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Lifelong Learning Programme Erasmus Intensive Programme

Granada, SPAIN, 2nd – 13th June, 2014

Towards a Scientific Career: an Introductory Course for Research in Biomedicine and Biotechnology (Second Edition) – BIOMED-TECH

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Towards a Scientific Career : an Introductory Course for Research in Biomedicine and Biotechnology (Second Edition) – BIOMED-TECH Editor: Rafael Salto González Diseño y maquetación: Luis Doña Toledo Ediciones Sider Granada, 2014 ISBN: 978-84-941343-7-1

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Summary

Foreword

Organizers

Organizing Committee

Participants

Meeting Venues

Course Schedule

Round Tables

Workshops

Scientfic visits

Students communications

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Foreword

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This course introduces students to a research career in the sphere of Biomedicine and Biotechnology, taking advantage of the particular strengths of the Campus of International Excellence of the University of Granada, Spain (CEI-BioTic) as coordinating institution. The partner institutions of the programme, working alongside the University of Granada, are the University of Nottingham (United Kingdom) and the University of Nova Lisboa (Portugal). The research companies participating are Abbott Laboratories SA, Bio -Iliberis R&D, Neuron Bio and the Fundación MEDINA. Also renowned guest lecturers from universities and research centers external to the consortium will

collaborate.

Objective: To help students achieve a professional research career in the sphere of Biomedicine and Biotechnology, taking advantage of the particular strengths of the Campus of International Excellence of the University of Granada (CEI-BioTic) as coordinating institution.

Target groups: 15 students (five students from each participating university) in the fin al year of their degree programme, which must be related, although not necessarily exclusively, to the areas of Biomedicine and Biotechnology.

Core activities: tutoring; workshops and practical sessions in the fields of Biomedicine and Biotechnology; programmes of visits to companies operating in these fields; talks and round -table events.

Learning outcomes: it is anticipated that, as a result of this intensive programme, students will be geared towards a professional research career in Biomedicine an d/or Biotechnology; they will understand the practical dimension of a scientific career (both academically and from the industry perspective); they will be aware of the professional opportunities in these scientific fields; and they will have established an initial network of contacts both in public institutions and in the industry.

Other anticipated outcomes: for teaching staff, the experience will facilitate university -industry cooperation, and will establish teaching approaches designed to motivate stud ents towards choosing a research career. The programme will also contribute to raising awareness of the Campus of International Excellence project, of which this Intensive Programme forms a part.

Foreword

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This activity is funded by the European Union as part of its action program in the field of Lifelong Learning, adopted

by the European Parliament and Council on the 15 th November 2006 (Decision No. 1720/2006/CE).

Organizers

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Organizing Committee Coordinator Rafael Salto González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Granada, Spain) Secretary Inmaculada Llamas Company (Department of Microbiology, School of Pharmacy, University of Granada, Granada, Spain) Technical Secretariat Noelia Terrón Arenas (CEI-BIOTIC, University of Granada, Granada, Spain) Members Fernando Henández Mateo (Department of Organic Chemistry, School of Sciences, University of Granada, Granada, Spain) María Dolores Girón González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of

Granada, Granada, Spain) Cecília Arraiano (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Lisboa, Portugal) Miguel Camara (Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United

Kingdom)

Organizing Committee

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Participants

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Professors

Miguel Alaminos Mingorance (Tissue Engineering Group, Department of Histology, School of Medicine, University of Granada, Spain)

Cecília Maria Arraiano (ITQB-Instituto de Tecnologia Química e Biológica / Universidade Nova de Lisboa, Oeiras, Portugal)

Steve Atkinson (Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom)

Elena Cabrera Cazorla (Department of Biochemistry and Molecular Biology II, School of Sciences, University of Granada, Spain)

Miguel Cámara (School of Molecular Medical Sciences, University of Nottingham, United Kingdom)

Victor Carriel (Tissue Engineering Group, Department of Histology, School of Medicine, University of Granada, Spain)

David Castro (Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain)

Manuel Espinosa Urgel (Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain)

José Luis García Pérez (Pfizer Center-Junta de Andalucia Center for Genomics and Oncological Research (Genyo), University of Granada, Granada, Spain)

Olga Genilloud (Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, Spain)

María Dolores Girón González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

Stephan Heeb (Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom)

Fernando Hernández Mateo (Department of Organic Chemistry, School of Sciences, University of Granada, Granada, Spain)

Tino Krell (Estación Experimental del Zaidin, CSIC, Granada, Spain)

Inmaculada Llamas Company (Department of Microbiology, School of Pharmacy, University of Granada, Spain)

José María López Pedrosa. (Department I + D, Abbott Laboratories, Granada, Spain)

Francisco Javier Marcos Torres (Department of Microbiology, School of Sciences, University of Granada, Spain)

José Marques Andrade (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Ignacio Molina Pineda de las Infantas (Department of Biochemistry and Molecular Biology II I & Immunology, School of Medicine, University of Granada, Spain)

José Muñoz Dorado (Department of Microbiology, School of Sciences, University of Granada, Spain)

Juana Pérez Torres (Department of Microbiology, School of Sciences, University of Granada, Spa in)

Vânia Pobre (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal)

Participants

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Emilia Quesada Arroquia (Department of Microbiology, School of Pharmacy, University of Granada, Spain)

José Carlos Reina Cabello (Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain)

Elena Requena Rodríguez (Neuron Bio, P. T. S. Granada, Granada, Spain)

Amalia Roca Hernández (Departamento de Agronomía- I+D+i, Bio-Iliberis R&D, Poligono Industrial Juncaril,

Peligros, Granada, Spain)

Rafael Salto González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of

Granada, Spain)

José Dámaso Vílchez Rienda (Department of Biochemistry and Molecular Biology II , School of Pharmacy, University of Granada, Spain)

Students

Christian Arenas López (University of Nottingham, Nottingham, UK)

Susana Barahona (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Natalie Barratt (University of Nottingham, Nottingham, UK)

Anabela Carvalho Vieira (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Lidia Delgado Calvo Flores (University of Granada, Granada, Spain)

Ricardo F. dos Santos (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Rebeka Krebesova (University of Granada, Granada, Spain)

Cátia María Morgado Peres (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Carlos Peris Torres (University of Granada, Granada, Spain)

Teresa Pinto (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Francisco José Reche Perez (University of Granada, Granada, Spain)

Francisco Ismael Román Moreno (University of Granada, Granada, Spain)

Amy Slater (University of Nottingham, Nottingham, UK)

Daniella Spencer (University of Nottingham, Nottingham, UK)

Carmen Tong (University of Nottingham, Nottingham, UK)

