Biochemistry & MCB.

The final aim at this area of BIFI is to understand and to control biological systems depending on proteins that have interest for chemical, biotechnological, pharmacological and biomedical applications. Knowledge of protein behaviour at molecular and cellular levels allows interpretation of the macroscopic mechanisms of the cellular functions in which they are involved, but many of the parameters controlling these processes still remain unknown. Proteins adopt an organized three-dimensional structure closely related with their function that can be regulated by the interaction with other biomolecules and/or small organic molecules. Defective structural arrangements can prevent protein interaction with other molecules, provoking many different illnesses in human beings.

Many other illnesses are also produced by infectious viruses and microorganisms, and a way of stopping them could be to block a step of their vital cycle involving a protein or biomolecule. Moreover, small molecules from the environment can also be either toxic inhibitors or activatiors of particular activities in different organisms, and in many cases they might be used as effectors of the expression some genes, and the production or action of particular proteins.  The research lines in Biochemistry and Molecular and Cellular Biology at BIFI study different biological systems involved in key metabolic routes that serve as model for other systems combining classic methodologies from this area with biophysical and computational methods. Applications of the obtained knowledge are additionally being used to control and to modulate the behaviour of particular systems with benefits for the society.

Mycobacterium Tuberculosis

Head of the Research Line:

Jesús Gonzalo-Asensio

Researchers:

Carlos Martín Montañés (Full Professor)
Esther Broset Blasco (FPI Grant)
Irene Pérez Sanchez (DGA Grant)
Ana Picó Marco (Lab Technician)

 

SUMMARY OF THE RESEARCH LINE

According to the last WHO report, Tuberculosis (TB) is responsible for 1.8 million deaths and causes 10.4 million new TB cases worldwide per year. Cumulative data indicate that TB has killed 1 billion people in the past 200 years, which turns it as the biggest killer compared to other infectious diseases as plague, influenza, smallpox, malaria, cholera or AIDS.

TB in humans is mainly caused by the Mycobacterium tuberculosis bacillus. However, other Mycobacterium species are also able to infect humans. These include M. canettii and M. africanum, responsible for human cases geographically restricted to East- and West-African countries respectively. In addition, a number of Mycobacterium species are also able to cause TB in mammals, which represents an economic problem as well as a zoonotic risk for humans. Overall, these species constitute the M. tuberculosis Complex (MTBC). (Figure 1)

1

Figure 1: Schematic phylogenetic relationships of the MTBC. Phylogenetic distribution of M. canettii, the L1-L7 lineages of M. tuberculosis and the animal-adapted species. The figure also shows the geographic distribution of each lineage and the preferred host. Adaptado de Broset et al. mBio 2015.

To gain a complete evolutionary perspective, it is highly recomended to extend genomic studies to the whole MTBC. Our cumulative knowledge in mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired during the natural evolution of mycobacterial species probably to ensure adaptation to different hosts (Gonzalo-Asensio et al, PNAS 2014). PhoPR is a well-known regulator of pathogenic phenotypes in the Mycobacterium tuberculosis complex (MTBC) including secretion of the virulent factor ESAT-6 (Broset et al. mBio 2015), biosynthesis of acyltrehalose-based lipids (Gonzalo-Asensio et al. JBC 2008) or modulation of antigen export (Solans et al. PLoS pathogens 2014) (Figure 2).

2

Figure 2. Molecular characterization of the PhoPR Two-Component System. A. Positioning of the PhoP transcription factor relative to the RNA polymerase and its target genes measured as the most probable values from ChIP-seq data. The PhoP consensus motif (TCACAG-N5-TCACAG) is also indicated. B. ChIP-seq reads from representative promoters of PhoP-regulated genes. Note the significant increase in ChIP-seq peaks in the wild-type strain relative to its phoP mutant indicative of a specific interaction of PhoP with these regions. C. PhoP binding motifs elucidated from ChIP-seq data shown in panel B. Distance to the start codon of target genes is also indicated. D. Genomic location of relevant phoP polymorphisms in the MTBC. Insertions of IS6110 in the promoter region and their positions relative to the phoP start codon are shown. Amino acid positions of the Asp71 residue involved in the phosphotransfer reaction and the Ser219Leu substitution in H37Ra are also indicated. E. Ribbon model of the DNA-binding domain of PhoP superimposed on the structure of a PhoB-DNA complex. Solid spheres show the wild-type serine 219 in H37Rv (left) or the leucine residue that appears in H37Ra (right) (Adapted from Gonzalo-Asensio et al. J. Bacteriol 2008). The mutant leucine residue is expected to interfere with DNA binding and/or recognition. F. Secondary structure of the PhoR sensor kinase indicating its membrane topology. Each domain has been coloured individually indicating the presence of α-helices (“racetrack” ovals) and β-strands (arrows). Note the presence of PhoR polymorphisms in the sensor loop located in the periplasmic space. Position of the Histidine involved in the phosphotransfer reaction is also indicated. Adapted from Broset et al. mBio 2015.

Evolutionarily conserved polymorphisms in PhoPR from M. africanum and animal-adapted species result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and likely to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream phoPR in a particular M. bovis strain able to transmit between humans or polymorphisms in M. africanum and M. bovis affecting the regulatory region of the espACD operon, which allow PhoPR independent ESAT-6 secretion (Gonzalo-Asensio et al. PNAS 2014) (Figure 3).

 

3

Figure 3. Comparative illustration of PhoPR-regulated phenotypes in M. tuberculosis, M. bovis, M. africanum L6 and a M. tuberculosis phoP mutant. M. tuberculosis carrying a functional PhoR is able to sense its cognate stimulus and subsequently phosphorylate PhoP. Phosphorylated PhoP regulates three well-known phenotypes including synthesis of SL and DAT/PAT (via pks2 and pks3 regulation), secretion of ESAT-6 (through espA regulation) and post-transcriptional regulation of tatC (mediated by the mcr7 non-coding RNA). M. bovis and M. africanum L6 carrying a defective PhoR G71I allele are expected to have defects in PhoP phosphorylation, as a consequence these strains lacks SL, DAT and PAT. However, ESAT-6 secretion in these strains is restored by compensatory mutations in the espACD promoter region that include RD8 deletion and species-specific polymorphisms (asterisks). M. tuberculosis phoP mutants lack the aforementioned PhoP-regulated phenotypes and consequently does not synthesize SL, DAT and PAT or secrete ESAT-6. These mutants also have a deregulated TAT system and consequently secrete higher amounts of TAT substrates that include the antigens Ag85A and Ag85C. Consequently, adequately attenuated M. tuberculosis phoP-based vaccine strains, such as MTBVAC, are expected to induce long-lasting immunogenicity in clinical trials. Adapted de Broset et al. mBio 2015

Our research line is focused on the rising significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation (Figure 3). In this context, the live-attenuated MTBVAC based on a phoP/fadD26 deletion mutant of M. tuberculosis is the first vaccine of this kind to successfully enter clinical development, representing a historic milestone in the field of vaccinology.

 

Relevant publications

1.- Evolutionary landscape of the Mycobacterium tuberculosis complex from the viewpoint of PhoPR: implications in virulence regulation and application to vaccine development. Esther Broset, Carlos Martín and Jesús Gonzalo-Asensio*. 2015. mBio, IF=6.786, D1 microbiology.

2.- Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator. Jesús Gonzalo-Asensio, Wladimir Malaga, Alexandre Pawlik, Catherine Astarie-Dequeker, Charlotte Passemar, Flavie Moreau, Françoise Laval, Mamadou Daffé, Carlos Martin, Roland Brosch, Christophe Guilhot. 2014 PROC. NATL. ACAD. SCI. USA, IF=9.809, D1 multidisciplinary sciences.

3.- A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component. Luis Solans, Nacho Aguiló, Sofía Samper, Alexandre Pawlik, Wafa Frigui, Carlos Martín, Roland Brosch, Jesús Gonzalo-Asensio*. 2014. Infection And Immunity, IF=4,156, Q1 Infectious Diseases.

4.- The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis. Luis Solans*, Jesús Gonzalo-Asensio*, Claudia Sala*, Andrej Benjak, Swapna Uplekar, Jacques Rougemont, Christophe Guilhot, Wladimir Malaga, Carlos Martín, Stewart T. Cole. 2014. PLoS PATHOGENS, IF=8,136 D1 Microbiology, Virology and Parasitology.

5.- Tuberculosis Vaccine. Carlos Martín, Brigitte Gicquel, Esther Pérez, Jesús Gonzalo Asensio, Ainhoa Arbués. INTERNATIONAL PATENT. PCT/ES 2007/070051. Spain (ES7730491) / Europe (1997881) / USA (US12/294,199) / Canada (CA2,647,287) / Japan (JP2009-500878) / China (CN200780010366.5) / Russia (RU2008142140 (0)) / India (IN8123/DELN/2008) / Brasil (BRPI-0709106-0).

 

Main research projects

1.- Advancing Novel and Promising TB Vaccine Candidates From Discovery to Preclinical and Early Clinical Development (Reference TBVAC2020) H2020, IP: Carlos Martín Montañés. Participant.

