Síntesis y Evaluación Biológica de Compuestos Antimitóticos

En esta conferencia se describe la síntesis de análogos de pironetina, un producto natural con actividad antitumoral por bloqueo de polimerización de tubuladas. El mecanismo de acción de la pironetina resulta novedoso al interaccionar covalentemente con alfa-tubulina lo que ha generado un gran interés químico y médico. Es por ello, que se describe la síntesis de análogos simples y se realiza la evaluación biológica frente a células tumorales lo que permitió obtener información sobre la relación estructura-actividadUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Vibrio vulnificus: from water to host

Vibrio vulnificus is an aquatic pathogen autochthonous from temperate, tropical and subtropical ecosystems where it lives either as a sessile cell, forming biofilms or as a free-swimming cell. From these locations, the pathogen can occasionally infect humans and fish causing a disease named vibriosis. The most severe form of human and fish vibriosis is associated with the pathogen’s ability to spread from the infection site to the bloodstream and multiply, process known as invasion. Before invasion, the pathogen has to colonize the mucosal host surface, process that involves not only bacterial attachment/adhesion but also resistance to mucosal immunity, commensal microbiota (competitors) and bacterial predators (mainly amoeba and phages). Recently, Amaro and cols. obtained evidence that supports that mucin, main protein in mucus, can activate horizontal gene transference in V. vulnificus, which could lead to the emergence of new virulent clones in natural mucosal environments. The objective of this thesis was to study the colonization and invasion processes under the global perspective that allow the “omic” technologies. In the first chapter, we focused our attention on a selected host for the pathogen in the aquatic environment, the eel, and analyzed its microbiome by using metagenomics. We describe for the first time the microbiome of the skin mucus of wild- and farmed-eels and compared it with that of the water. We discovered that mucus concentrates, selectively, bacteria present in water and identified the genes involved in a successful colonization process, most of which could be considered virulence genes. Then, we developed a protocol to identify MGE and prophages in the metagenomes and described a series of putative ICEs, pathogenicity islands and prophages some of which contained virulence and antibiotic resistance genes. Finally, we were able to describe multiple lytic phages, which could be considered as a part of the mucosal immunity. The second chapter of this thesis is focused on the invasion, and, in particular, in the ability of this pathogen to grow in blood. The objective was to discover all the pathogen’s genes involved in growth in human blood by applying transposon insertion sequencing (TIS). TIS is a powerful method that couples high-density transposon mutagenesis with next-generation sequencing to comprehensively assess the fitness of thousands of transposon mutants across a genome. TIS considers that the essentiality of a gene in a condition should be proportional to its presence in the library. Accordingly, genes present in both control (LB-1 or inactivated HS) and tested (fresh HS) libraries are not essential or neutral, and genes present only in the control library are essential for growth in blood and, consequently, for invasion. We identified the genes, obtained the corresponding mutants and complemented strains and performed a series of experiments in vitro and in vivo to corroborate the essentiality of the gene and its function. We identified a series of genes involved in the CPS formation and others with unknown function as essential for human invasion. Moreover, a series of functions have been proposed as important for growth in human blood.Vibrio vulnificus is an aquatic pathogen autochthonous from temperate, tropical and subtropical ecosystems where it lives either as a sessile cell, forming biofilms or as a free-swimming cell. From these locations, the pathogen can occasionally infect humans and fish causing a disease named vibriosis. The most severe form of human and fish vibriosis is associated with the pathogen’s ability to spread from the infection site to the bloodstream and multiply, process known as invasion. Before invasion, the pathogen has to colonize the mucosal host surface, process that involves not only bacterial attachment/adhesion but also resistance to mucosal immunity, commensal microbiota (competitors) and bacterial predators (mainly amoeba and phages). Recently, Amaro and cols. obtained evidence that supports that mucin, main protein in mucus, can activate horizontal gene transference in V. vulnificus, which could lead to the emergence of new virulent clones in natural mucosal environments. The objective of this thesis was to study the colonization and invasion processes under the global perspective that allow the “omic” technologies. In the first chapter, we focused our attention on a selected host for the pathogen in the aquatic environment, the eel, and analyzed its microbiome by using metagenomics. We describe for the first time the microbiome of the skin mucus of wild- and farmed-eels and compared it with that of the water. We discovered that mucus concentrates, selectively, bacteria present in water and identified the genes involved in a successful colonization process, most of which could be considered virulence genes. Then, we developed a protocol to identify MGE and prophages in the metagenomes and described a series of putative ICEs, pathogenicity islands and prophages some of which contained virulence and antibiotic resistance genes. Finally, we were able to describe multiple lytic phages, which could be considered as a part of the mucosal immunity. The second chapter of this thesis is focused on the invasion, and, in particular, in the ability of this pathogen to grow in blood. The objective was to discover all the pathogen’s genes involved in growth in human blood by applying transposon insertion sequencing (TIS). TIS is a powerful method that couples high-density transposon mutagenesis with next-generation sequencing to comprehensively assess the fitness of thousands of transposon mutants across a genome. TIS considers that the essentiality of a gene in a condition should be proportional to its presence in the library. Accordingly, genes present in both control (LB-1 or inactivated HS) and tested (fresh HS) libraries are not essential or neutral, and genes present only in the control library are essential for growth in blood and, consequently, for invasion. We identified the genes, obtained the corresponding mutants and complemented strains and performed a series of experiments in vitro and in vivo to corroborate the essentiality of the gene and its function. We identified a series of genes involved in the CPS formation and others with unknown function as essential for human invasion. Moreover, a series of functions have been proposed as important for growth in human blood

