Estudio computacional de la afinidad de dendrímeros PAMAN G0 por iones metálicos presentes en aguas contaminada

80 p.Los nanopolímeros dendríticos han sido potenciados en diversos ámbitos, siendo uno de ellos la remoción de metales en agua. Estos materiales presentan afinidad por diversas moléculas dependiendo de su grupo terminal que es capaz de modificar la especificidad de atrapamiento por ciertas moléculas. En esta tesis, se utilizaron aproximaciones químico-computacionales aplicadas al estudio de las interacciones entre metales y dendrímeros PAMAM G0 sin funcionalizar y funcionalizados con un conjunto de grupos terminales (asparagina, lisina y arginina). Los metales estudiados son iones bivalentes Cu, Ni y Zn que se presentan comúnmente como residuos tóxicos en agua. Las metodologías computacionales utilizadas corresponden a aproximaciones DTF (Teoría de Funcionales de la Densidad) y semi-empíricas. Los métodos utilizados permitieron recabar información relevante con respecto a la geometría y afinidad de éstos complejos. Los resultados obtenidos por medio de técnicas computacionales de la TFD indican que las coordinaciones más estables entre dendrímero-metal se encuentran en el sector del núcleo del dendrímero, para lo cual se obtuvieron geometrías del tipo cuadrado planar distorsionadas. Por otra parte, la afinidad encontrada por dichos estudios indica el siguiente orden: Ni(II) > Cu(II) > Zn(II), lo cual difiere de la información obtenida experimentalmente, la cual mostró la tendencia Cu(II) > Ni(II) y Zn(II). Sin embargo, estos estudios podrían ser complementados adicionando solvente implícito al sistema, lo que podría conseguir la misma tendencia en ambos estudios. Los cálculos por métodos semi-empíricos por su parte sugieren que se obtendría una mejor extracción de éstos metales a través de una funcionalización de PAMAM G0 con el grupo asparagina./ABSTRACT: Dendritic nanopolymers have been successfully applied in different technological solutions; one of them is the removal of metals in water. Dendrimers exhibit high affinity for different molecules depending of their terminal groups that are capable of modifying the capture specificity for certain molecules. In this thesis, computational chemistry approaches were used to study the interactions between metals and the non-functionalized dendrimer PAMAM G0, and PAMAM G0 functionalized with a set of chemical groups (asparagine, lysine and arginine). The selected metal ions corresponded to the divalent cations Cu, Ni and Zn, commonly present as toxic waste in water. Relevant information about the geometry and affinity of these metal complexes was obtained through DFT and semi-empirical approximations. DFT results indicated that the core area of the dendrimer corresponds to the most stable coordination site exhibiting a distorted square planar geometry. Moreover, the affinity of the ligand for each metal showed the following tendency: Ni(II)> Cu(II)> Zn(II). This sequence differs from the experimental information; however, the introduction of implicit solvation models could complement and improve these studies. Analyses of the complexation of metals with the functionalized dendrimer at semi-empirical level of theory showed that PAMAM G0 modified with asparagine is the best candidate for water remediatio

Functional Analysis of the <i>Brassica napus</i> L. Phytoene Synthase (PSY) Gene Family

<div><p>Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including <i>Brassica napus</i> L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In <i>Arabidopsis thaliana</i>, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in <i>B. napus</i> all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six <i>B. napus</i> PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six <i>B. napus</i> PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six <i>B. napus</i> PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.</p></div

<i>B. napus</i> PSY gene expression during seed development.

<p>A. <i>BnaX.PSY</i> gene expression was determined by RT-PCR using homologue-specific primers. B. <i>B</i>. <i>napus 18S</i> gene expression (loading control). C. Sampling stages of <i>B. napus</i> seed development (from left to right: 20, 35, 40 and 60 days post anthesis) and leaf tissue. L: 100 bp ladder; WC: water control; gDNA: <i>B</i>. <i>napus</i> gDNA control; C+: PSY homologue-specific plasmid controls (positive); C-: PSY homoelogue plasmid controls (negative). RT-PCR (40 cycles) was performed in two biological replicates, only one is shown for simplicity.</p

Phylogenetic relationship of <i>B. napus</i> and selected monocot and dicot PSY enzymes.