2.- Multiplex Iterative Genome Engineering (MIGE) in Mycobacterium. Applications to development of genetic tools for testing new antibiotics Myco-MIGE (Reference BFU2015-72190-EXP). Ministerio de Economía y Competitividad, IP: Jesús Gonzalo-Asensio.

3.- Análisis de las diferencias de IS6110 entre los miembros del complejo Mycobacterium tuberculosis y el papel de su localización en el origen de replicación. (Reference PI15/00317) FIS-Instituto de Salud Carlos III, IP: Sofía Samper Blasco. Participant.

4.- Polimorfismos genómicos y transcriptómicos en M. tuberculosis complex y su significado en la clínica. (Reference PI12/01970) FIS-Instituto de Salud Carlos III. IP: Sofía Samper. Participant

 

Collaborators

  • Roland Brosch. Institut Pasteur, Paris
  • Christophe Guilhot. IPBS-CNRS, Toulouse
  • Marcelo Guerin. Ikerbasque, Spain
  • Inmaculada Yruela, BiFi, Spain
  • Bruno Contreras-Moreira. BiFi, Spain

http://genmico.unizar.es

Development of Antimicrobials
and Mechanisms of Resistance

jose_ainsa_group_2

Head of the Research Line:

José Antonio Aínsa Claver

Researchers:

José Antonio Aínsa Claver, PI
Ainhoa Lucía Quintana, Postdoc
Clara Aguilar Pérez, PhD student
Ernesto Anoz Carbonell, PhD student
Begoña Gracia Díaz, Laboratory techniccian

 

SUMMARY OF THE RESEARCH LINE

Research line Development of Antimicrobials and Mechanisms of Resistance is committed to study mechanisms of resistance to antimicrobial agents in diverse microbial pathogens and to use this information for identifying novel molecules with antimicrobial activity and characterise their mechanisms of action and resistance. This work is funded by public grants got in competitive calls at the national and international level.

In recent years, we have characterised several efflux pumps from Mycobacterium tuberculosis and we have contributed to identify compounds that evading resistance mediated by efflux pumps have an increased antimicrobial activity. We are characterising novel drug targets, not only in Mycobacterium but also in other bacterial pathogens, and exploring novel molecules (peptides,…) as alternatives to conventional antibiotics.

During the last 7 years, we have published 15 scientific articles, we have got a patent related with the diagnostic value of a specific resistance gene and have made a number of communications to national and international conferences.

Our research is getting insight into novel perspectives, such as the use of nanoparticles for administering antibiotics, or combination of molecules with antimicrobial activity.

mtt_assayb

 

Relevant publications

1.- Identification of Aminopyrimidine-Sulfonamides as Potent Modulators of Wag31-mediated Cell Elongation in Mycobacteria. Vinayak Singh, Neeraj Dhar, János Pató, Gaëlle S. Kolly, Jana Korduláková, Martin Forbak, Joanna C. Evans, Rita Székely, Jan Rybniker, Zuzana Palčeková, Júlia Zemanová, Isabella Santi, François Signorino-Gelo, Liliana Rodrigues, Anthony Vocat, Adrian S. Covarrubias, Monica G. Rengifo, Kai Johnsson, Sherry Mowbray, Joseph Buechler, Vincent Delorme, Priscille Brodin, Graham W. Knott, José A. Aínsa, Digby F. Warner, György Kéri, Katarína Mikušová, John D. McKinney, Stewart T. Cole, Valerie Mizrahi, Ruben C. Hartkoorn. Molecular Microbiology, in press.

2.- Antituberculosis drugs: reducing efflux = increasing activity. Liliana Rodrigues, Tanya Parish, Meenakshi Balganesh, José A. Ainsa. Drug Discovery Today, in press.

3.- Lipid transport in Mycobacterium tuberculosis and its implications in virulence and drug development. Bailo R, Bhatt A, Aínsa JA. Biochem Pharmacol. 2015 Aug 1;96(3):159-67. doi: 10.1016/j.bcp.2015.05.001. PMID: 25986884.

4.- Measuring efflux and permeability in mycobacteria. Rodrigues L, Viveiros M, Aínsa JA. Methods Mol Biol. 2015;1285:227-39. doi: 10.1007/978-1-4939-2450-9_13. PMID: 25779319.

5.- Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux. Lee RE, Hurdle JG, Liu J, Bruhn DF, Matt T, Scherman MS, Vaddady PK, Zheng Z, Qi J, Akbergenov R, Das S, Madhura DB, Rathi C, Trivedi A, Villellas C, Lee RB, Rakesh, Waidyarachchi SL, Sun D, McNeil MR, Ainsa JA, Boshoff HI, Gonzalez-Juarrero M, Meibohm B, Böttger EC, Lenaerts AJ. Nat Med. 2014 Feb;20(2):152-8. doi: 10.1038/nm.3458. PMID: 24464186.

6.- Analysis of mutations in streptomycin-resistant strains reveals a simple and reliable genetic marker for identification of the Mycobacterium tuberculosis Beijing genotype. Villellas C, Aristimuño L, Vitoria MA, Prat C, Blanco S, García de Viedma D, Domínguez J, Samper S, Aínsa JA. J Clin Microbiol. 2013 Jul;51(7):2124-30. doi: 10.1128/JCM.01944-12. PMID: 23616454.

7.- Zanthoxylum capense constituents with antimycobacterial activity against Mycobacterium tuberculosis in vitro ex vivo within human macrophages. Luo X, Pires D, Aínsa JA, Gracia B, Duarte N, Mulhovo S, Anes E, Ferreira MJ. J Ethnopharmacol. 2013 Mar 7;146(1):417-22. doi: 10.1016/j.jep.2013.01.013. PMID: 23337743.

8.- Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis. Rodrigues L, Villellas C, Bailo R, Viveiros M, Aínsa JA. Antimicrob Agents Chemother. 2013 Feb;57(2):751-7. doi: 10.1128/AAC.01482-12. PMID: 23165464.

9.- Inhibitors of mycobacterial efflux pumps as potential boosters for anti-tubercular drugs. Viveiros M, Martins M, Rodrigues L, Machado D, Couto I, Ainsa J, Amaral L. Expert Rev Anti Infect Ther. 2012 Sep;10(9):983-98. doi: 10.1586/eri.12.89. PMID: 23106274.

10.- Mycobacterial shuttle vectors designed for high-level protein expression in infected macrophages. Eitson JL, Medeiros JJ, Hoover AR, Srivastava S, Roybal KT, Aínsa JA, Hansen EJ, Gumbo T, van Oers NS. Appl Environ Microbiol. 2012 Oct;78(19):6829-37. doi: 10.1128/AEM.01674-12. PMID: 22820329.

11.- Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG. Ramón-García S, Mick V, Dainese E, Martín C, Thompson CJ, De Rossi E, Manganelli R, Aínsa JA. Antimicrob Agents Chemother. 2012 Apr;56(4):2074-83. doi: 10.1128/AAC.05946-11. PMID: 22232275.

12. A prodrug approach for improving antituberculosis activity of potent Mycobacterium tuberculosis type II dehydroquinase inhibitors. Tizón L, Otero JM, Prazeres VF, Llamas-Saiz AL, Fox GC, van Raaij MJ, Lamb H, Hawkins AR, Ainsa JA, Castedo L, González-Bello C. J Med Chem. 2011 Sep 8;54(17):6063-84. doi: 10.1021/jm2006063. PMID: 21780742.

13.- Antimycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in Mozambique. Luo X, Pires D, Aínsa JA, Gracia B, Mulhovo S, Duarte A, Anes E, Ferreira MJ. J Ethnopharmacol. 2011 Sep 1;137(1):114-20. doi: 10.1016/j.jep.2011.04.062. PMID: 21571059.

14.- Inhibition of drug efflux in mycobacteria with phenothiazines and other putative efflux inhibitors. Rodrigues L, Aínsa JA, Amaral L, Viveiros M. Recent Pat Antiinfect Drug Discov. 2011 May;6(2):118-27. PMID: 21517739.

15.- Design, synthesis and inhibitory activity against Mycobacterium tuberculosis thymidine monophosphate kinase of acyclic nucleoside analogues with a distal imidazoquinolinone. Familiar O, Munier-Lehmann H, Aínsa JA, Camarasa MJ, Pérez-Pérez MJ. Eur J Med Chem. 2010 Dec;45(12):5910-8. doi: 10.1016/j.ejmech.2010.09.056. PMID: 20951473.

 

Main research projects

1.- NAREB – Nanotherapeutics for antibiotic resistant emerging bacterial pathogens. Unión Europea. Universidad de Zaragoza. 01/02/2014 – 31/01/2018. IP: José Antonio Aínsa Claver.

2.- SAF-2013-48971-C2-2-R: Aplicaciones biomédicas de AS-48: una proteína con amplio espectro de actividad antimicrobiana. MINECO – Ministerio de Economia y Competitividad. Universidad de Zaragoza. 01/01/2014 – 31/12/2016. IP: José Antonio Aínsa Claver.