Participants

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Meeting Venues

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Salón de Actos

Residencia Universitaria Corrala de Santiago, Universidad de Granada

Calle Santiago, 518009 – Granada

Opening of the Erasmus Course - Round Tables - Clausure

School of Sciences, University of Granada

Calle Doctor Severo Ochoa, s/n 18071 Granada

Workshop 2 - Workshop 4

Meeting Venues

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School of Pharmacy, University of Granada

Campus de Cartuja sn

18071 Granada

Workshop 1 - Workshop 3

Meeting Venues

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June, 2nd-6th

2nd June 3th June 4th June 5th June 6th June 9:30-10:00 Opening of the Erasmus Course

9:30-14:00 Round Table 2: Regulation of gene expression in bacteria

9:30-14:00 Round Table 3: Microbiology, Signal Transduction and Biotechnology

9:30-14:00 Visit 1: Bio-Iliberis

9:30-14:00 Round Table 4: Frontiers in Biotechnology

10:00-14:00 Round Table 1: Signaling in Bacteria, a promising field in Biotechnology 14:00-16:00 Lunch

14:00-16:00 Lunch

14:00-16:00 Lunch

14:00-16:00 Lunch

14:00-16:00 Lunch

16:00-19:30 Workshop 1: Quorum Sensing and Quorum Quenching (I)

16:00-19:30 Workshop 1: Quorum Sensing and Quorum Quenching (II)

16:00-19:30 Workshop 2: Bioinformatic analysis of bacterial signal transduction systems

16:00-19:30 Workshop 1: Quorum Sensing and Quorum Quenching (III)

16:00-19:30 Workshop 1: Quorum Sensing and Quorum Quenching (IV)

Course Schedule

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June, 9th-13th

9th June 10th June 11th June 12th June 13th June

9:30-12:00

Round Table 5:

Advanced Chemistry

for Biomedicine

9:30-12:00

Visit 2:

Abbott Laboratories

9:30-11:00

Round Table 6:

Employment and

Research

opportunities

9:30-12:00

Visit 3: Fundación Medina

9:30-14:00

Round Table 7:

Frontiers in

Biomedicine

12:00-14:00

Students Poster

Presentations 1

12:00-14:00

Students Poster

Presentations 2

11:00-14:00

Students Poster

Presentations 3

12:00-14:00

Visit 4:

Neuron Bio 13:00-14:00

Closure

14:00-16:00 Lunch

14:00-16:00 Lunch

14:00-16:00 Lunch

14:00-16:00 Lunch

14:00-16:00 Lunch

16:00-19:30 Workshop 3: Site directed mutagenesis as a tool for the analysis of the structure-function in proteins (I)

16:00-19:30 Workshop 3: Site directed mutagenesis as a tool for the analysis of the structure-function in proteins (II)

16:00-19:30 Workshop 3: Site directed mutagenesis as a tool for the analysis of the structure-function in proteins (III)

16:00-19:30 Workshop 4: Synthesis and Characterization of neoglycoproteins by Click Chemistry and the Chemistry of Vinyl Sulphones (I)

16:00-19:30 Workshop 4: Synthesis and Characterization of neoglycoproteins by Click Chemistry and the Chemistry of Vinyl Sulphones (II)

Course Schedule

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Round Tables

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Round table 1 June 2nd, 10:00-14:00

Signalling in Bacteria, a Promising Field in Biotechnology and Biomedicine Chairwoman: Inmaculada Llamas Company (Department of Microbiology, School of Pharmacy, University of Granada, Spain)

The complexity of bacterial communication through quorum sensing Steve Atkinson (Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.)

Impact of quorum sensing in different industrial sectors Miguel Cámara (Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.)

Biofilm formation and intercellular signaling in plant -bacteria interactions Manuel Espinosa Urgel (Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain)

Round tab le 1

Round Tables

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The complexity of bacterial communication through quorum sensing Steve Atkinson. Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

Impact of quorum sensing in different industrial sectors Miguel Cámara. Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom. OBJECTIVES

Describe how quorum sensing controls gene expression in bacteria

Understand the mechanisms by which quorum sensing causes disease in different bacteria

Understand the impact of quorum sensing signal molecules on different hosts

Discuss the exploitation of quorum sensing in different industrial sectors Bacteria can colonise different environmental niches and to do so they need very sophisticated mechanisms of adaptation which enable them to sense these changes and respond in a coordinated manner. For a long time it was considered that bacteria, as unicellular organisms, existed in isolation. However, it has now been known for some time that bacteria possess intercellular sensing mechanisms which facilitate adaptive responses to changes in the external environment enabling them to exhibit multicellular behaviour. The ability of a single bacterial cell to communicate with its neighbours to mount a unified response that is advantageous to its survival makes considerable sense. A prime example is the co -ordinated differentiation and migration of groups of highly specialised swarmer cells in the rapid colonisation of a surface. Similarly, in the process of conjugal plasmid

transfer, the ability of a potential donor cell to monitor the availability of recipient ho st cells, would appear crucial before committing itself to an energetically costly process. For opportunistic pathogens, the outcome of the interaction between the host and the bacterium is strongly affected by bacterial population density. Coupling the production of virulence factors with bacterial population density ensures the host has insufficient time to mount an effective defence against the consolidated attack from the invading bacterial population. Bacteria are now known to be able to sense diffu sible signal molecules, generated by other bacterial cells to modulate specific adaptive responses that enhance survival. These small, extracellular molecules are termed microbial “autoinducers” or “pheromones”. This autoinducers are used by individual b acterial cells generally to gain information from the status of other members of the same species. The term “quorum sensing” has been adopted to describe the accumulation of a diffusible low molecular weight signal molecule to enable an individual bacterial cell to sense when a minimum numbe , or “quorum” of bacteria has been attained to facilitate a co -ordinate adaptive response. Quorum sensing (QS) is thus an example of multicellular behaviour in bacteria and regulates diverse physiological processes including bioluminescence, swarming, antibiotic biosynthesis, plasmid conjugal transfer and the production of virulence determinants in animal, fish and plant pathogens.

