The Role of Second Generation Antiretroviral Drugs in HIV-1 Subtype B and non-B Variants Harboring Natural Polymorphisms and Drug Resistance Mutations.

Cette thèse traite de la résistance du VIH-1 aux antirétroviraux, en particulier de l'activité antivirale de plusieurs inhibiteurs non nucléosidiques de la transcriptase inverse (INNTI) ainsi que des inhibiteurs de protéase (IP). Nous avons exploré l’émergence et la spécificité des voies de mutations qui confèrent la résistance contre plusieurs nouveaux INNTI (étravirine (ETR) et rilpivirine (RPV)) (chapitres 2 et 3). En outre, le profil de résistance et le potentiel antirétroviral d'un nouvel IP, PL-100, est présenté dans les chapitres 4 et 5. Pour le premier projet, nous avons utilisé des sous-types B et non-B du VIH-1 pour sélectionner des virus résistants à ETR, et ainsi montré que ETR favorise l’émergence des mutations V90I, K101Q, E138K, V179D/E/F, Y181C, V189I, G190E, H221H/Y et M230L, et ce, en 18 semaines. Fait intéressant, E138K a été la première mutation à émerger dans la plupart des cas. Les clones viraux contenant E138K ont montré un faible niveau de résistance phénotypique à ETR (3,8 fois) et une diminution modeste de la capacité de réplication (2 fois) par rapport au virus de type sauvage. Nous avons également examiné les profils de résistance à ETR et RPV dans les virus contenant des mutations de résistance aux INNTI au début de la sélection. Dans le cas du virus de type sauvage et du virus contenant la mutation unique K103N, les premières mutations à apparaître en présence d’ETR ou de RPV ont été E138K ou E138G suivies d’autres mutations de résistance aux INNTI. À l’inverse, dans les mêmes conditions, le virus avec la mutation Y181C a évolué pour produire les mutations V179I/F ou A62V/A, mais pas E138K/G. L'ajout de mutations à la position 138 en présence de Y181C n'augmente pas les niveaux de résistance à ETR ou RPV. Nous avons également observé que la combinaison de Y181C et E138K peut conduire à un virus moins adapté par rapport au virus contenant uniquement Y181C. Sur la base de ces résultats, nous suggérons que les mutations Y181C et E138K peuvent être antagonistes. L’analyse de la résistance au PL-100 des virus de sous-type C et CRF01_AE dans les cellules en culture est décrite dans le chapitre 4. Le PL-100 sélectionne pour des mutations de résistance utilisant deux voies distinctes, l'une avec les mutations V82A et L90M et l'autre avec T80I, suivi de l’addition des mutations M46I/L, I54M, K55R, L76F, P81S et I85V. Une accumulation d'au moins trois mutations dans le rabat protéique et dans le site actif est requise dans chaque cas pour qu’un haut niveau de résistance soit atteint, ce qui démontre que le PL-100 dispose d'une barrière génétique élevée contre le développement de la résistance. Dans le chapitre 5, nous avons évalué le potentiel du PL-100 en tant qu’inhibiteur de protéase de deuxième génération. Les virus résistants au PL-100 émergent en 8-48 semaines alors qu’aucune mutation n’apparaît avec le darunavir (DRV) sur une période de 40 semaines. La modélisation moléculaire montre que la haute barrière génétique du DRV est due à de multiples interactions avec la protéase dont des liaison hydrogènes entre les groupes di-tétrahydrofuranne (THF) et les atomes d'oxygène des acides aminés A28, D29 et D30, tandis que la liaison de PL-100 est principalement basée sur des interactions polaires et hydrophobes délocalisées à travers ses groupes diphényle. Nos données suggèrent que les contacts de liaison hydrogène et le groupe di-THF dans le DRV, ainsi que le caractère hydrophobe du PL-100, contribuent à la liaison à la protéase ainsi qu’à la haute barrière génétique contre la résistance et que la refonte de la structure de PL-100 pour inclure un groupe di-THF pourrait améliorer l’activité antivirale et le profil de résistance.