<p>The evolutionary history was inferred using the neighbor-joining method using MEGA4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114878#pone.0114878-Tamura1" target="_blank">[38]</a>. The percentage of replicate trees in which the associated taxa clustered together in the 500-bootstrap replication test is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units (number of amino acid substitutions per site) as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114878#pone.0114878-Zuckerkandl1" target="_blank">[39]</a>. Arabidopsis (AtPSY, AAA32836), cassava (MePSY1, ACY42666; MePSY2; ACY42670), maize (ZmPSY1, P49085; ZmPSY2, AAQ91837; ZmPSY3, ABC75827), pepper (CaPSY1, ACE78189.1), rice (OsPSY1, AAS18307; OsPSY2, AAK07735; OsPSY3, ABC75828), sorghum (SbPSY1, AAW28996; SbPSY2, XP002442578; SbPSY3, AAW28997) and tomato (SlPSY1, P08196.2; SlPSY2, ABV68559.1; SlPSY3, XP_004228928.1). The cassava MePSY3 protein sequence was obtained from Phytozome (<a href="http://www.phytozome.net/cassava.php" target="_blank">http://www.phytozome.net/cassava.php</a>) as described in Arango <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114878#pone.0114878-Arango1" target="_blank">[12]</a>.</p

Functional complementation of <i>B. napus</i> PSY genes.

<p><i>E. coli</i> cells were transformed with: (A) pDS1B, a pBAD33 vector carrying <i>E. uredovora</i> carotenogenic genes <i>crtE</i>, <i>crtB</i>, <i>crtI</i>, <i>crtY</i> and <i>CrtX</i>; (B) pDS1B-Δ<i>crtB</i> which has a deletion of the <i>Eu crtB</i> gene + pETBlue1 (empty vector); (C) pDS1B-Δ<i>crtB</i> + pETBnaC.PSY.a; (D) pDS1B- Δ<i>crtB</i> + pETBnaA.PSY.b; (E) pDS1B- Δ<i>crtB</i> + pETBnaA.PSY.c; (F) pDS1B- Δ<i>crtB</i> + pETBnaA.PSY.d; (G) pDS1B- Δ<i>crtB</i> + pETBnaC.PSY.e; and (H) pDS1B- Δ<i>crtB</i> + pETBnaC.PSY.f and HPLC chromatograms obtained at 450 nm are shown for each transformation. The spectral fine spectrum for beta carotene (peak 1) is shown as an example in the control panel (A). The amount (mg) of β-carotene produced by each complementation assay expressed as per gram of dry weight (DW) is shown in (I). Bars represent standard deviation calculated from three replications. For each vector combination, different letters indicate significant differences at p<0.05 determined by Tukey's HSD test.</p

Assessment of the Activity of Nitroisoxazole Derivatives against <i>Trypanosoma cruzi</i>

The development of new compounds to treat Chagas disease is imperative due to the adverse effects of current drugs and their low efficacy in the chronic phase. This study aims to investigate nitroisoxazole derivatives that produce oxidative stress while modifying the compounds’ lipophilicity, affecting their ability to fight trypanosomes. The results indicate that these compounds are more effective against the epimastigote form of T. cruzi, with a 52 ± 4% trypanocidal effect for compound 9. However, they are less effective against the trypomastigote form, with a 15 ± 3% trypanocidal effect. Additionally, compound 11 interacts with a higher number of amino acid residues within the active site of the enzyme cruzipain. Furthermore, it was also found that the presence of a nitro group allows for the generation of free radicals; likewise, the large size of the compound enables increased interaction with aminoacidic residues in the active site of cruzipain, contributing to trypanocidal activity. This activity depends on the size and lipophilicity of the compounds. The study recommends exploring new compounds based on the nitroisoxazole skeleton, with larger substituents and lipophilicity to enhance their trypanocidal activity