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The genetic basis of QS-mediated gene regulation:

Figure 1 : Basic quorum sensing regulatory circuit At the genetic level, any QS system is constituted by two basic genetic elements (Fig.1): (i) the gene(s) involved in the biosynthesis of the signal molecule (S), in some Gram -negatives known as the “I” gene (for inducer), and ( ii) the gene(s) involved in the signal transduction (T), in some Gram -negatives known as the “R” gene (for regulator). The signal synthetase is responsible for the synthesis of the autoinducer molecule which, upon accumulation in the environment, activates the signal transducer or transcriptional regulator protein. This in turns will result in the autoinduction of more signal synthesis (amplification loop), and the activation of multiple gene expression in a coordinated fashion within the whole bacterial p opulation. QS is used by many organisms

Until more than two decades ago, N-acylhomoserine lactone (AHL)-mediated regulation of gene expression was considered to be restricted to just a few esoteric bioluminescent marine bacteria related to Vibrio fischer i. However, the Gram-negative plant pathogen Erwinia carotovora was unexpectedly discovered to use N-3-oxohexanoyl-homoserine lactone (3OC6-HSL) to control both the production of a carbapenem antibiotic and virulence. Mutants which could not synthesize 3 OC6-HSL were avirulent in a plant model of infection, and virulence could be restored in these mutants by the addition of synthetic 3OC6 -HSL. This discovery was followed by a major surge of interest in the role of AHLs in bacterial gene expression. A fam ily of AHL signal molecules has now been identified which varies predominantly in the presence or absence of an acyl chain substituent group at C3 (oxo - or hydroxy-) and the length of the N-acyl side chain. In addition, genes encoding homologues of the V. fischeri LuxI and LuxR proteins have been cloned from many different Gram -negative bacteria including Aeromonas hydrophila , Agrobacterium tumefaciens, Burkholderia cepacia, E. carotovora, Pseudomonas aeruginosa, P. aureofaciens, Yersinia enterocolitica, Y. pseudotuberculosis, Y. pestis and Rhizobium leguminosarum.

QS signal molecules are chemically diverse AHLs are not the only class of QS signal molecules (QSSMs). P. aeruginosa for example also produces a quinolone signal molecule (Fig. 2) and even a cis-3-decenoic acid, which belongs to the family of the Diffusible Signal Molecules (DSF) originally found in Xanthomonas campestris to control virulence and now also identified in other organisms such as Burkholderia spp. Gram-positive bacteria also quorum sense, although the nature of the signal

Signal

Synthesis

ST

Signal

Transduction

Amplification

Loop

T

Multiple Gene Expression

• Antibiotic Production

• Symbiosis

• Virulence

• Swimming & Swarming

• Conjugation

• Biofilm Development

• Growth Inhibition

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molecules differs from those of Gram-negative bacteria, generally preferring to use peptides. Peptide -based

adaptation is now known to be utilised by a variety of bacteria to regulate a number of physiological processes. For example, both Bacillus subtil is and Streptococcus pneumoniae employ peptides as QS molecules to regulate development of bacterial competence as well as sporulation. Moreover, in Staphylococcus aureus and Enterococcus faecalis , peptide-mediated QS is involved in the regulation of viru lence genes. More recently, quorum sensing regulatory mechanisms have also been found in eukaryotic cells including yeast, such as Candida albicans, which uses farnesol as a quorum sensing signal and fungi, such as Aspergillus flavus, which uses oxolipids to control sporulation and toxin production.

Figure 2. The diversity of quorum sensing signal molecules (QSSMs) The multi-functionality of QS molecules form Gram -negative bacteria: • QS and virulence gene regulation An indication of a role for AHLs in the pathogenesis of human infection initially came from studies of the opportunistic pathogen P. aeruginosa. This environmental bacterium can infect almost any body site given the right predisposing conditions. Individuals at greatest risk of P. aeruginosa infection include those with burn wounds and cystic fibrosis . P. aeruginosa secretes many different extracellular toxic factors including exotoxins which inhibit protein synthesis in mammalian cells and various tissue -damaging exoenzymes including an alkaline protease and an elastase. Most of these virulence determinants are con trolled via a QS-dependent “hierarchical”

cascade which consists of at least two major AHL signal molecules, namely N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), together with two pairs of I/R homologue s (LasI-LasR and RhlI-RhlR). P. aeruginosa also produces another QS signal molecule, the Pseudomonas quinolone signal (PQS) which is also released into the extracellular environment. PQS belongs to an extensive family of 4 -hydroxy-2-alkyl-quinolines (AQs) produced by this organism. The biosynthesis of AQs is more complex than that of AHLs and requires a series

Gram-negative Gram-positive

CSP-1

γ-Butyrolactones

O

HO

H

H

OO

Cyclic Peptides

Tyr Ser Thr NH

H

O

S

O

Asp

PheIle

NH

SMe

C18-FAME

H3CO

OHO

2-Alkyl-4-

QuinolonesNH

R2

O

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N-Acylhomoserine Lactones

O

O

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R2

OR1 HO

O

OH

CH3

O

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O

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CH3

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2HN-EMRLSKFFRDFILQRKK-COOH

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Farnesol Oxylipids

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of gene known as the pqs genes. PQS has been shown to be essential for virulence although some of the key

mechanisms behind this regulation remain unknown. Furthermore PQS also modulates AHL -mediated responses in this organism. AHL and AQ-mediated QS signalling has been found be active during lung infections of cystic fibrosis patients as these molecules have been detected in their sputum. In animal models, P. aeruginosa QS mutants show a marked reduction in virulence, emphasising the role of QS in disease. Acute P. aeruginosa infections are caused by free-swimming planktonic cells that multiply and spread rapidly within the host tissues. However, it is also apparent that P. aeruginosa is capable of growing as surface-associated biofilms and this mode of growth is a strategy for causing chronic, persistent infections since biofilms are highly resistant to antibiotics, disinfectants and the actions of the immune system. Evidence for the occurrence of P. aeruginosa biofilms in the cystic fibrosis lung has been obtained using both optical and electron microscopy of sputum and post-mortem samples. For some conditions, QS may play a crucial role in the development of biofilm communities. Yet in others, biofilm formation itself ma y represent a means to achieve a quorum. The production of many different QS-regulated functions have been shown to affect biofilm development in P. aeruginosa such as the sugar-binding lectins LecA and LecB, the surfactant rhamnolipids, etc. Whilst AQ type molecules are restricted mainly to Pseudomonas aeruginosa and some Burkholderia spp, AHL production is very much wide spread mainly amongst Gram -negative bacteria. One example of these is Yersinia pseudotuberculosis and Yersinia enterocolitica, which are capable of causing self-limiting gastric infections in humans, and Yersinia pestis which is the causative agent of plague, a rapidly spreading lethal sepsis. Recent genome sequencing has revealed that Y. pseudotuberculosis and Y. pestis share almost 99% identity at the DNA level, with Y. pestis being a recently evolved sub -clone of Y. pseudotuberculosis, arising approximately 20,000 years ago. For a number of years Y. pseudotuberculosis and Y. enterocolitica have also been used as model organisms to investigate the molecular mechanisms which underpin QS and have revealed the presence of quorum sensing I/R orthologues of in Y. pseudotuberculosis (ypsR/I, YtbR/I) and Y. enterocolitica (yenI/R ycoR). Both organisms synthesise a series of AHLs which, through genetic mutation or signal degradation, have been shown to be important for regulating a diverse range of virulence descriptors such as biofilm formation, motility, adhesion, and the regulation of components the type three secretion system. •QS in Gram-positive bacteria One of the best characterised QS systems in Gram -positive species is that of Staphylococcus aureus (Fig. 3). In S. aureus, AIP signal transduction involves a two-component system comprising a sensor kinase and a response regulator. The agr locus consists of two divergent transcriptional units. One operon is expressed from the P2 promoter and consists of agrBDCA, which encode the proteins needed for generatin g and sensing the peptide

