts in a straightforward manner for the empirically known correlation between the atomic ionization potential (IP) and the metal work function (WF), IP/WF$\sim$2. We formulate simple expressions for the model parameters, requiring only a single property (the atomic polarizability or the nearest neighbor distance) as input. Without any additional adjustable parameters, the model yields both the IP and the WF within $\sim$10% for all metallic elements, as well as matches the size evolution of the ionization potentials of finite metal clusters for a large fraction of the experimental data. The parametrization takes advantage of a remarkably constant numerical correlation between the nearest-neighbor distance in a crystal, the cube root of the atomic polarizability, and the image force cutoff length. The paper also includes an analytical derivation of the relation of the outer radius of a cluster of close-packed spheres to its geometric structure.Comment: Original submission: 8 pages with 7 figures incorporated in the text. Revised submission (added one more paragraph about alloy work functions): 18 double spaced pages + 8 separate figures. Accepted for publication in PR

Proton transfer or hemibonding? The structure and stability of radical cation clusters

The basin hopping search algorithm in conjunction with second-order Moller-Plesset perturbation theory is used to determine the lowest energy structures of the radical cation clusters (NH_3)_n^+, (H_2O)_n^+, (HF)_n^+, (PH_3)_n^+, (H_2S)_n^+ and (HCl)_n^+, where n=2-4. The energies of the most stable structures are subsequently evaluated using coupled cluster theory in conjunction with the aug-cc-pVTZ basis set. These cationic clusters can adopt two distinct structural types, with some clusters showing an unusual type of bonding, often referred to as hemibonding, while other clusters undergo proton transfer to give an ion and radical. It is found that proton transfer based structures are preferred by the (NH_3)_n+, (H_2O)_n^+, and (HF)_n^+ clusters while hemibonded structures are favoured by (PH_3)_n^+, (H_2S)_n^+ and (HCl)_n^+. These trends can be attributed to the relative strengths of the molecules and molecular cations as Brønsted bases and acids, respectively, and the strength of the interaction between the ion and radical in the ion-radical clusters