3.- MM4TB – More medicines for tuberculosis. Unión Europea. Universidad de Zaragoza. 01/02/2011 – 31/01/2016. IP: José Antonio Aínsa Claver.

4.- BIO-2009-09405. Estudio de transportadores de membrana de Mycobacterium tuberculosis: implicaciones en resistencia y virulencia. Ministerio de Ciencia e Innovación. Universidad de Zaragoza. 01/01/2010 – 31/12/2012. IP: José Antonio Aínsa Claver.

 

Collaborators

  • Katarina Mikusova, Comenius University (Bratislava, Slovakia). TrxR as a novel drug target in M. tuberculosis.
  • Rita Skelezy, Stewart T. Cole, École Polytechnique Fédérale de Lausanne (Laussane, Switzerland). Antituberculosis activity and efflux susceptibility of compounds.
  • Valakunja Nagaraja, Indian Institute of Science (Bangalore, India). A genetic system for evaluating topoisomerase inhibitors in M. tuberculosis.
  • Adela G. De la Campa, Centro Nacional de Microbiología, Instituto de Salud Carlos III (Majadahonda, Madrid, Spain). Development of inhibitors against M. tuberculosis topoisomerase.
  • Mercedes Maqueda, Universidad de Granada (Granada, Spain). Antimicrobial activity of bacteriocin AS-48.

http://genmico.unizar.es

Genetic regulation
and physiology of cyanobacteria

Head of the Research Line:

María F. Fillat Castejón

Researchers:

María Luisa Peleato Sánchez
Teresa Bes Fustero
Emma Sevilla Miguel
Andrés Sandoval
Cristina Sarasa Buil

 

SUMMARY OF THE RESEARCH LINE

Cyanobacteria are microorganisms that perform oxygenic photosynthesis and are able to colonize the most extreme environments. Because of its abundance and ubiquity, cyanobacteria play a key role in the overall carbon and nitrogen cycles and constitute the basis of the food chain in aquatic ecosystems. Some species of cyanobacteria are able to fix atmospheric nitrogen and have been used in fertilizer production. Likewise, strategies to use cyanobacteria as biotechnological choice in the biodiesel production or in the removal of heavy metals from sewage are under development. However, cyanobacteria can also be harmful. Due to the increasing eutrophization of water reservoirs, it is becoming frequent the appearance of cyanobacterial blooms in water intended for consumption or recreative uses. Several cyanobacterial species proliferating in these blooms can produce toxins that have deleterious effects on the human health as well as in animals. Although at present the factors unleashing the cyanotoxin synthesis are unknown, iron availability seems to be a determinant factor.

Our works is aimed to get a better understanding of the regulation of iron metabolism in cyanobacteria and their relationship with nitrogen metabolism, oxidative stress, cyanotoxin production and the formation of biofilms. All these processes are interrelated by a family of transcriptional regulators called FUR (ferric uptake regulator). Most of cyanobacteria express three FUR paralogues called FurA (Fur), Zur (FurB) and PerR (FurC). While most studies about these proteins deal with its regulatory character in cyanobacteria FUR are multifunctional, acting through various strategies not well characterized, together with their activity as transcriptional regulators.

Moreover, FUR proteins are also involved in the expression of virulence factors and biofilm formation in many pathogens, such as Escherichia coli, Pseudomonas aeruginosa or Clostridium difficile, among others. Since Fur is an essential protein for many of these microorganisms it has been proposed that could constitute a new therapeutic target, as an alternative to traditional antibiotics mechanisms.

Our research group has two main objectives:

– Performance of a functional study of FUR proteins in cyanobacteria including their potential biotechnological applications. Note that although the best studied of these proteins facet is its regulatory action in cyanobacteria FUR proteins have a multifunctional character, acting through various strategies not well characterized, together with its activity as transcriptional regulators.

– Characterization of the FUR regulators from the pathogens Pseudomonas aeruginosa and Clostridium difficile. Evaluation of these proteins as potential therapeutic targets by altering their activity by screening chemical libraries.

 

Relevant publications

1.- Pivotal Role of Iron in the Regulation of Cyanobacterial Electron Transport. González A, Sevilla E, Bes MT, Peleato ML, Fillat MF. Journal: Adv Microb Physiol. 2016;68:169-217.

2.- The genome-wide transcriptional response to FurA depletion unveils new roles for this essential cyanobacterial global regulator. González A, Bes MT, Peleato ML and Fillat MF. Journal: PlosOne. 2016 Mar 11;11(3):e0151384. doi: 10.1371/journal.pone.0151384. eCollection

3.- Cysteine mutational studies provide insight into a thiol-based redox switch mechanism of metal and DNA binding in FurA from Anabaena sp. Botello-Morte L, Pellicer S, Contreras LM, Neira JL, Abian O, Velázquez-Campoy A, Peleato ML, Fillat MF and Bes MT. PCC 7120. Journal: Antioxid Redox Signal (PMID:26414804) Fecha: 2015.

4.- The Pkn22 Ser/Thr kinase in Nostoc PCC 7120: role of FurA and NtcA regulators and transcript profiling under nitrogen starvation and oxidative stress. Yingping F, Lemeille S, González A, Risoul V, Denis Y, Richaud P, Lamrabet O, Fillat MF, Zhang CC and Latifi A. Journal: BMC Genomics  2015 Jul 29;16:557. doi: 10.1186/s12864-015-1703-1.

5.- Pivotal role of iron in the regulation of cyanobacterial electron transport. González A, Sevilla E, Bes MT, Peleato ML and Fillat MF. Adv Microb Physiol. 2016;68:169-217. doi: 10.1016/bs.ampbs.2016.02.005. Epub 2016 Mar 15.

6.- Iron homeostasis and environmental responses in cyanobacteria: regulatory networks involving Fur. Peleato ML, Bes MT and Fillat MF. Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria. Chapter 19. pp. 1067-1078. Frans de Bruijn ed., Wiley-Blackwell.

7.- Zur (FurB) is a key factor in the control of the oxidative stress response in Anabaena sp. PCC 7120. Sein-Echaluce VC, González A, Napolitano M, Luque I, Barja F, Peleato ML, Fillat MF. Journal: Environ Microbiol. 17(6): 2006-2017 (2015). doi: 10.1111/1462-2920.12628.

8.- Mesoscopic Model and Free Energy Landscape for Protein-DNA Binding Sites: Analysis of Cyanobacterial Promoters. Tapia-Rojo R, Mazo JJ, Hernández JÁ, Peleato ML, Fillat MF, Falo F. JOURNAL: PLoS Comput Biol. 2014 Oct 2;10(10):e1003835. doi: 10.1371/journal.pcbi.1003835.

9.- The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators. Fillat MF. Journal: Arch Biochem Biophys. 546:41-52 (2014). doi: 10.1016/j.abb.2014.01.029

10.- The FurA regulon in Anabaena sp. PCC 7120: in silico prediction and experimental validation of novel target genes. González A, Angarica VE, Sancho J, Fillat MF. Journal: Nucleic Acids Research. 42(8):4833-46 (2014). doi: 10.1093/nar/gku123

11.- Unraveling the Redox Properties of the Global Regulator FurA from Anabaena sp. PCC 7120: Disulfide Reductase Activity Based on Its CXXC Motifs. Botello-Morte L, Bes MT, Heras B, Fernández-Otal A, Peleato ML, Fillat MF. Journal: Antioxid Redox Signal. 20(9):1396-406 (2014). doi: 10.1089/ars.2013.5376

12.- FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120. González, A., Bes, M.T., Valladares, A., Peleato, M.L. and Fillat M.F. Journal: Environ Microbiol. 14(12):3175-87 (2012) doi: 10.1111/j.1462-2920.2012.02897.x.

13.- Site-directed mutagenesis and spectral studies suggest a putative role of FurA from Anabaena sp. PCC 7120 as heme sensor protein. Pellicer S, González A, Peleato ML, Martínez JI, Fillat MF and Bes MT. Journal: The FEBS Journal, 279(12):2231-46 (2012). doi: 10.1111/j.1742-4658.2012.08606.x.

14.- Unravelling the regulatory function of FurA in Anabaena sp. PCC 7120 through 2-D DIGE proteomic analysis. González, A., Bes, M.T., Peleato, M.L. and Fillat M.F. Journal: Journal of Proteomics, 74:660-71 (2011) doi: 10.1016/j.jprot.2011.02.001.

15.- Overexpression of FurA in Anabaena sp. PCC 7120 reveals new targets for this regulator involved in photosynthesis, iron uptake and cellular morphology. González, A., Bes, M.T., Barja, F., Peleato, M.L. and Fillat M.F. Journal: Plant and cell Physiology. Vol 51 (11):1900-1914 (2010). doi: 10.1093/pcp/pcq148.

 

Main research projects

1.- Multifuncionalidad de las proteínas FUR en cianobacterias: mecanismos alternativos de regulación del metabolismo y contribución a la formación de biofilms. MINECO. Start-End date: 01/01/2017 To: 31/12/2019. Total amount: 140.000 euros. PI: María F. Fillat Castejón. Researchers: 5.