signal molecule. The divergent operon, transcribed from the P3 promoter, encodes δ-haemolysin and produces

the non-coding RNAIII. The QS peptide is derived from the product of the agrD gene by the action of the AgrB protein, which is also responsible for the export of the peptide out of the cell. As the bacterial population increases, the peptide accumulates in the extracellular environment. Once a critical concentration is reached, the peptide interacts with the AgrC membrane-associated sensor protein (a histidine protein kinase) causing it to autophosphorylate. The high-energy phosphate is then transferred to the response regulator and in its phosphorylated state AgrA then induces the transcription of RNAIII, the effector molecule, which mediates the changes in expression of multiple target genes resulting in the agr response. Since the genes responsible for

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peptide production (agrBD) are also induced, this results in the production of more peptide signal and the

generation of an autoinduction feedback loop.

Figure 3. Quorum sensing in Staphyloccoccus aureus. •QS molecules can be key for cross-talk during AHLs and AQs can also influence the behavior of other bacteria. Although there are several examples in the literature of this phenomenon, the majority refer to cross -talk between Gram-negative bacteria. However, it has been shown that AHLs produced by P. aeruginosa can inhibit toxin production in Staphylococcus aureus and the mechanism is by inhibiting the QS system of this Gram -positive bacterium. This could explain why in some lung infections when P. aeruginosa colonises, the levels of S. aureus are reduced. • Impact of QS molecules and in the host bacterial interactions and inter -kingdom signalling In addition, some QS signalling molecules may function as virulence determinants per se (some examples of which are given in Table 1). For example, 3OC12-HSL produced by P. aeruginosa has potent immuno-modulatory activity and is capable of influencing both macrophage and T helper cell responses by suppressing the lipopolysaccharide -stimulated release of interleukin-PQS produced by this organism has been shown to affect the pro liferation of human monocytes without affecting cell viability. Since T-cell responses are an important component of the host defence against P. aeruginosa in experimental animal models of infection, QS molecules may function as a virulence determinant pe r se by modulating host inflammatory responses to a degree which promotes the survival and growth of the pathogen. Additionally, both signal molecules 3OC12-HSL and PQS have been found to have potent cardiovascular properties by inducing vasorelaxation of arteries. This may enable the pathogen to stimulate blood flow to the site of infection with a view to increase the supply of nutrients. Futhermore, 3OC12 -HSL has been shown to be involved in the

disruption of tight junctions in epithelial cells which wo uld facilitate the process of invation.

Signal detection Signal synthesis Effector RNA

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Table 1. Examples of interactions between QS molecules and their hosts

Interaction Reference 3OC12-HSL inhibits growth of Gram -positive bacteria Qazi et al (2006) Infect Immun 74:910

3OC12-HSL and PQS modulate T and B cell function Telford et al. , (1998) Infect. Immun. 66:36

Hooi et al (2004) 72:6463 Ritchie et al. (2005) Infect. Immun. 73:1648

3O-C12-HSL promotes IL-8 release from respiratory epithelial cells Di Mango et al., (1995) J. Clin. Invest. 96:2204 Smith et al., (2001) J. Immunol. 167: 366

3OC12-HSL induces cox-2 expression in skin cells Smith et al., (2002) J. Bacteriol. 184: 1132

3OC12-HSL accelerates macrophage and neutrophil apoptosis Tateda et al. (2003) Infect. Immun. 71:5785 3OC12-HSL induces marked bradycardia in live conscious rats Gardiner et al (2001) B.J. Pharmacol 133:1047

3OC12-HSL and PQS have vasodilatory activity Lawrence et al (1999) B.J. Pharmacol 128:845; unpublished(for PQS) 3OC12-HSL disrupts epithelial barrier integrity Vikstrom et al (2012)PLoS Pathog 8(10)p.e.1002953

3OC10-HSL accelerates auxin-dependent root formation in bean Bai et al (2012) Plant Physiol 158:725

3OC8-HSL affects gene expression in Miao et al (2012) Biochem. Biophys. Res. Commun. 427:293 3OC8/C6-HSL induce root elongation in Arabidopsis Liu et al (2012) Mo.l Plant. Microbe. Interact. 25:677

3OC14-HSL induces biocontrol traits in plants Schikora et al (2011) Plant Physiol 157:1407 Schuhegger et al. Plant Cell Environ (2006):29:909

Schenk et al (2012) Plant Signal. Behavior 7:178

Figure 4. Impact of quorum sensing molecules and their plant mimics on plant -microbial interactions In plants, AHLs produced by various Gram-negative bacteria are recognised and elicit multiple responses depending on the molecule and the plant species. These plant responses include the modulation of the systemic resistance against pathogens or hormonal changes that lead to the reprogramming plant gene expression. In addition, plants have also been shown to produce quorum sensing mimics which can interfere with bacterial quorum sensing systems. The multiple effects of the impact of quorum sensing molecule s in the relationship between microbial communities and the interactions with plants are shown in Figure 4. In the recent years significant interest in the exploitation of QS in agriculture to increase crop production has developed. As more of

AHL

Plant QS mimics

AHL-producer/responder

AHL-degrader

AHL/plant signal-responder

Plant responses To QS signalsDefensesPrimary metabolismStress responsesGene regulationProtein dynamicsCytoskeleton

Bacterial responses to QS signalsVirulenceBiofilm formationPlasmid transferMotilityStress responsesProtein foldingEPS synthesis

Bacterial responses to host mimicsVirulence Plant nodulation 2,4-DAPMotility Antimicrobial production AdhesionNutritional and stress response Chitinase

Bauer & Mathesius (2004) Curr Op Plant Biol 7:429; Gonzalez & Venturi (2013) Trends in Plant Sci 18:167

Aryl-HSL producer

Aryl-HSL

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these mechanisms are regularly being discovered, it is becoming clear that interkingdom signalling is widespread

in nature. Exploitation of quorum sensing as an antimicrobial target There are two broad strategies for the control of bacterial infection, either (i) kill the pathogen or (ii) attenuate the virulence of the infecting organism such that it fails to adapt to the host environment allowing it to be readily cleared by the innate host defences. The latter approach has, however, lacked specific targets for rational drug design. The discovery that bacteria employ small molecules to globally regulate the production of virulence determinants now offers just such novel targets. Int erference with either the synthesis or transmission of a QS

signal by a molecular antagonist –a quorum sensing blocker– is an attractive strategy for switching off virulence gene expression and thereby attenuating pathogenicity. The three main strategies for exploiting QS as an antibacterial target are shown in Fig. 5. QS blockers can either target (i) the signal generation including the inhibition of the expression of the signal synthetase; (ii) the signal itself by promoting its degradation; (iii) the signal receptor by antagonising the effect of the signal molecule itself.

