Caractérisation des structures de septines hautement organisées chez la drosophile et leur interaction avec le cytosquelette d’actine

Les septines sont des GTPases conservées dérégulées dans le cancer et les maladies neurodégénératives. Elles servent de protéines d’échafaudage et forment une barrière de diffusion à la membrane plasmique et au corps central lors de la cytokinèse. Elles interagissent avec l’actine et s’organisent en complexes qui polymérisent et forment des structures hautement organisées (anneaux et filaments). Leur dynamique d’assemblage et leur rôle dans la cellule restent à être élucidés. La Drosophile est un modèle simple pour l’étude des septines puisqu’on n’y retrouve que 5 gènes (sep1, sep2, sep4, sep5, peanut) comparativement aux 13 gènes chez l’humain. À l’aide d’un anticorps contre Pnut, nous avons identifié des structures tubulaires dans 30% des cellules S2 de Drosophile. Mon projet a comme but de caractériser ces tubes en élucidant leurs constituants, leur comportement et leurs propriétés pour mieux clarifier le mécanisme par lequel les septines forment des structures hautement organisées et interagissent avec le cytosquelette d’actine. Par immunofluorescence, j’ai pu démontrer que ces tubes sont cytoplasmiques, en mitose ou interphase, ce qui suggère qu’ils ne sont pas régulés par le cycle cellulaire. Pour investiguer la composition et les propriétés dynamiques de ces tubes, j’ai généré une lignée cellulaire exprimant Sep2-GFP qui se localise aux tubes et des ARNi contre les cinq septines. Trois septines sont importantes pour la formation de ces tubes et anneaux notamment Sep1, Sep2 et Pnut. La déplétion de Sep1 cause la dispersion du signal GFP en flocons, tandis que la déplétion de Sep2 ou de Pnut mène à la dispersion du signal GFP uniformément dans la cellule. Des expériences de FRAP sur la lignée Sep2-GFP révèlent un signal de retour très lent, ce qui indique que ces structures sont très stables. J’ai aussi démontré une relation entre l’actine et les septines. Le traitement avec la Latrunculin A (un inhibiteur de la polymérisation de l’actine) ou la Jasplakinolide (un stabilisateur des filaments d’actine) mène à la dépolymérisation rapide (< 30 min) des tubes en anneaux flottants dans le cytoplasme, même si ces tubes ne sont pas reconnus suite à un marquage de la F-actine. L’Actin05C-mCherry se localise aux tubes, tandis que le mutant déficient de la polymérisation, Actin05C-R62D-mCherry perd cette localisation. On observe aussi que la déplétion de la Cofiline et de l’AIP1 (ce qui déstabilise l’actine) mène au même phénotype que le traitement avec la Latrunculine A ou la Jasplakinolide. Alors on peut conclure qu’un cytosquelette d’actine dynamique est nécessaire pour la formation et le maintien des tubes de septines. Les futures études auront comme but de mieux comprendre l’organisation des septines en structures hautement organisées et leur relation avec l’actine. Ceci sera utile pour l’élaboration du réseau d’interactions des septines qui pourra servir à expliquer leur dérégulation dans le cancer et les maladies neurodégénératives.

Molecular Origins of Higher Harmonics in Large-Amplitude Oscillatory Shear Flow: Shear Stress Response

Recent work has focused on deepening our understanding of the molecular origins of the higher harmonics that arise in the shear stress response of polymeric liquids in large-amplitude oscillatory shear flow. For instance, these higher harmonics have been explained by just considering the orientation distribution of rigid dumbbells suspended in a Newtonian solvent. These dumbbells, when in dilute suspension, form the simplest relevant molecular model of polymer viscoelasticity, and this model specifically neglects interactions between the polymer molecules [R.B. Bird et al., J Chem Phys, 140, 074904 (2014)]. In this paper, we explore these interactions by examining the Curtiss-Bird model, a kinetic molecular theory designed specifically to account for the restricted motions that arise when polymer chains are concentrated, thus interacting and specifically, entangled. We begin our comparison using a heretofore ignored explicit analytical solution [Fan and Bird, JNNFM, 15, 341 (1984)]. For concentrated systems, the chain motion transverse to the chain axis is more restricted than along the axis. This anisotropy is described by the link tension coefficient, ε, for which several special cases arise: ε = 0 corresponds to reptation, ε > 1/8 to rod-climbing, 1/2 ≥ ε ≥ 3/4 to reasonable predictions for shear-thinning in steady simple shear flow, and ε = 1 to the dilute solution without hydrodynamic interaction. In this paper, we examine the shapes of the shear stress versus shear rate loops for the special cases ε = (0,1/8, 3/8,1) , and we compare these with those of rigid dumbbell and reptation model predictions.