2.- BFU2012-31458: La superfamilia de reguladores Fur: análisis funcional en cianobacterias, potenciales aplicaciones en biotecnología y como diana terapeútica en patógenos. FONDOS FEDER. MINECO. MINISTERIO DE ECONOMIA Y COMPETITIVIDAD. Start-End date: 01/01/2013 – 31/12/2015. Total amount: 114.660 euros. PI: María Francisca Fillat Castejón. Researchers: 5.

3.- B18 BIOLOGÍA ESTRUCTURAL. Gobierno de Aragón. Start-End date: 01/01/2014 – 31/12/2016. Total amount: 20.609 euros. PI: María Luisa Peleato Sánchez. Researchers: 16.

4.- 2012/GA LC 003. Evaluación del riesgo asociado al impacto del cambio climatico en aguas: proliferación de patógenos oportunistas y cianobacterias potencialmente tóxicas y alteración de la fijación de CO2 atmosférico. DGA-LA CAIXA. Start-End date: 01/05/2012 – 30/09/2013. Total amount: 42.208,18 euros. PI: María Francisca Fillat Castejón. Researchers: 9.

5.- BFU2009-07424. Transducción de señales redox mediadas por FurA (ferric uptake regulator) en cianobacterias. Consecuencias en la fotosíntesis y la fijación de nitrógeno. FONDOS FEDER. MINISTERIO DE CIENCIA E INNOVACIÓN. Start-End date: 01/01/2010 – 31/12/2012. Total amount: 139.150 euros. PI: María Francisca Fillat Castejón. Researchers: 5.

6.- B18 BIOLOGÍA ESTRUCTURAL. Gobierno de Aragón. Start-End date: 01/01/2011 – 31/12/2012. Total amount: 38.192 euros. PI: Carlos Gómez-Moreno. Researchers: 23.

7.- Identificación de cianobacterias potencialmente tóxicas y microorganismos patógenos en amebas de vida libre en aguas de Aragón. Diputación general de Aragón-DGA. Start-End date: 01/10/2009 – 30/09/2011. Total amount: 49.000 euros. PI: María Francisca Fillat Castejón. Researchers: 10.

8.- Equipo para análisis y cuantificación de interacciones moleculares mediante resonancia de plasmón de superficie (SPR) (UNZA08-4E-021). Gobierno de Aragón- FEDER. Start-End date: Jan 2009 – Dec 2011. Total amount: 384.569,51 euros. PI: José Felix Saenz.

9.- INF2008-BIO-05. INCUBADOR ORBITAL CON ILUMINACIÓN. D.G.A./U.Z. Start-End date: 10/07/2008 – 31/12/2008. Total amount: 14.373 euros. PI: María Francisca Fillat Castejón. Researchers: 1.

10.- Respuesta de un tapete microbiano de cianobacterias a la contaminación por hidrocarburos. Proyectos Interreg. Departamento de Economía, Hacienda y Empleo de la Diputación General de Aragón. Start-End date: 01/02/2006 – 31/12/2007. PI: María Francisca Fillat Castejón. Researchers: 8.

 

Collaborators

  • F. Barja (Universidad de Ginebra)
  • B. Heras (Universidad de LaTrobe, Melbourne)
  • S. Goñi (Université de Pau et des Pays de l’Adour, France)
  • B. Landeros (Universidad de Baja California)
  • J.M. Mulet (Univ. Politécnica de Valencia)
  • J. Salinas (Universidad de Alicante)
  • I. Luque (Instituto de Bioquímica vegetal y Fotosíntesis, CSIC, Sevilla)
  • A. Lostao (Instituto de Nanociencia de Aragón, Universidad de Zaragoza)
  • A. Lanas (Instituto Aragonés de Ciencias de la Salud, Zaragoza)
Genetics and pig metabolism

Head of the Research Line:

Pascual López Buesa

Researchers:

José Alberto Carrodeguas Villar
Carmen Burgos Serrano
Pedro Latorre Muro
Jorge Hidalgo Gracia

 

SUMMARY OF THE RESEARCH LINE

  1. Analysis of mutations in pigs
  2. Study of mutation´s effects on productive traits and pig carcass and pork quality traits
  3. Search for mutations that influence fat content and pork quality
  4. Study of biochemical mechanisms of mutations effects in pigs and pork

 

Relevant publications

1.- Joint analysis of additive, dominant and first order epsitatic effects of four genes (IGF2, MC4R, PRKAG3, LEPR) with known effects on fat content and fat distribution in pigs. Lopez Buesa P, Burgos C, Galve A, Varona L. Animal Genetics (2014) 45, 133-137.

2.- Allelic frequencies of NR6A11 and VRTN, two genes that affect vertebrae number in diverse pig breeds. Burgos C, Latorre P, Altarriba J, Carrodeguas JA, Varona L, Lopez Buesa P. Meat Science (2015) 100, 150-155.

3.- A2456 substitution in PCK1 gene changes the enzyme kinetic and functional properties modifying fat distribution in pigs. Latorre P, Burgos C, Hidalgo J, Varona L, Carrodeguas JA, Lopez Buesa P. Scientific Reports (2016) 6, 19617.

4.- Inhibition of pig phosphoenolpyruvate carboxykinase isoenzymes by 3-mercaptopicolinic acid and novel inhibitors. Hidalgo J, Latorre P, Carrodeguas JA, Velazquez Campoy A, Sancho J, Lopez Buesa P. Plos ONE (2016) 11(7) e0159002.

 

Main research projects

1.- Modulación de las características del músculo esquelético por la Pepck. AGL 2015, 66177-R (Modulation of skeletal muscle characteristics by Pepck).

 

Collaborators

  • Luis Varona Aguado, Universidad de Zaragoza.
  • Jesús Ventanas and Carmen García, Universidad de Extremadura.
Functional genomics
of the OXPHOS system (GENOXPHOS)

Head of the Research Line:

Patricio Fernández Silva

Researchers:

Patricio Fernández Silva
Patricia Meade Huerta
Raquel Moreno Loshuertos

 

SUMMARY OF THE RESEARCH LINE

Our group is dedicated to the study of biogenesis, structural organization and pathology of the oxidative phosphorylation system (OXPHOS system) using molecular biology and functional genetics techniques. Our main objectives are:

  • Functional genetics of mouse mitochondrial DNA (mtDNA). In order to have a greater number of cellular models with functional alterations in the OXPHOS system, we have developed a mutagenesis technology which has allowed us to obtain a collection of mouse cell lines harbouring mutations in their mtDNA. Thus, we have managed to generate mutants in all respiratory complexes with mtDNA encoded subunits (complexes I, III, IV and V) and also a mutant in mitocondrial protein synthesis. These models allow studying the role of the affected genes by structural and functional analysis.
  • Animal models of mtDNA associated pathologies and potential therapies. We are developing mouse models carrying mtDNA mutations by transferring mitochondria harbouring mutations selected from those generated in our laboratory to embryos. Moreover, we have generated knock-in mice expressing the exogenous protein AOX (alternative oxidase, from fungus Emericella nidulans) and we are crossing them with knock-out mice for complex IV nuclear genes in muscle, which present severe myopathy. In this way, we try to evaluate the potential of the AOX protein as a therapeutic approach for failures in respiratory complexes III and IV.
  • Effect of mtDNA polymorphic variants. To evaluate the influence of mtDNA genotypes (mitocondrial haplogroups) on complex phenotypes such as aging, we have generated conplastic mice, which carry different mtDNA variants in the same nuclear background. Through different methodological approaches such as transcriptomics or metabolomics, we have demonstrated how different combinations of mitochondrial and nuclear genomes are responsible for differences in metabolism or quality of aging in these individuals.
  • Structural organization of the OXPHOS system. We have proposed a new model of organization of the mitochondrial electron transport chain (the plasticity model) and we have identified the first supercomplexes assembly factor (SCAF1). Currently, we are interested in analyzing both genetic and environmental factors involved in the formation of these superstructures as well as in the regulation of their levels and their functional implications on energy metabolism.

 

Relevant publications

1.- Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing. Latorre-Pellicer A, Moreno-Loshuertos R, Lechuga-Vieco AV, Sánchez-Cabo F, Torroja C, Acín-Pérez R, Calvo E, Aix E, González-Guerra A, Logan A, Bernad-Miana ML, Romanos E, Cruz R, Cogliati S, Sobrino B, Carracedo Á, Pérez-Martos A, Fernández-Silva P, Ruíz-Cabello J, Murphy MP, Flores I, Vázquez J, Enríquez JA. Nature 2016 Jul 28; 535(7613):561-5.

2.- The CoQH2/CoQ ratio serves as a sensor of respiratory chain efficiency. Guarás, E. Perales-Clemente, E. Calvo, R. Acín-Pérez, E. Nuñez, C. Pujol, I. Martínez-Carrascoso, M. Loureiro-Lopez, F. García-Marqués, M. A. Rodríguez-Hernández, A. Cortés, F. Diaz, A. Pérez-Martos, C. T. Moraes, P. Fernández-Silva, A. Trifunovic, P. Navas, J. Vázquez and J.A. Enríquez. Cell Reports 2016 Apr 5;15 (1), 197-209.