Figure 5. The 3 main strategies under study to exploit QS inhibition as a novel antibacterial target. REFERENCES (ordered by year of publication)

Cámara M, Williams P and Hardman A (2002) Controlling infection by tuning in and turning down the volume of

bacterial small-talk. Lancet Infectious Diseases 2:667 -676. — This review, although written a few years ago,

is still clinically relevant covering the very basics of QS including both Gram-positive and Gram-negative organisms.

Chhabra SR, Philipp B, Eberl L, Givskov M, Williams P and Cámara M (2005) Extracellular communication in

bacteria. In Chemistry of Pheromones and Other Semiochemicals Ii. 240:27 9-315. — This review will be of

interest to those who would like to learn more about the chemistry of QS signal molecules and QS inhibitors.

Diggle SP, Cornelis P, Williams P and Cámara M (2006a) 4 -Quinolone signalling in Pseudomonas aeruginosa:

Old molecules, new perspectives. International Journal of Medical Microbiology 296: 83 -91. — This is an

easy reading review on PQS and its precursors but not the most up to date.

Diggle SP, Stacey RE, Dodd C, Cámara M, Williams P and Winzer K (2006b) The galactophil ic lectin, LecA,

contributes to biofilm development in Pseudomonas aeruginosa. Environmental Microbiology 8:1095-1104. — This paper describes the role of the QS-regulated lectin (LecA) in biofilm formation and how the blockage of this lectin with sugars shows great potential for antibacterial therapy in chronic infections.

Signals synthetase

Signals molecule

Signals receptor

1.Inhibition of signal generation

2. Signal degradation

3.Inhibition of signal generation

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Harraghy N, Kerdudou S and Herrmann M (2007) Quorum -sensing systems in staphylococci as therapeutic

targets. Analytical and Bioanalytical Chemistry 387: 437 -444. — Very good review on QS in Staphylococci

with clear diagrams

Qazi S, Middleton B, Muharram SH, Cockayne A, Hill P, O'Shea P, Chhabra SR, Cámara M and Williams P (2006) N-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in Staphylococcus

aureus. Infect. Immun. 74: 910-919. — This is a paper where the interference of AHLs with the S. aureus QS

system was shown. It also shows evidence suggesting the presence of a specific receptor for these signal molecules in S. aureus.

Rasmussen TB and Givskov M (2006) Quorum sensing inhibitors: a bargain of effects. Microbiology -Sgm 152:

895-904. — This review shows the different ways QS can be exploited as an antibacterial target.

Venturi V (2006) Regulation of quorum sensing in Pseudomonas. FEMS Microbiology Reviews 30: 274-291. —

Very detailed review of the molecular mechanisms controlling QS in P. aeruginosa. Excellent for those interested in the genetics of QS.

Williams P, Winzer K, Chan WC and Cámara M (2007) Look who's talking: communication and quorum sensing

in the bacterial world. Philosophical Transactions of the Royal Society B: Biological Sciences 362: 1119 -1134.

— Very broad review of QS covering different bacteria and the origins of this important discovery. This is

the introductory chapter of a very interesting special issue on QS.

Novick R and Geisinger E (2008) Quorum Sensing in Staphylococci . Annual Review of Genetics 42:541-564. —

Excellent review covering many different aspects of QS in S. aureus. Ideal for those wanted to explore more into one of the best described QS models in Gram -positives.

Dong Y-H, Wang LH and Zhang LH (2007) Quorum-quenching microbial infections: mechanisms

andimplications. Phil. Trans. R. Soc. B 362:1201 -1211. — This is one of the few reviews on the exploitation of

quorum sensing as an antimicrobial target in which the use of enzymes that degrade quorum sensing molecules is well covered.

Njotoge J and Sperandio V (2009): Jamming bacterial communication: new approaches for the treatment of

infectious diseases. EMBO Mol Med. 1:201-210. — This review gives an excellent overview on strategies to

inhibit quorum sensing in both Gram positive and Gram negative bacteria.

Williams P and Cámara M (2009) Quorum sensing and environmental adaptation in Pseudomonas aeruginosa:

a tale of regulatory networks and multifunctionalsignal molecules. Current Opinion in Microbiol 12:1 -10. —

This review shows an update of the intricate quourum sensing regulatory networks of P. aeruginosa and the impact different signal molecules from this organism have on the host.

Adam Schikora, Sebastian T. Schenk, Elke Stein, Alexandra Molitor, Alga Zuccaro and Karl-Heinz Kogel (2011) N-acyl-homoserine lactone confers resistance toward biotrophic and hemibiotrophic pathogens via altered

activation of AtMPK6. Plant Physiology 157:1407 -1418. — This study reveals the mechanism by which AHL

elicits systemic resistance agains pathogens in Arabidopsis thaliana

Heeb S, Fletcher MP., Chhabra SR, Diggle SP, Williams P and Cámara M (2011) Quinolones: from antibiotics to

autoinducers. FEMS Microbiol Rev. 35:247-274. — This is the most recent review on the role of quinol one

signal molecules in Pseudomonas and other organisms. Interesting for additional reading for those that would

like to explore a bit further.

Amara N, Krom BP, Kaufmann GF, and Meijler MM (2011) Macromolecular inhibition of quorum sensing:

enzymes, antibodies, and beyond. Chemical Reviews 111:195-208. — This is one of the few reviews where

the use of immunotherapy against QS is addressed. In general very up to date with some interesting new concepts.

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Hartmann A and Schikora A . Quorum sensing of bacteria and trans-kingdom interactions of N-acyl

homoserine lactones with eukaryotes. Journal of Chemical Ecology 38:704 -713. — This is a comprehensive

review on quorum sensing with an emphasis on interkingdom signalling.