Pyrosequencing survey of intestinal microbiota diversity in cultured sea bass (Dicentrarchus labrax) fed functional diets.

The routine use of chemotherapy to control bacterial diseases in aquatic populations has resulted in the development and spread of antibiotic resistance. The inclusion of immunostimulants in fish diets (functional diets) is one of the main strategies to solve this threat. This study aimed to analyse the intestinal microbiota of cultured European sea bass (Dicentrarchus labrax) fed two functional diets applying pyrosequencing of PCR-amplified 16S rRNA gene. Quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. The autochthonous intestinal microbiota of sea bass consisted of two dominant bacterial genera: Dysgonomonas (Bacteroidetes) and Ralstonia (Betaproteobacteria), but effects of diet on this dominance were observed. In fact, the genus Dysgonomonas significantly decreased in samples from fish fed functional diets, recovering control levels at the end of the study. However, Ralstonia proportion significantly raised in samples from fish fed diet C and maintained this high level along the study period. The developed protocol could be used to study the composition of bacterial communities in the fish intestine under different nutritional and environmental conditions and its impact on infection, immune system and general fitness of fish

Desarrollo de una aplicación de realidad aumentada para dispositivos móviles

El trabajo realizado en este proyecto se enmarca dentro del ámbito de los dispositivos móviles y de la realidad aumentada. La memoria de este proyecto final de carrera puede servir para hacerse una idea de cual es el estado actual del uso de esta tecnología, que se puede esperar de ella en un futuro y que herramientas están al alcance de los desarrolladores para poder usarla en sus aplicaciones. Sobre todo nos ha interesado como está influyendo en los dispositivos móviles y que utilidades puede tener para los usuarios. En el siguiente documento se explicará, por tanto, de que se trata esta tecnología, como funciona y qué usos se le puede dar. A continuación, veremos también cuales son las herramientas actuales de las que disponen los programadores, centrándonos en aquellas que se difunden bajo la Licencia Publica General de GNU. Éste es el denominado software libre cuya principal característica es que el código fuente de las aplicaciones está disponible para ser modificado y distribuido, bajo unas ciertas condiciones. En cuanto al tema de los dispositivos móviles nos centraremos en el Nokia N900, ya que es el modelo del terminal con el que hemos estado trabajando. Se incluye en este documento una descripción de sus características y prestaciones, así como la explicación de como portar algunas de las herramientas a este dispositivo para hacerlas funcionar. En concreto, haremos hincapié en la instalación, uso y funcionamiento de una aplicación de realidad aumentada para este dispositivo llamada ¿Araap¿.Medina Carda, M. (2011). Desarrollo de una aplicación de realidad aumentada para dispositivos móviles. http://hdl.handle.net/10251/12198.Archivo delegad

Synthesis of Combretastatin A-4 and 3 0 -Aminocombretastatin A-4 derivatives with Aminoacid Containing Pendants and Study of their Interaction with Tubulin and as Downregulators of the VEGF, hTERT and c-Myc Gene Expression

Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3 0 -aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in order to compare their biological actions with those of their parent compounds. Thus, inhibition of cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4 and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-549 cells. The best results were obtained in the inhibition of gene expression, particularly on the VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by the parent compound

Stereoselective synthesis of the published structure of synargentolide A and of one of its stereoisomers

A stereoselective synthesis is described of the structure published for the naturally occurring synargentolide A, an α,β-unsaturated lactone. The key steps of the synthesis were a Brown´s asymmetric allylation and a ring closing metathesis. The spectroscopic data of the synthetic product were very close to those of the natural product but did not exactly match them. An epimer of the published structure was then synthesized according to a similar strategy. Again, the data of the synthetic product did not match those reported for the natural product. It is thus likely that the actual structure of synargentolide A differs from the published one in more than mere stereochemical aspects.Marco Ventura, Juan Alberto, [email protected]

Synthesis, In Silico Studies, Antiprotozoal and Cytotoxic Activities of Quinoline‐Biphenyl Hybrids