3.- ROS-triggered phosphorylation of complex II by Fgr kinase regulates cellular adaptation to fuel use.  Acín-Pérez R, Carrascoso I, Baixauli F, Roche-Molina M, Latorre-Pellicer A, Fernández-Silva P, Mittelbrunn M, Sanchez-Madrid F, Pérez-Martos A, Lowell CA, Manfredi G, Enríquez JA. Cell Metab. 2014 Jun 3;19(6):1020-33.

4.- Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado M, Cipolat S, Costa V, Casarin A, Gomes LC, Perales-Clemente E, Salviati L, Fernandez-Silva P, Enriquez JA, Scorrano L. Cell, 2013 Sep 26;155(1):160-71.

5.- Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Lapuente-Brun E, Moreno-Loshuertos R, Acín-Pérez R, Latorre-Pellicer A, Colás C, Balsa E, Perales-Clemente E, Quirós PM, Calvo E, Rodríguez-Hernández MA, Navas P, Cruz R, Carracedo Á, López-Otín C, Pérez-Martos A, Fernández-Silva P, Fernández-Vizarra E, Enríquez JA. Science, 2013 Jun 28;340(6140):1567-70.

6.- Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations. Moreno-Loshuertos R, Ferrín G, Acín-Pérez R, Gallardo ME, Viscomi C, Pérez-Martos A, Zeviani M, Fernández-Silva P, Enríquez JA. PLoS Genet. 2011 Apr;7(4):e1001379.

7.- A genome-wide shRNA screen for new OxPhos related genes. Bayona-Bafaluy MP, Sánchez-Cabo F, Fernández-Silva P, Pérez-Martos A, Enríquez JA. Mitochondrion. 2011 May; 11(3):467-75.

8.- Tissue-specific differences in mitochondrial activity and biogenesis. Fernández-Vizarra E, Enríquez JA, Pérez-Martos A, Montoya J, Fernández-Silva P. Mitochondrion. 2011 Jan; 11(1):207-13.

9.- Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task? Perales-Clemente E, Fernández-Silva P, Acín-Pérez R, Pérez-Martos A, Enríquez JA.  Nucleic Acids Res. 2011 Jan; 39(1):225-34.

10.- Five entry points of the mitochondrially encoded subunits in mammalian complex I assembly. Perales-Clemente E, Fernández-Vizarra E, Acín-Pérez R, Movilla N, Bayona-Bafaluy MP, Moreno-Loshuertos R, Pérez-Martos A, Fernández-Silva P, Enríquez JA. Mol Cell Biol. 2010 Jun; 30(12):3038-47.

11.- Respiratory active mitochondrial supercomplexes. Acín-Pérez R, Fernández-Silva P, Peleato ML, Pérez-Martos A, Enriquez JA. Mol Cell. 2008 Nov 21; 32(4):529-39.

12.- Functional genetic analysis of the mammalian mitochondrial DNA encoded peptides: a mutagenesis approach. Bayona-Bafaluy MP, Movilla N, Pérez-Martos A, Fernández-Silva P, Enriquez JA. Methods Mol Biol. 2008; 457:379-90.

13.- Restoration of electron transport without proton pumping in mammalian mitochondria. Perales-Clemente E, Bayona-Bafaluy MP, Pérez-Martos A, Barrientos A, Fernández-Silva P,Enriquez JA. Proc Natl Acad Sci U S A. 2008 Dec 2; 105(48):18735-9.

14.- Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants. Moreno-Loshuertos R, Acín-Pérez R, Fernández-Silva P, Movilla N, Pérez-Martos A, Rodriguez de Cordoba S, Gallardo ME, Enríquez JA. Nat Genet. 2006 Nov; 38(11):1261-8.

15.- Respiratory complex III is required to maintain complex I in mammalian mitochondria. Acín-Pérez R, Bayona-Bafaluy MP, Fernández-Silva P, Moreno-Loshuertos R, Pérez-Martos A, Bruno C, Moraes CT, Enríquez JA. Mol Cell. 2004 Mar 26; 13(6):805-15.

 

Main research projects

1.- Generación de modelos y ensayo de terapia génica para enfermedades oxphos. FIS (PI12/0129). Investigador principal: Patricio Fernández Silva.

2.- Estudio de factores genéticos y ambientales implicados en el ensamblaje y la estabilidad de los complejos y supercomplejos respiratorios. Universidad de Zaragoza/Ibercaja(JIUZ-2015-BIO-06). Investigador principal: Raquel Moreno Loshuertos.

3.- Estudio del efecto de la modulación del estado Redox y los ROS sobre la formación y estabilidad de los supercomplejos respiratorios. Universidad de Zaragoza (UZ2016-BIO-04). Investigador principal: Raquel Moreno Loshuertos.

4.- B55 genómica funcional del sistema de fosforilación oxidativa (GENOXPHOS). Diputación General de Aragón 2013. Investigador principal: Patricio Fernández Silva.

5.- Grupo Consolidado Biología Estructural (B18). Diputación General de Aragón (B18). Investigador principal: María Luisa Peleato.

6.- Ensayo de la xenoexpresión como terapia génica para las enfermedades mitocondriales. Fundación Ramón Areces (212328). Investigador principal: Patricio Fernández Silva.

7.- Terapia génica de las enfermedades mitocondriales mediante xenoexpresión. FIS (PI09/00946). Investigador principal: Patricio Fernández Silva.

8.- CONSOLIDER. Papel funcional del estrés oxidativo y nitrosativo en grandes sistemas biológicos. Ministerio de Ciencia y Tecnología (CSD2007-00020). Investigador principal: José Antonio Enríquez.

9.- Efecto de los fallos en el sistema OXPHOS sobre la expresión génica y la diferenciación de células ES. Instituto Aragonés de Ciencias de la Salud (PIPAMER09/05). Investigador principal: Patricio Fernández Silva.

10.- EUMITOCOMBAT: rational treatment strategies combating mitocondrial oxidative phosphorilation (OXPHOS) disorders. Unión Europea (LSHM-CT-2004-503116). Investigador principal: José Antonio Enríquez.

 

Collaborators

  • Dr. José Antonio Enriquez. Centro Nacional de Investigaciones Cardiovasculares (CNIC). Madrid-Spain
  • Massimo Zeviani. MBU-MRC. Cambridge-UK
  • Eva Monleón. Dtpo. de Anatomía e Histología Humanas- UZ
Studies on Microcystin
and its Technology

Head of the Research Line:

María Luisa Peleato Sánchez

Researchers:

María Teresa Bes Fustero
Andrés González Rodríguez
Laura Calvo Beguería
Laura Ceballos Laita
Noemi Bervis Semilianelue

 

SUMMARY OF THE RESEARCH LINE

We are a group interested in two main goals: factors affecting the expression of genes involved in microcystin synthesis, and role of microcystins in the cyanobacteria.

Toxic cyanobacterial blooms are more and more widespread in surface waters of the earth, and a serious health concern in many areas due to the production of several toxins, such as high levels of microcystins. Freshwater eutrophyzation has lead to frequent occurrence of blooms. However, there is widespread debate about the effect of the environment on microcystin production, since the toxicity of blooms can vary between years under apparently the same environmental conditions.

Cyanobacteria produce a broad range of secondary metabolites known as cyanotoxins that have toxic effects on eukaryotes. Among the cyanotoxins, there is a group of potent hepatoxins called microcystins. Several genera of cyanobacteria, such as Microcystis, Anabaena, Planktothrix, and Nostoc can produce the cyclic heptapeptide microcystin. Microcystins are potent inhibitors of protein phosphatases 1 and 2A in eukaryotes. Studies of the regulation and function of cyanotoxins are interconnected and have focused on local ecosystems and the effect of environmental parameters on toxin production. In nature, cyanobaterial blooms can be unpredictable toxic or non-toxic from one year to the next, and even under laboratory conditions, results are highly variable. The effects of environmental factors such as light intensity, temperature, nitrogen, phosphorous, and trace metals on microcystins production have been studied under field and laboratory, and the results are conflicting. There are two main open questions concerning the microcystins:

1.- The regulation of the expression of the genes involved in microcystin synthesis, and the environmental factors affecting the toxicity of the cyanobacterial populations.

2.- The physiological role of microcystis.

 

toxiccyanobacterialblooms

 

FACTORS AFFECTING THE MICROCYSTIN SYNTHESIS

  • Results suggest that iron availability is a key factor

Although at present the factors unleashing the cyanotoxin synthesis are unknown, iron availability seems to be a determinant factor in the expression of the gene cluster involved in the microcystin synthesis (mcy operon).

  • Nitrate promote growth, but not microcystin synthesis

Our work indicates that M. aeruginosa growth in laboratory conditions in abundance of nitrate in the medium suffer a bloom similar to the observed in field conditions, but quantification of microcystin-LR and mcyD expression per cell indicated that they were not changed during the bloom.