Galloway WR, Hodgkinson JT, Bowden S, Welch M and Spring DR (2012) Applications of small molecule

activators and inhibitors of quorum sensing in Gram -negative bacteria Trends Microbiol20:449-58. — This

review not only gives a brief overview on quorum sensi ng and the molecules used by Gram-positive and Gram-negative bacteria but also provides some interesting insights into the applications of quorum sensing and its inhibition into various fields.

Gonzalez JF and Venturi V (2013). A novel widespread interking dom signaling circuit. Trends in Plant Science

18:167-174. — A recent, short review on interkingdom signalling between plants and bacteria.

La Sarre B. and Federle MJ (2013). Exploiting quorum sensing to confuse bacterial pathogens. Microbiol. Mol. Biol. Rev. 77:73-111. A very thorough review on different ways to inhibit quorum sensing in a wide range of organisms.

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Biofilm formation and intercellular signaling in plant -bacteria interactions

Manuel Espinosa Urgel. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain

Single- or multispecies communities associated to a surface and surrounded by an exopolymeric matrix, referred to as biofilms, are formed by a wide variety of bacteria under different environm ental conditions and on diverse surfaces, both biotic and abiotic. The establishment of these multicellular communities attached to solid surfaces is one of the main persistence strategies of bacteria in the environment, and is the cause of serious problem s in medicine and in industrial settings. However, the ability of microorganisms to adhere to solid surfaces may have useful biotechnological applications. One example is agriculture; colonization of seed and root surfaces by beneficial bacteria is key to the success of biological control or plant growth promotion processes.

Biofilm formation responds to a complex regulatory network that is only partially understood. Among the elements

that can play a role in the process, signal exchange between bacterial c ells has received significant attention. The synthesis of virulence factors and certain functions involved in niche colonization by different bacteria is coordinated in a growth- and population density-dependent manner via cell-to-cell communication or quorum sensing (1). Many bacteria are known to produce small diffusible molecules, termed autoinducers, which mediate cell-cell communication, inducing changes in gene expression when a certain concentration of the signal molecule is reached (1,2). Communication through quorum sensing signals is not limited to bacteria of the same species, but can also involve interspecific cross-talk, as well as signaling between bacteria and their eukaryotic hosts (3,4). There is an increasing body of evidente suggesting a c omplex exchange of signals between plants and plant -colonizing bacteria.

A variety of chemically diverse molecules have been described as part of quorum sensing systems, and often a bacterium can produce more than one type of signal. As an example, in the oportunistic human pathogen Pseudomonas aeruginosa, quorum sensing relies on two N -acylhomoserine lactone (AHL) regulatory circuits (las

and rhl) linked to a 2-alkyl-4-quinolone system. The later includes 2-heptyl-3-hydroxy-4-quinolone, commonly referred to as the Pseudomonas quinolone signal or PQS. The biosynthetic precursor of PQS, 2 -heptyl-4(1H)-quinolone (HHQ) has also been demonstrated to function as a signal in P. aeruginosa (5). While AHL-dependent signaling is widespread among Gram-negative bacteria, 4-alkyl-quinolones are more restricted and have so far only been detected in P. aeruginosa and certain Burkholderia and Alteromonas species. Fatty acids and fatty acid derivatives have also been described as intercellular signals, in Xanthomonas and other plant-pathogenic bacteria (6,7).

Pseudomonas putida KT2440 is a plant root-colonizing bacterium that has phytoprotective and plant -growth promoting properties (8). Determinants involved in the colonization of root surfaces and in biofilm formation by this strain have been described (9). They include two la rge adhesins, LapA and LapF, and certain exopolysaccharides, among others. One of the genes identified as involved in root colonization is ddcA. While its precise function is still unclear, expression of ddcA responds to signal(s) present in the supernatan ts of grown cultures of P. putida and other Pseudomonas, as well as in plant root exudates (10). In turn, P. putida KT2440 alters the profile of Arabidopsis root exudates (8), although the specific nature of this modification has not been characterize d. KT2440 does not have the genetic elements involved in the synthesis of AHL or PQS (11,12), although PQS has an influence on motility and biofilm formation by this strain. Using a biosensor based on ddcA, we have identified certain fatty acids as molecules involved in intercellular signalling in Pseudomonas putida, and between Pseudomonas aeruginosa and P. putida.

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As is the case in other microorganisms, in the genome of KT2440 there is a gene coding for a putative

transcriptional regulator of the LuxR protein family. This protein, named PpoR, modulates swarming motility and plays a role in survival in the presence of competitors (13). PpoR could participate in both inter - and intraspecific processes relevant to the fitness of P. putida related to iron acquisition, and not necessarily mediated by typical quorum sensing signal molecules.

References

[1] Fuqua C, Parsek MR, Greenberg EP. 2001. Annu Rev Genet. 35: 439 -468.

[2] Venturi V. 2006. FEMS Microbiol. Rev. 30, 274–291.

[3] Antunes LC, Ferreira RB. 2009. Crit Rev Microbiol 35: 69-80.

[4] Pacheco AR, Sperandio V. 2009. Curr Opin Microbiol. 12:192 -198.

[5] Dubern JF, Diggle SP. 2008. Mol. Biosyst. 4: 882 -888.

[6] Dow M. 2008. Sci Signal. 1(21):pe23.

[7] Deng Y, Wu J, Eberl L, Zhang LH. 2010. Appl Environ Microbiol. 76: 4675-4683.

[8] Matilla MA, Ramos JL, Bakker PAHM, Doornbos R, Badri DV, Vivanco JM, Ramos -González MI. 2010. Environ Microb Rep. 2: 381–388.

[9] Martínez-Gil M, Ramos-González, MI, Espinosa-Urgel M (in preparation).

[10] Espinosa-Urgel M, Ramos JL. 2004. Appl Environ Microbiol. 70: 5190 -5198.

[11] Fernández-Piñar R, Cámara M, Dubern JF, Ramos JL, Espinosa -Urgel M. 2011. Res Microbiol. 162: 773-781

[12] Fernández-Piñar R, Cámara M, Soriano MI, Dubern J, Heeb S, Ramos JL, Espinosa -Urgel M. 2011. Env Microb Rep. 3: 79-85.

[13] Fernández-Piñar R, Espinosa-Urgel, M, Dubern JF, Heeb S, Ramos JL, Cámara M. 2012. Environ Microb Rep. 4: 417-423.

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Round table 2

June 3th, 9:30-14:00

Regulation of gene Expression in Bacteria Chairman: Miguel Cámara (Centre of Biomolecular Sciences, School of Life Sciences, University of Nottingham,

Nottingham, United Kingdom.)