This is the pre-peer reviewed version of the following article: Synthesis, In Silico Studies, Antiprotozoal and Cytotoxic Activities of Quinoline‐Biphenyl Hybrids, which has been published in final form at https://doi.org/10.1002/slct.201903835. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThe synthesis, in silico studies, antiprotozoal and cytotoxic activities of eleven quinoline‐biphenyl hybrids are described herein. The structure of the synthesized products was elucidated by a combination of spectrometric analyses. The synthesized compounds were evaluated against Plasmodium falciparum, and amastigotes forms both Leishmania (V) panamensis and Trypanosoma cruzi. Cytotoxicity was evaluated against human U‐937 macrophages. 8‐phenylquinoline (4 a) showed similar activity than meglumine antimoniate and 4‐(quinolin‐8‐yl)phenol (4 b) exhibited an activity similar to that of benznidazole. 8‐(3,4‐dimethoxyphenyl) quinoline (4 k) showed the best activity against P. falciparum. Although these compounds were toxic for mammalian U‐937 cells, however they may still have potential to be considered as candidates for drug development because of their antiparasite activity. Molecular docking was used to determine the in silico inhibition of some of the designed compounds against PfLDH and cruzipain, two important pharmacological targets involved in antiparasitic diseases. All hybrids were docked to the three‐dimensional structures of PfLDH and T. cruzi cruzipain as enzymes using AutoDock Vina. Notably, the docking results showed that the most active compounds 4‐(quinolin‐8‐yl)phenol (4 b, CE50: 11.33 μg/mL for T. cruzi) and 8‐(3,4‐dimethoxyphenyl) quinoline (4 k, CE50: 8.84 μg/mL for P. falciparum) exhibited the highest scoring pose (−7.5 and −7.7 kcal/mol, respectively). This result shows a good correlation between the predicted scores with the experimental data profile, suggesting that these ligands could act as competitive inhibitors of PfLDH or T. cruzi cruzipain enzymes, respectively. Finally, in silico ADME studies of the quinoline hybrids showed that these novel compounds have suitable drug‐like properties, making them potentially promising agents for antiprotozoal therapy

Convergent, stereoselective syntheses of the glycosidase inhibitors broussonetines D and M

The first syntheses of the polyhydroxylated alkaloids (iminosugars) broussonetines D and M glycosidase inhibitors of the pyrrolidine class, have been performed in a convergent, stereocontrolled way from D-serine as the chiral starting material. A cross metathesis step was one key feature of the synthesis. The versatility of the synthetic concept chosen permits the access to many members of this compound family, both natural ones and analogues thereo

Synthesis and evaluation of biphenyl derivatives as potential downregulators of VEGF protein secretion and telomerase-related gene expressions

A group of 47 biphenyl functionalized compounds, prepared by means of Suzuki couplings, has been investigated for their cytotoxicity on two tumoral cell lines (HT-29 and MCF-7) and one non tumoral cell line (HEK-293). 29 selected compounds have been investigated for their ability to inhibit the production of the vascular endothelial growth factor (VEGF). Subsequently, the capacity of the compounds to downregulate the expression of the VEGF, h-TERT and c-Myc genes, the two latter involved in the control of the activation of telomerase, has also been determined.Financial support has been granted to M.C. by the Ministerio de Economía y Competitividad of Spain (project CTQ2014- 52949-P), by the Consellería d´Empresa, Universitat i Ciencia de la Generalitat Valenciana (projects PROMETEO/2013/027 and ACOMP/2014/ 274) and by the University Jaume I (project PI- 1B2011-37). M. S.-P. thanks the University Jaume I for a predoctoral fellowship

Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution

BACKGROUND: Fish skin mucosal surfaces (SMS) are quite similar in composition and function to some mammalian MS and, in consequence, could constitute an adequate niche for the evolution of mucosal aquatic pathogens in natural environments. We aimed to test this hypothesis by searching for metagenomic and genomic evidences in the SMS-microbiome of a model fish species (Anguilla Anguilla or eel), from different ecosystems (four natural environments of different water salinity and one eel farm) as well as the water microbiome (W-microbiome) surrounding the host. RESULTS: Remarkably, potentially pathogenic Vibrio monopolized wild eel SMS-microbiome from natural ecosystems, Vibrio anguillarum/Vibrio vulnificus and Vibrio cholerae/Vibrio metoecus being the most abundant ones in SMS from estuary and lake, respectively. Functions encoded in the SMS-microbiome differed significantly from those in the W-microbiome and allowed us to predict that successful mucus colonizers should have specific genes for (i) attachment (mainly by forming biofilms), (ii) bacterial competence and communication, and (iii) resistance to mucosal innate immunity, predators (amoeba), and heavy metals/drugs. In addition, we found several mobile genetic elements (mainly integrative conjugative elements) as well as a series of evidences suggesting that bacteria exchange DNA in SMS. Further, we isolated and sequenced a V. metoecus strain from SMS. This isolate shares pathogenicity islands with V. cholerae O1 from intestinal infections that are absent in the rest of sequenced V. metoecus strains, all of them from water and extra-intestinal infections. CONCLUSIONS: We have obtained metagenomic and genomic evidence in favor of the hypothesis on the role of fish mucosal surfaces as a specialized habitat selecting microbes capable of colonizing and persisting on other comparable mucosal surfaces, e.g., the human intestine