  • Light and darkness

Microcystin synthesis requires light. In darkness, marked diminution of mcyD expression was detected, similar to the obtained blocking the photosynthetic electron transfer chain using DCMU. Also, mcyD expression was induced as consequence of a light shift to high light conditions.

  • Oxidative stress

Methyl viologen and hydrogen peroxide decrease the expression of mcyD and microcystin synthesis.

 

ROLE OF MICROCYSTINS IN THE CYANOBATERIA

  • Microcystins can bind proteins

In vitro, and probably as consequence of cells disruption, microcystins bind proteins unspecifically.

  • Microcystin-LR can bind some metals

EPR experiments indicated that microcystins complex with Cu and Fe 3+.

 

TECHNOLOGY OF MICROCYSTINS

In collaboration with the company Zeu-Inmunotec we have developed a simple, rapid in vitro bioassy kit to quantify microcystins. The assay is for the detection of microcystins and nodularins in water. It is based on the mechanism of action of microcystins on protein phosphatase PP2A. The test measures the activity of PP2A in water samples potentially contaminated with these toxins. It is therefore an in vitro assay that quantifies the toxicity of all MCs present in the sample and check whether the toxin concentration is over the maximum allowed levels (1 µg/L, WHO 1998).

 

Relevant publications

1.- FurA modulates gene expression of alr3808 a DpsA homologue in Nostoc PCC7120. Hernández, J.A., Pellicer, S., Huang L., Peleato, M.L. and Fillat M.F.  FEBS Letters, Volume 581,1351-1356 (2007).

2.- Cross-talk between iron and nitrogen regulatory networks in Anabaena (Nostoc) sp. PCC 7120: Identification of overlapping genes in FurA and NtcA regulons. López-Gomollón, S; Hernández, JA; Pellicer, S; Espinosa Angarica, V; Peleato, ML; Fillat, MF. J. Mol. Biol., Volume 374:267-81 (2007).

3.- Iron availability affects mcyD expression and microcystin-LR synthesis in Microcystis aeruginosa PCC7806. Sevilla, E., Martin-Luna, B., Vela, L., Bes, M.T., Fillat, M.F. y Peleato, M.L. Environmental Microbiology, 10, 2476-2483 (2008).

4.- Exploring the interaction of microcystin-LR with proteins and DNA. Vela, L., Sevilla, E., González, C., Bes, M.T., Fillat, M.F. y Peleato, M.L. Toxicology in Vitro, 22: 1714-1718 (2008).

5.- Respiratory Active Mitocondrial Supercomplexes. Acin-Perez, R., Fernández-Silva, P., Peleato, M.L., Perez-Martos, A., Enriquez, J.A. Molecular Cell, 32: 529-539 (2008).

6.- New insights into the role of Fur proteins: FurB (All2473) from Anabaena sp. PCC 7120 protects DNA and increases cell survival under oxidative stress. López-Gomollón, S; Sevilla, E., Bes, M.T.; Peleato, ML; Fillat, MF. Biochem. J., Vol. 418:201-7 (2009).

7.- High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment. Pellicer, S., Bes, M.T.; González, A., Neira, J.L., Peleato, ML; Fillat, MF. Process Biochemistry, 45: 292-296 (2010).

8.- Optimization of intracellular microcystin-LR extraction for its analysis by protein phosphatase inhibition assay. E. Sevilla, H. Smienk, P. Razquin, L. Mata and M. L. Peleato. Water Science and Technology, 60: 1903-1909. 2009.

9.- Mutants of Anabaena sp. PCC 7120 lacking of alr1690 and a-furA antisense RNA show a pleiotropic phenotype and altered photosynthetic machinery. Hernández J.A., Alonso I., Pellicer, S., Peleato, M.L., Cases, R., Strasser, R.J., Barja,F. and Fillat M.F. Journal of Plant Physiology, 167: 430-437 (2010).

10.- Microcystin-LR synthesis as response to nitrogen: transcriptional analysis of the mcyD gene in Microcystis aeruginosa PCC7806. Sevilla, E, Martin-Luna, B., Vela, L., Bes, M.T., Fillat, M.F. and Peleato M.L. Ecotoxicology 19:1167-1173 (2010).

11.- Oligomerization properties of cyanobacterial FurA: Direct visualization by in situ atomic force microscopy under different redox conditions. Lostao A., Peleato M.L., Gómez-Moreno, C. and Fillat, M.F. Biochim Biophys Acta, Proteins and Proteomics. Vol. 1804(9): 1723-9 (2010).

12.- Overexpression of FurA induces morphological and physiological changes in Anabaena sp. PCC 7120. González, A., Bes, M.T., Barja, F., Peleato, M.L. and Fillat M.F. Plant and cell Physiology, Vol 51 (11):1900-1914 (2010).

13.- Unravelling the regulatory function of FurA in Anabaena sp. PCC 7120 through 2-D DIGE proteomic analysis. González, A., Bes, M.T., Peleato, M.L. and Fillat M.F. Journal of Proteomics 74:660-71 (2011).

14.- Expression of fur and its antisense a-fur from Microcystis aeruginosa PCC7806 as response to light and oxidative stress. Martin-Luna B, Sevilla E, Gonzalez A, Bes MT, Fillat MF and Peleato ML. J. Plant Physiol (2011).

15.- Identification of three new antisense RNAs in the fur locus from unicellular cyanobacteria. Sevilla E, Martín-Luna B, González A, Peleato ML and Fillat MF. Microbiology (2011).

 

Main research projects

1.- Identification of potentially toxic cyanobacteria and pathogens in free-living amoebae in the waters of Aragón. Government of Aragón-DGA, 01/10/2009 TO: 30/10/2011, IP: Mary F. Fillat Castejón.

2.- Signal transduction mediated by Fura redox (ferric uptake regulator) in cyanobacteria. Effects on photosynthesis and nitrogen fixation. Ministry of Science and Innovation, 01/01/2010 TO 31/12/2012, IP: Mary F. Fillat Castejón.

Contracts and I + D + I with companies:

3.- Food Quality and Safety: Development of new diagnostic test. ITA (Regional Government of Aragon), Zeu Inmunotec-Aragon Regional, University of Zaragoza Otri, February 2003-August 2004. Project Director: Maria Luisa Sánchez Peleato.

4.- Effect of peroxides on the cyanotoxin microcystin. TTO-University of Zaragoza and OX-CTA SA (Huesca) water treatment company, February 2004- May2004. Project Director: Maria Luisa Sánchez Peleato.

5.- Development of test for the detection of biotoxins. TTO-University of Zaragoza and Zeu-Inmunotec, July 2004 – July 2006. Project Director: Maria Luisa Sánchez Peleato.

6.- Development of a rapid test for detection of the toxin microcystin in waters. TTO-University of Zaragoza and Zeu-Inmunote, February 2006-July 2006. Project Director: Maria Luisa Sánchez Peleato.

7.- Development of a new biodegradable composite biocide for control and elimination of pathogens in the water. CDTI-OX S.L. water treatment company, 1 November 2005 to December 31, 2007, Project Director: Maria Luisa Sánchez Peleato.

8.- Validation of a test for the detection of microcystin in waters of mouth. Zeu OTRI Inmunotec, November 6, 2006 to May 5, 2007. Project Director: Maria Luisa Sánchez Peleato.

9.- Developing Technology for determination of the cyanotoxins microcystin in tissues and biological matrices. Araid Foundation, check technology, January-September 2011,Business Partner: Zeu-Inmunotec.

10.- Validation test for detection of microcystin in water in the mouth. January to December 2011, Business Partner: Zeu-Inmunotec.

 

Collaborators

  • Lígia M. Saraiva, ITQB Molecular Genetics of Metalloproteins Group, Instituto de Tecnología Química e Biológica, Portugal.
  • Santos Susin, Apoptose et Système Immunitaire, Institut Pasteur, Francia.
  • José Luis Neira. Centro de Biología Molecular y Celular, Elche, Alicante, España.
  • Antonia Herrero, Instituto de Bioquímica Vegetal y Fotosíntesis (CSIC), Sevilla, España.
  • Enrique Flores, Instituto de Bioquímica Vegetal y Fotosíntesis CSIC.
  • Carlos González, Instituto de Química-Física Rocasolano, CSIC.

 

Contact

Dr. María Luisa Peleato Sánchez
Department of Biochemistry and Molecular and Cell Biology
Faculty of Sciences, University of Zaragoza, Spain

Apoptosis and metabolism

Head of the Research Line:

José Alberto Carrodeguas Villar

Researchers:

Nerea Novo
Guayente Latorre
Alba Beltrán

 

SUMMARY OF THE RESEARCH LINE

Our major research lines deal with, on one side, the characterization of relatively novel proteins, such as Mtch1, involved in apoptosis and, on the other side, the characterization of novel functions in the well-known Bcl-2 family of proteins, with a focus not only in apoptosis but also in metabolism. We are also participating in a project that intends to determine the phenotypic effects of genetic polymorphisms in enzymes involved in intermediate metabolism, such as PEPCK.