MEF2C: expression, regulation and interaction with target genes in health and diseases

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Insights into (S)-rivastigmine inhibition of butyrylcholinesterase (BuChE): Molecular docking and saturation transfer difference NMR (STD-NMR)

Rivastigmine is a very important drug prescribed for the treatment of Alzheimer's disease (AD) symptoms. It is a dual inhibitor, in that it inhibits both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). For our screening program on the discovery of new rivastigmine analogue hits for human butyrylcholinesterase (hBuChE) inhibition, we investigated the interaction of this inhibitor with BuChE using the complimentary approach of the biophysical method, saturation transfer difference (STD)-NMR and molecular docking. This allowed us to obtain essential information on the key binding interactions between the inhibitor and the enzyme to be used for screening of hit compounds. The main conclusions obtained from this integrated study was that the most dominant interactions were (a) H-bonding between the carbamate carbonyl of the inhibitor and the NH group of the imidazole unit of H434, (b) stacking of the aromatic unit of the inhibitor and the W82 aromatic unit in the choline binding pocket via pi-pi interactions and (c) possible CH/pi interactions between the benzylic methyl group and the N-methyl groups of the inhibitor and W82 of the enzyme.

Identification of differentially expressed genes associated with pathogen-host interaction in 'Villard Blanc' and its expression analysis in grapevine cultivars resistant and susceptible to downy mildew.

2011

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: Part 14. The infrared and Raman characteristics of phthalocyanine in “pinwheel-like” homoleptic bis[1,8,15,22-tetrakis(3-pentyloxy)phthalocyaninato] rare earth(III) double-decker complexes

The infrared (IR) spectroscopic data and Raman spectroscopic properties for a series of 13 “pinwheel-like” homoleptic bis(phthalocyaninato) rare earth complexes M[Pc(α-OC5H11)4]2 [M = Y and Pr–Lu except Pm; H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected and comparatively studied. Both the IR and Raman spectra for M[Pc(α-OC5H11)4]2 are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues, namely M(Pc)2 and M[Pc(OC8H17)8]2, but resemble (for IR) or are a bit more complicated (for Raman) than those of heteroleptic counterparts M(Pc)[Pc(α-OC5H11)4], revealing the decreased molecular symmetry of these double-decker compounds, namely S8. Except for the obvious splitting of the isoindole breathing band at 1110–1123 cm−1, the IR spectra of M[Pc(α-OC5H11)4]2 are quite similar to those of corresponding M(Pc)[Pc(α-OC5H11)4] and therefore are similarly assigned. With laser excitation at 633 nm, Raman bands derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. The IR spectra reveal that the frequencies of pyrrole stretching and pyrrole stretching coupled with the symmetrical CH bending of –CH3 groups are sensitive to the rare earth ionic size, while the Raman technique shows that the bands due to the isoindole stretchings and the coupled pyrrole and aza stretchings are similarly affected. Nevertheless, the phthalocyanine monoanion radical Pc′− IR marker band of bis(phthalocyaninato) complexes involving the same rare earth ion is found to shift to lower energy in the order M(Pc)2 > M(Pc)[Pc(α-OC5H11)4] > M[Pc(α-OC5H11)4]2, revealing the weakened π–π interaction between the two phthalocyanine rings in the same order.

Time-Dependent Density Functional Molecular Orbital and Excited State Calculations on Bis(porphyrinyl)butadiynes in the Monocationic, Neutral, Monoanionic, and Dianionic Oxidation States

We report a theoretical study of the multiple oxidation states (1+, 0, 1−, and 2−) of a meso,meso-linked diporphyrin, namely bis[10,15,20-triphenylporphyrinatozinc(II)-5-yl]butadiyne (4), using Time-Dependent Density Functional Theory (TDDFT). The origin of electronic transitions of singlet excited states is discussed in comparison to experimental spectra for the corresponding oxidation states of the close analogue bis{10,15,20-tris[3‘,5‘-di-tert-butylphenyl]porphyrinatozinc(II)-5-yl}butadiyne (3). The latter were measured in previous work under in situ spectroelectrochemical conditions. Excitation energies and orbital compositions of the excited states were obtained for these large delocalized aromatic radicals, which are unique examples of organic mixed-valence systems. The radical cations and anions of butadiyne-bridged diporphyrins such as 3 display characteristic electronic absorption bands in the near-IR region, which have been successfully predicted with use of these computational methods. The radicals are clearly of the “fully delocalized” or Class III type. The key spectral features of the neutral and dianionic states were also reproduced, although due to the large size of these molecules, quantitative agreement of energies with observations is not as good in the blue end of the visible region. The TDDFT calculations are largely in accord with a previous empirical model for the spectra, which was based simplistically on one-electron transitions among the eight key frontier orbitals of the C4 (1,4-butadiyne) linked diporphyrins.