Prevalence, distribution and drug resistance of motile aeromonads in freshwater ornamental fishes

The objective of the present study was to assess the prevalence of various motile aeromonads in freshwater ornamental fishes and to elucidate the antibiogram and beta hemolytic activity among the isolates. A total of 120 ornamental fish samples were screened and analyzed for Aeromonas spp. Motile aeromonads were isolated from 37.5% of the ornamental fish samples. Various species of motile aeromonads such as Aeromonas caviae, Aeromonas hydrophila, Aeromonas jandaei, Aeromonas schubertii, Aeromonas sobria, Aeromonas trota and Aeromonas veronii were detected. All the isolates were sensitive to ceftazidime, chloramphenicol, ciprofloxacin and gentamicin. Multiple antibiotic resistance was observed in 58% of the isolates.

Distribution, Extracellular Virulence Factors and Drug Resistance of Motile Aeromonads in Fresh Water Ornamental Fishes and Associated Carriage Water

During last decades there has been a continuous growth of aquaculture industries all over the world and taking into consideration the spurt in freshwater ornamental fish aquaculture and trade in Kerala, the present study was aimed to assess the prevalence of various motile Aeromonas spp. in fresh water ornamental fishes and associated carriage water. The extracellular virulence factors and the antibiogram of the isolates were also elucidated. Various species of motile aeromonads such as Aeromonas caviae, A. hydrophila, A. jandaei, A. schubertii, A. sobria, A. trota and A. veronii were detected. Aeromonas sobria predominated both fish and water samples. Extracellular enzymes and toxins produced by motile aeromonds are important elements of bacterial virulence. The production of extracellular virulence factors - proteases, lipase, DNase and haemolysin by the isolates were studied. All the isolates from both fish and water samples produced gelatinase and nuclease but the ability to produce lipase, caseinase and haemolysins was found to vary among isolates from different sources. Among the 15 antibiotics to which the isolates were tested, all the isolates were found to be sensitive to chloramphenicol, ciprofloxacin and gentamicin and resistant to amoxycillin. Local aquarists maintain the fish in crowded stressful conditions, which could trigger infections by the obligate/ opportunistic pathogenic members among motile aeromonads

Protein Folding Simulations: Confinement, External Fields and Sequence Design

The present Thesis looks at the problem of protein folding using Monte Carlo and Langevin simulations, three topics in protein folding have been studied: 1) the effect of confining potential barriers, 2) the effect of a static external field and 3) the design of amino acid sequences which fold in a short time and which have a stable native state (global minimum). Regarding the first topic, we studied the confinement of a small protein of 16 amino acids known as 1NJ0 (PDB code) which has a beta-sheet structure as a native state. The confinement of proteins occurs frequently in the cell environment. Some molecules called Chaperones, present in the cytoplasm, capture the unfolded proteins in their interior and avoid the formation of aggregates and misfolded proteins. This mechanism of confinement mediated by Chaperones is not yet well understood. In the present work we considered two kinds of potential barriers which try to mimic the confinement induced by a Chaperon molecule. The first kind of potential was a purely repulsive barrier whose only effect is to create a cavity where the protein folds up correctly. The second kind of potential was a barrier which includes both attractive and repulsive effects. We performed Wang-Landau simulations to calculate the thermodynamical properties of 1NJ0. From the free energy landscape plot we found that 1NJ0 has two intermediate states in the bulk (without confinement) which are clearly separated from the native and the unfolded states. For the case of the purely repulsive barrier we found that the intermediate states get closer to each other in the free energy landscape plot and eventually they collapse into a single intermediate state. The unfolded state is more compact, compared to that in the bulk, as the size of the barrier decreases. For an attractive barrier modifications of the states (native, unfolded and intermediates) are observed depending on the degree of attraction between the protein and the walls of the barrier. The strength of the attraction is measured by the parameter $\epsilon$. A purely repulsive barrier is obtained for $\epsilon=0$ and a purely attractive barrier for $\epsilon=1$. The states are changed slightly for magnitudes of the attraction up to $\epsilon=0.4$. The disappearance of the intermediate states of 1NJ0 is already observed for $\epsilon =0.6$. A very high attractive barrier ($\epsilon \sim 1.0$) produces a completely denatured state. In the second topic of this Thesis we dealt with the interaction of a protein with an external electric field. We demonstrated by means of computer simulations, specifically by using the Wang-Landau algorithm, that the folded, unfolded, and intermediate states can be modified by means of a field. We have found that an external field can induce several modifications in the thermodynamics of these states: for relatively low magnitudes of the field ($<2.06 \times 10^8$ V/m) no major changes in the states are observed. However, for higher magnitudes than ($6.19 \times 10^8$ V/m) one observes the appearance of a new native state which exhibits a helix-like structure. In contrast, the original native state is a $\beta$-sheet structure. In the new native state all the dipoles in the backbone structure are aligned parallel to the field. The design of amino acid sequences constitutes the third topic of the present work. We have tested the Rate of Convergence criterion proposed by D. Gridnev and M. Garcia ({\it work unpublished}). We applied it to the study of off-lattice models. The Rate of Convergence criterion is used to decide if a certain sequence will fold up correctly within a relatively short time. Before the present work, the common way to decide if a certain sequence was a good/bad folder was by performing the whole dynamics until the sequence got its native state (if it existed), or by studying the curvature of the potential energy surface. There are some difficulties in the last two approaches. In the first approach, performing the complete dynamics for hundreds of sequences is a rather challenging task because of the CPU time needed. In the second approach, calculating the curvature of the potential energy surface is possible only for very smooth surfaces. The Rate of Convergence criterion seems to avoid the previous difficulties. With this criterion one does not need to perform the complete dynamics to find the good and bad sequences. Also, the criterion does not depend on the kind of force field used and therefore it can be used even for very rugged energy surfaces.