1.- Functional characterization of PSAP/Mtch1 (Presenilin 1-associated protein/mitochondrial carrier homolog 1). PSAP interacts with presenilin-1, which is part of the gamma secretase complex, involved in Alzheimer´s disease. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1), since it contains a domain conserved in carriers of the inner mitochondrial membrane, although it is located in the outer membrane. Mtch1 induces apoptotic cell death when expressed in cultured cells.

In this line, we have reported that Mtch1 has two proapoptotic isoforms generated by alternative splicing, which are targeted to the mitochondrial outer membrane by means of several internal targeting signals. Both isoforms contain two proapoptotic domains. Each of these domains is capable of inducing apoptotic cell death when targeted to the outer mitochondrial membrane by fusion to the carboxyl terminal transmembrane domain of Bcl-XL (which is involved both in outer membrane targeting and insertion). Mtch1 can induce apoptotic cell death in the absence of Bax and Bak, proapoptotic members of the Bcl-2 family of proteins. It does not appear to have a carrier function but it could function as a receptor with so-far unknown ligands in the surface of mitochondria.

We are characterizing self-interactions in Mtch1 by a combination of cross-linking and blue-native electrophoretic assays. We are also dissecting the function of different Mtch1 regions. Furthermore, we are analyzing the effect of knocking out Mtch1 in Drosophila, in the whole organism as well as in cultured fly cells.

2.- Bcl-2 proteins. Bcl-2 proteins are major players in the commitment of the cell towards death, by integrating several cell signals. Their known major functions rely on protein-protein interactions for induction or inhibition of the initial steps of cell death. Nevertheless there is increasing evidence that they may have alternative functions related to metabolism regulation. We are working in novel functions of one antiapoptotic protein of this family, Bcl-XL.

In this line, we have reported the involvement of the transmembrane domain of Bcl-XL in the dimerization of the protein. We are now studying the implication of this protein in metabolism.

3.- PEPCK. Together with Dr. Pascual López Buesa, we have been studying several genetic polymorphisms that affect pig meat quality. We are now focusing in both the cytosolic and the mitochondrial phosphoenolpyruvate kinase, not just with respect to phenotypic implications, but also from the point of view of metabolic regulation and links to metabolic pathologies.

In this line, we have reported a polymorphism that alters the kinetic properties of the enzyme producing a phenotypic change in pigs, by increasing fat infiltration in skeletal muscle. We have also characterized kinetically the recombinant mitochondrial isoform of the enzyme.

We are starting to unravel different functionalities of all these proteins that may relate them to the control of cell proliferation, death and metabolism.

 

Relevant publications

1.- c.A2456C-substitution in Pck1 changes the enzyme kinetic and functional properties modifying fat distribution in pigs. Latorre P, Burgos C, Hidalgo J, Varona L, Carrodeguas JA, López-Buesa P. Sci Rep. 2016 Jan 21;6:19617. doi: 10.1038/srep19617.

2.- Early growth response 1 (EGR-1) is a transcriptional regulator of mitochondrial carrier homolog 1 (MTCH 1)/presenilin 1-associated protein (PSAP). Nelo-Bazán MA, Latorre P, Bolado-Carrancio A, Pérez-Campo FM, Echenique-Robba P, Rodríguez-Rey JC, Carrodeguas JA. Gene. 2016 Mar 1;578(1):52-62. doi: 10.1016/j.gene.2015.12.014. Epub 2015 Dec 9.

3.- Reducing the standard deviation in multiple-assay experiments where the variation matters but the absolute value does not. Echenique-Robba P, Nelo-Bazán MA, Carrodeguas JA. PLoS One. 2013 Oct 30;8(10):e78205. doi: 0.1371/journal.pone.0078205. eCollection 2013.

4.- Protein oligomerization mediated by the transmembrane carboxyl terminal domain of Bcl-XL. Ospina A, Lagunas-Martínez A, Pardo J, Carrodeguas JA. FEBS Lett. 2011 Oct 3;585(19):2935-42. doi: 10.1016/j.febslet.2011.08.012. Epub 2011 Aug 16.

5.- Identification of specific pluripotent stem cell death–inducing small molecules by chemical screening. Conesa C, Doss MX, Antzelevitch C, Sachinidis A, Sancho J, Carrodeguas JA. Stem Cell Rev. 2012 Mar;8(1):116-27. doi: 10.1007/s12015-011-9248-4.

6.- Exposure of any of two proapoptotic domains of presenilin 1-associated protein/mitochondrial carrier homolog 1 on the surface of mitochondria is sufficient for induction of apoptosis in a Bax/Bak-independent manner. Lamarca V, Marzo I, Sanz-Clemente A, Carrodeguas JA. Eur J Cell Biol. 2008 May;87(5):325-34. doi: 10.1016/j.ejcb.2008.02.004. Epub 2008 Mar

7.- Two isoforms of PSAP/MTCH1 share two proapoptotic domains and multiple internal signals for import into the mitochondrial outer membrane. Lamarca V, Sanz-Clemente A, Pérez-Pé R, Martínez-Lorenzo MJ, Halaihel N, Muniesa P, Carrodeguas JA. Am J Physiol Cell Physiol. 2007 Oct;293(4):C1347-61. Epub 2007 Aug 1.

8.- DNA binding properties of human pol gammaB. Carrodeguas JA, Pinz KG, Bogenhagen DF. J Biol Chem. 2002 Dec 20;277(51):50008-14. Epub 2002 Oct 11.

9.- Crystal structure and deletion analysis show that the accessory subunit of mammalian DNA polymerase gamma, Pol gamma B, functions as a homodimer. Carrodeguas JA, Theis K, Bogenhagen DF, Kisker C. Mol Cell. 2001 Jan;7(1):43-54.

10.- Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase. Carrodeguas JA, Bogenhagen DF. Nucleic Acids Res. 2000 Mar 1;28(5):1237-44.

 

Main research projects

1.- Modulación de las características del músculo esquelético por la fosfoenolpiruvato carboxiquinasa. Facultad de Veterinaria – Universidad de Zaragoza. Pascual López Buesa y José Alberto Carrodeguas Villar. CICYT. 2016-1018.

2.- Modulación de las características del músculo esquelético por la fosfoenolpiruvato carboxiquinasa. Instituto Universitario De Investigación De Biocomputación y Física De Sistemas Complejos – Universidad de Zaragoza. José Alberto Carrodeguas Villar. VIC. INV. – APOYO INV. 2016.

3.- PEPCK y sus efectos sobre el metabolismo, los caracteres productivos y la calidad de la carne y la canal del ganado porcino. Facultad De Veterinaria – Universidad de Zaragoza. Pascual Luis López Buesa. VIC. INV. – APOYO INV. 2015.

4.- Identificación de moléculas bioactivas en células troncales mediante cribado funcional de quimiotecas: herramientas para terapias seguras. Instituto de Biocomputación y Física de Sistemas Complejos. José Alberto Carrodeguas Villar. Universidad de Zaragoza/Ibercaja. 2012-2013.

5.- Proteínas Mtch: regulación transcripcional en humanos y efectos fenotípicos del mutante en Drosophila. Instituto Universitario De Investigación De Biocomputación y Física De Sistemas Complejos. José Alberto Carrodeguas Villar. Universidad de Zaragoza. 2011.

6.- Genética química para la identificación de compuestos bioactivos que promueven diferenciación específica, proliferación o apoptosis en células madre. Instituto de Biocomputación y Física de Sistemas Complejos. Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. Universidad de Zaragoza. José Alberto Carrodeguas Villar. Instituto Aragonés de Ciencias de la Salud. 2011.

7.- Regulación de la actividad de proteínas proapoptóticas mitocondriales. Instituto de Biocomputación y Física de Sistemas Complejos. Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. Universidad de Zaragoza. José Alberto Carrodeguas Villar. Ministerio de Ciencia e Innovación. 2010.

8.- Identificación de compuestos químicos que inducen diferenciación celular específica o muerte celular apoptótica en células madre embrionarias de ratón (continuación). Instituto de Biocomputación y Física de Sistemas Complejos. Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. José Alberto Carrodeguas Villar. Instituto Aragonés de Ciencias de la Salud. 2009-2010.

9.- Identificación de compuestos químicos que inducen diferenciación celular específica o muerte celular apoptótica en células madre embrionarias de ratón (continuación). Instituto de Biocomputación y Física de Sistemas Complejos. Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. José Alberto Carrodeguas Villar. Instituto Aragonés de Ciencias de la Salud. 2008-2010.

10.- Mecanismos moleculares de proteínas de la membrana externa mitocondrial similares a transportadores implicadas en apoptosis. Papel en enfermedades degenerativas y en cáncer. Facultad de Veterinaria. Universidad de Zaragoza. José Alberto Carrodeguas Villar. MEC. 2006-2009.