RNA, RNases and Control of Gene Expression . Cecília Maria Arraiano (ITQB-Instituto de Tecnologia Química e Biológica / Universidade Nova de Lisboa, Oeiras, Portugal)

Next Generation Sequencing and RNA Bioinformatics . Vânia Pobre (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal)

The non-coding genome and regulatory RNAs. Ignacio José Andrade (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal)

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RNA, RNases and Control of Gene Expression

Cecília Maria Arraiano. ITQB-Instituto de Tecnologia Química e Biológica / Universidade Nova de Lisboa, Apt 127, 2781-901 Oeiras, Portugal. cecilia@itqb.unl.pt

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites wit h different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher -order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression.

RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are 420 different RNases. RNase E is a single -strand-specific endonuclease critical for mRNA decay in E. coli.

The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double -stranded RNA and family members are

involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay ofmany dif ferent mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

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Next Generation Sequencing and RNA Bioinformatics

Vânia Pobre. Instituto de Tecnologia Química e Biológica, UNL, Av. da República, EAN, 2781-901 Oeiras, Portugal. vaniapobre@itqb.unl.pt

Next Generation Sequencing (NGS) is a high-throughput technology that came to revolutionize the fields of Genomics and most particularly Transcriptomics. NGS is based on modern sequencing technologies and there are several applications available. For example, RNA -Seq (analysis of all the RNA existent in a sample) or Small RNA Sequencing (analysis of only the small RNA fraction in a sample). Depending on the objective of the sequencing project there are also several platforms available. The most well -known are the HiSeq from Illumina, the 454 from Roche and the Ion Torrent from Life technologies. I will discuss all t he differences and similarities between the several platforms. Moreover I will show an NGS pipeline Workflow since the sample preparation until the data analysis.

Bioinformatics knowledge is particularly important in the analysis of NGS results but also f or other RNA areas like prediction of RNA secondary structures and RNA -RNA interaction. An overview of different bioinformatics programs and their applications will also be presented.

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The non-coding genome and regulatory RNAs

José Andrade. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, 2781-901 Oeiras, Portugal. andrade@itqb.unl.pt

The non-coding regions of the genome have undergone an expansion throughout evolution. Whereas in prokaryotes the genome size and gene number are strongly correlated, in eukaryotes the vast majority of nuclear DNA is non-coding, i.e. does not encode for a protein sequence. Surprising as it may sound, it is estimated that only

uman nuclear genome actually consists of coding DNA. Over the recent years, the non -coding DNA has moved from being considered “junk” to be recognised as a powerful reservoir of regulatory elements of gene expression. Promoter sequences, intergenic regions , introns and regulatory non-coding RNAs (ncRNAs) are examples of the non-coding genome.

Transcribed non-coding regions give rise to various classes of non -coding RNAs which can even outnumber the protein-coding genes. Although a non-coding RNA is not translated into a protein, it is a functional molecule with regulatory activity. ncRNAs can be either activators or repressors of gene expression but they typically inhibit translation, usually by causing the destruction of specific mRNA molecules in a pro cess known as RNA interference (RNAi). From the tiny microRNAs to long non -coding RNAs, regulatory RNA-based networks seem to virtually control all biological processes within the cell. Furthermore, engineered ncRNAs systems are already revolutionizing genetic manipulation by providing game-changing tools for biotechnology. Examples of these regulatory RNAs and their diverse modes of action in the control of gene expression will be presented.

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Round table 3 June 4th, 9:30-14:00

Microbiology, Signal Transduction and Biotechnology Chairwoman: Inmaculada Llamas Company (Department of Microbiology, School of Pharmacy, University of

Granada, Spain)

Bacterial signal transduction: introduction, one component sytems and eukaryotic-like protein kinases and phosphatases. José Muñoz Dorado (Department of Microbiology, School of Sciences, University of Granada, Spain)

Two-component systems and ECF sigma factors. Juana Pérez Torres (Department of Microbiology, School of Sciences, University of Granada, Spain)

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Bacterial signal transduction: introduction, one component sytems and eukaryotic-like protein kinases and phosphatases.

José Muñoz Dorado. Department of Microbiology, School of Sciences, University of Granada, Avda. Fuentenueva s/n. E-18071 Granada. Spain Tel. +34 958243183. Fax. +34 958249486. E-mail: jdorado@ugr.es

Prokaryotic cells are smaller in size than eukaryotic cells. This small size, in the upper limit of the colloidal particles, entails a number of peculiar features, one which being a high surface/volume ratio, which increases the exposures of cells to environmental changes. In order to survive in a fluctuating environment, bacterial cells have evolved a high number of adaption mechanisms, which are able to detect a variety of physical and chemical signals to rapidly trigger a proper response. These mechanism s are termed “signal-transduction mechanisms”. Currently, abundant information about these mechanisms is available, and many of these adaptation processes are among the biological processes characterized in more detail. Bacterial signal -transduction mechanisms are also varied, and they are classified in four different groups: one -component systems, two-component systems,

extracytoplasmic function (ECF) σ factors, and eukaryotic-like protein kinases and phosphatases. With the

exception of ECF σ factors, these mechanisms are found not only in Bacteria, but also in Archaea. ECF σ factors, in

contrast, are unique of Bacteria. Nevertheless, not all these mechanisms are present in all the bacterial genomes that have been sequenced. Furthermore, the number of genes that participate in all these signal -transduction mechanisms is related to the lifestyle of each individual. These signal-transduction mechanisms can detect signals as different as nutrient concentrations, O2, light, metals, etc. But in the case of some pathogens, they can also detect the presence of the host, regulating th e expression of genes involved in virulence. Furthermore, some of these mechanisms are essential for the viability of some pathogens. For these two reasons, searching chemical compounds that specifically can block these regulatory mechanisms in pathogens has received much attention, because they can be potential targets to treat some infectious diseases. Actually, several chemicals have now been identified to act in these pathways, and they are under investigation to be used in humans. Undoubtedly, this res earch area has a great future in the area of biotechnology and biomedicine. This presentation will focus on two of the four bacterial signal -transduction mechanisms: one component systems and eukaryotic-like protein kinases and phosphatases.

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Two-component systems and ECF sigma factors.

Juana Pérez Torres. Department of Microbiology, School of Sciences, Avda. Fuentenueva s/n. Universidad de Granada. 18071 Granada. Spain. Tel. +34 958243183. Fax. +34 958249486. E-mail: jptorres@ugr.es

Many organisms use as signaling pathways, phosphotransfer schemes that are known as Two Component Systems (TCS). They involve a histidine kinase (HK) receptor and a response regulator (RR). HKs contain a variable input domain or sensor domain, often membrane associa ted, which detects the external stimulus. Upon detection of the signal, different changes within the sensor domain, promote that the kinase autophosphorylates a conserved histidine and subsequently transfers this phosphoryl group to an aspartate on its cog nate cytoplasmic RR. The phosphorylated regulator protein then typically binds to DNA and promotes transcription of different genes.