Identification of plasmodium vivax proteins with potential role in invasion using sequence redundancy reduction and profile hidden markov models

Background: This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results: A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions: The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.

Language teaching : theoretical basis and curricular design.

Este libro intenta ofrecer una panorámica diferente de la enseñanza de idiomas y llenar el vacío con el que con mucha frecuencia se encuentran los opositores al cuerpo de Enseñanza de Escuelas Oficiales de Idiomas y de Secundaria cuando deciden buscar información sobre un amplio temario. Esta obra combina una base teórica, que cubre aspectos sobre las teorías del lenguaje y de la enseñanza, con un diseño curricular práctico. La primera parte consta de cuatro bloques: 1) El proceso de comunicación, funciones del lenguaje y comunicación oral y escrita. 2) Competencia comunicativa y enseñanza comunicativa del inglés. 3) Teorías sobre el aprendizaje y la adquisición de un idioma extranjero, el concepto de interlenguaje y el tratamiento de error. 4) Evolución de la enseñanza de idiomas. La segunda parte trata del diseño curricular: los objetivos principales que se persiguen en la enseñanza de un idioma, los contenidos, la metodología a seguir con los estudiantes y criterios de evaluación. Es un tratado basado en la propia experiencia personal de la autora como profesora de inglés.

Teaching and learning design and technology : a guide to recent research and its applications.

Este manual presenta un resumen de algunas de las investigaciones más importantes en diseño y tecnología de la última década. Los resultados están relacionados con la enseñanza y el aprendizaje de la tecnología y el diseño en el aula. La investigación es relevante desde el nivel de la enseñanza primaria, secundaria y formación profesional. Abarca la enseñanza de estas materias como un tema separado o como integrado con otras áreas del currículo. Discute el desarrollo de libros de texto, el desarrollo curricular, la organización departamental, el aprendizaje de los niños, y el criterio de evaluación.

Parental permissiveness, control, and affect and drug use among adolescents

Resumen tomado de la publicación

''Rainbow Designer'' : for global and multicultural design

What do the designers tend to achieve? To relate themselves to the reality by producing visual registers of emotions and thoughts, or by projecting and producing objects that are functional, adapting technologies to daily needs. That requires that a designer be a keen observer of his physical surroundings and have a fine sensibility to cultures, enabling him to disassemble the latent forms of the reality and cultural symbolisms in order to perceive the order underlying them and the principles of their composition and unity. Only then could he reproduce the nature and respond to cultural callings. In this process of understanding the surrounding reality of nature and cultures, a designer always moves, generally without being aware of it, between two processes: identity search and self-identification.