 

Collaborators

  • Miguel Fernández Moreno and Juan José Arredondo. Instituto de Investigaciones Biomédicas. CSIC-UAM. Madrid.
  • Javier Sancho, Milagros Medina, Adrián Velázquez Campoy, Patricio Fernández Silva and Raquel Moreno Loshuertos. Instituto de Biocomputación y Física de Sistemas Complejos. Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. Universidad de Zaragoza.
  • Ramón Hurtado Guerrero. Instituto de Biocomputación y Física de Sistemas Complejos. Universidad de Zaragoza.
  • José Carlos Rodríguez-Rey. Department of Molecular Biology, University of Cantabria, IDIVAL, Santander, Cantabria, Spain.
  •  Flor Pérez Campo. Department of Internal Medicine, Hospital U. Marqués de Valdecilla-IDIVAL University of Cantabria, 39008 Santander, Cantabria, Spain.

Plant Evolutionary Biology

Head of the Research Line:

Pilar Catalán Rodríguez

Researchers:

Ernesto Pérez
Isabel Marques
Rubén Sancho

SUMMARY OF THE RESEARCH LINE

Our research focuses on molecular systematics, population-genetics, bio/phylogeography, comparative genomics, diversity and conservation of plants. The group of species that constitute our primary interest are the temperate grasses (Poaceae, Festuca, Brachypodium), distributed in most continents and including many ecologically and economically important species, and other mountain, steppe and Mediterranean-type plant species. Our research emphasize on studies of functional genomics, phylogeny, speciation, hybridization, polyploidization, island and continental colonizations, reproductive biology, ecological adaptation, niche modelling, conservation genetics and taxonomy of wild plants. Our lab has implemented new approaches of genomic inheritance analysis in plant polyploids and cutting-edge plant phylogenomics and landscape genomics analyses. In collaboration with our colleagues of the Joint Genome Institute and the International Brachypodium Consortium, we use model plants of the grass genus Brachypodium to investigate the evolution and regulatory mechanisms of relevant biological and adaptive traits such as switches in annuality/perenniality and tolerance to environmental stresses.

 

Relevant publications

1.- Past climate changes facilitated homoploid speciation in three mountain spiny fescues (Festuca, Poaceae). Marques I, Draper D, López-Herranz ML, Segarra-Moragues JG, Catalán P. Sci Rep. 2016. 6:36283. doi: 10.1038/srep36283.

2.- Transcriptome-derived evidence supports recent polyploidization and a major phylogeographic division in Trithuria submersa (Hydatellaceae, Nymphaeales). Marques I, Montgomery S, Barker MS, Macfarlane T, Conran J, Catalán P, Rieseberg L, Rudall PJ, Graham SW. New Phytol. 2016. 210: 310-323. doi: 10.1111/nph.13755.

3.- Late Cretaceous – Early Eocene origin of yams (Dioscorea, Dioscoreaceae) in the Laurasian Palearctic and their subsequent Oligocene – Miocene diversification. Viruel J, Segarra-Moragues JG, Raz L, Forest F, Wilkin P, Sanmartin I, Catalán P. J Biog. 2016. 43: 750-762. doi: 10.1111/jbi.12678.

4.- Evolution of the beta-amylase gene in the temperate grasses: non-purifying selection, recombination, semiparalogy, homeology and phylogenetic signal. Minaya M, Díaz-Pérez AJ, Mason-Gamer R, Pimentel M, Catalán P. Mol Phylogenet Evol. 2015. 91: 68-85. doi: 10.1016/j.ympev.2015.05.014. Epub 2015 May 29.

5.- Ant pollination promotes spatial genetic structure in the long-lived Borderea pyrenaica (Dioscoreaceae). Pérez-Collazos E, Segarra-Moragues JG, Villar L, Catalán P. Biol J Linnean Soc . 2015. 116: 144-155. doi: 10.1111/bij.12562.

6.- Update on genomics and basic biology of Brachypodium. Catalan P, Chalhoub B, Chochois V, Garvin DF, Hasterok R, Manzaneda AJ, Mur LAJ, Pecchioni N, Rasmussen SK, Vogel JP, Voxeur A. Trends Plant Sci 2014. 19:414-418. doi: 10.1016/j.tplants.2014.05.002. Epub 2014 Jun 7.

7.- Latitudinal environmental niches and riverine barriers shaped the phylogeography of the central Chilean endemic Dioscorea humilis (Dioscoreaceae). Viruel J, Catalán P, Segarra-Moragues JG. PLoS ONE 2014. 9(10): e110029. doi:10.1371/journal.pone.0110029.

8.- Mediterranean origin and Miocene-Holocene Old World diversification of meadow fescues and ryegrasses (Festuca subgen. Schedonorus and Lolium). Inda L.A., Sanmartin I, Buerki S, Catalán P. J Biog. 2014. 41: 600-614. doi: 10.1111/jbi.12211.

9.- A DNA barcoding method to discriminate between the model plant Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae). López-Alvarez D, López-Herranz ML, Betekhtin A, Catalán P. PLoS ONE. 2012 7(12): e51058. doi:10.1371/journal.pone.0051058.

10.- Divergence and biogeography of the recently evolved Macaronesian red Festuca (Gramineae) species inferred from coalescence-based analyses. Diaz-Pérez A, Sequeira M, Santos-Guerra A, Catalán P. Mol Ecol. 2012 21: 1702-1726. doi: 10.1111/j.1365-294X.2012.05495.x. Epub 2012 Feb 21.

 

Main research projects

1.- Evolución de caracteres biológicos y procesos de especiación en el género modelo Brachypodium (Poaceae) mediante análisis de genómica comparada y funcional. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez, Ernesto Pérez Collazos y Rubén Sancho Cohen. MINECO. 2017-2019.

2.- Perenniality, abiotic stress tolerance, and biomass allocation in Brachypodium, a model grass genus for bioenergy. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez. JOINT GENOME INSTITUTE – CSP. 2017-2021.

3.- Evolución de caracteres biológicos (perennialidad, alopoliploidia, adaptación al nicho ambiental) en el género modelo de gramíneas Brachypodium mediante análisis genómicos, citogenéticos y de modelización ecológica. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez, Ernesto Pérez Collazos y Rubén Sancho Cohen. VIC. INV. – APOYO INV. 2016.

4.- Origin: The model plant system Trithuria (Hydatellaceae), a new window into the origin of flowering plants and gene function. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Isabel Marques y Pilar Catalán Rodríguez. MARIE CURIE IOF. 2013-2016.

5.- Genómica comparada, biogeografía y evolución floral y adaptativa de gramíneas modelo. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez, Ernesto Pérez Collazos y Rubén Sancho Cohen. CICYT. 2013-2015.

6.- Brachypodium adaptation to drought stress across different geographic and ecological clines. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez y Ernesto Pérez Collazos. EPPN. 2013-2014.

7.- Genética y ecología del paisaje de pastos subalpinos pirenaico-cantábricos (Festuca, Gramineae): Conservación de la biodiversidad y restauración vegetal. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez y Ernesto Pérez Collazos. MMAMRM-OAPN. 2010-2012.

8.- Evolución multigenómica de las gramíneas templadas (Pooideae, Poaceae). Biogeografía y filogeografía de especies modelo de pooideas. Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez y Ernesto Pérez Collazos. CICYT. 2010-2012.

9.- Sistemática, evolución y biogeografía de los linajes basales de la subtribu Loliinae y transferencia horizontal de genes en la supertribu Aveneae-Poeae (Gramineae). Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez y Ernesto Pérez Collazos. CICYT. 2006-2009.

10.- Convergencia evolutiva transcontinental y genética de la conservación de los ñames enanos (Dioscoreaceae) críticamente amenazados (Borderea, Epipetrum). Departamento de Ciencias Agrarias y del Medio Natural. Escuela Politécnica Superior de Huesca – Universidad de Zaragoza. Pilar Catalán Rodríguez y Ernesto Pérez Collazos. FBBVA. 2006-2009.

 

Collaborators

  • John Vogel and Sean Gordon. Joint Genome Institute. Walnut Creek CA USA.
  • David Des Marais. Arnold Arboretum. University of Harvard. Boston MA USA.
  • Richard Amasino and Daniel Woods. University of Wisconsin-Madison. Madison WI USA.
  • Bruno Contreras-Moreira. Estación Experimental de Aula Dei – CSIC. Zaragoza Spain.
  • Antonio Manzaneda. Universidad de Jaén. Jaén Spain.
  • Teresa Garnatje. Instituto Botánico de Barcelona – CSIC. Barcelona Spain.
  • Boulous Chalhoub. Centre Versailles-Grignon INRA. Versailles France.
  • Robert Hasterok and Alexander Betekhtin. University of Silesia. Katowice Poland.
  • Luis Mur, John Draper and John Doonan. Aberystwyth University. Aberystwyth UK.
  • Antonio Díaz-Pérez. Universidad Central de Venezuela. Maracay Venezuela.
  • Liliana Giussani. Instituto Botánico Darwinion – CONICET. Buenos Aires Argentina.
  • Marina Olonova. Tomsk State University. Tomsk Russia.

 

http://bifi.es/bioflora/

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