TCSs are found in all domains (Bacteria, Archaea and Eukarya). However their abundance in each domain is quite different. These His-Asp phosphotransfer systems are involved in the majority of the signaling pahthways in Bacteria, but are very rare in Eukarya, where the cascades involving Thr, Ser and Tyr are predominant. But both systems have been described in the three domain s, and in many cases both signaling systems are interconnected. According to different databases, more than two hundred thousands of TCS have been identified in Bacteria and Archaea. Many of them have been characterized and they are involved in many import ant cellular functions. Each TCS respond to a specific environmental stimulus, such as pH, nutrient level, redox state, osmotic pressure, quorum signals, metals and antibiotics. Some TCSs control gene clusters that contribute to cell growth, biofilms formation, bacterial cell attachment, nitrogen fixation, quorum sensing, chemiotaxis, changes in osmolarity, antibiotics production, xenobiotic degradation, metal homeostasis and other. Additionally, some of them are involved in virulence. For survival, the pat hogenic bacteria need to adapt to environmental changes upon entry into the host (different pH, osmotic pressure, nutrient availability, etc.) and they must resist the attacks from the host. Many of these pathogens use effective strategies for colonization such as the formation of biofilm and entry into a dormant state such as sporulation. It has been demonstrated that many pathogenic bacteria express these factors as a specific response to the particular host environment, and it is known that those respons es in many cases are dependent on TCSs. This fact reinforces the TCSs as potential target for antimicrobial therapy.

Another means that bacteria use to connect the extracellular stimuli with appropriated cellular responses are the Extracytoplasmic functions factors (ECFs). These regulatory proteins are the third abundant signaling systems after the one-component and the TCSs. The catalytic core of the bacterial RNA polymerase requires a subunit known as sigma factor for the recognition of the promoters. All bacteria encode a housekeeping sigma factor, and

most of them a number encode also alternative σ factors, which redirect the RNA polymerase to other promoters

and modulate gene expression under certain stress conditions. There are two families of sigma factors: sigma 70 and sigma 54. ECFs are sigma factors that belong to the sigma 70 family. They are small proteins that contain only two regions (sigma 2 and sigma 4), both required for interaction with the RNA polymerase core enzyme and recognition of the promoter. The regulation of the activity in most of them is carried out by a cognate anti -sigma factors, which are often membrane-anchored proteins encoded in an operon with their sigma factor. In the absence of external stimulus, the anti-sigma factor is sequestered by the sigma factor, keeping it inactive. Upon simulation, the anti-sigma factor, releases the sigma factor, which in turn becomes active and recruits RNA polymerase core enzyme to direct transcription initiation to its target promoters. Most of the bacterial genomes encode for various ECF and the number increases in complex bacteria. Recently, comparativ e genomic studies are allowing the description of novel very interesting mechanisms of ECF -dependent signaling.

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Round table 4 June 6th, 9:30-12:00

Frontiers in Biotechnology Chairman: Emilia Quesada Arroquia (Department of Microbiology, School of Pharmacy, University of Granada, Spain)

Microbial natural products as sources of novel drugs and high value biotechnological products. Olga Genilloud (Scientific Director, Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, Spain)

Isothermal calorimetry: a response to challenges in modern biomedicine and biotechnology Tino Krell (Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection. Granada, Spain)

Exopolysaccharides from bacteria inhabiting hypersaline environments . Emilia Quesada Arroquia (Department of Microbiology, School of Pharmacy, University of Granada, Spain)

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Microbial natural products as sources of novel drugs and high value biotechnological products

Olga Genilloud. Scientific Director, Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, Spain.

Microbial natural products have been one of the most important sources of new l eads in modern drug discovery, and especially in the case of antimicrobials (e.g. penicillins, cephalosporins, aminoglycosides, tetracyclines and macrolides), with a large number of drugs and analogs successfully introduced in the market. The dis covery that microorganisms are a source of novel molecules with therapeutic potential fostered the development of natural products-based research operations both in academia and industry. Existing natural products libraries represent untapped sources of novel molecules and the large diversity of microbial sources that remain largely underexplored open new avenues for future drug discovery. The success of natural products relies in part on the unique chemical space and architectural complexity that can be found in natural products libraries, often an inspiration for medicinal chemists to design innovative therapeutic agents when synthetic libraries fail to deliver the desired scaffolds. Natural products present a potency and selectivity against biological targets resulting from an extended evolutionary selection. The collections of natural products are more diverse than synthetic or combinatorial libraries with a chemical space sharing more similarities with drug molecules. The complexity of NPs structures represents privileged starting points for the synthesis of novel natural product -like molecules and drug development possibilities.

In spite of this past success in delivering novel structures, the development of new drugs from natural products has traditionally experienced major old limitations related with the oral availability and structural complexity not amenable to medicinal chemistry improvement programs. Despite the uniqueness of these molecules, existing rediscovery problem of old known molecules, and th e lower outputs, the isolation limitations, and the difficulties to develop better candidates from the original molecule was faced to the strong competition of synthetic and combinatorial libraries prone to High Throughput Screening (HTS) scenarios. Furthe rmore natural products screening programs had to compete for resources and for targets and the crude extracts collection were not amenable to be used in centralized HTS facilities. Nevertheless, natural products, their semi -synthetic derivatives and natural-products inspired compounds still represent one of the most important sources of chemical diversity and bioactive novel structures ever described. From a therapeutic perspective, and more especially in the case of antibiotics, there is still an urgent need for new molecules with a major threat of resistant emerging pathogens for which the demand has not been fulfi lled yet.

Despite these limitations that are intrinsic to the nature of these compounds, new advances in the field have revolutionized NPs research and are opening new avenues for the discovery of novel natural product scaffolds. Most natural products are share common biosynthetic pathways that determine common structural backbones such as polyketides, nonribosomal peptides or terpenes that can b e subsequently modified and functionalized by diverse types of decorating enzymes. Key issues in traditional natural product screening programs as the need to

select the right microbial strain with the capacity to produce a new compound, the appropriate n utritional conditions to produce the desired novel compound and the use of the right target in the assay, have evolved into a more complex scenario where emerging technologies such as microbial and chemical ecology, genomics, metabolomics, systems biology and synthetic biology, are combined ensuring that the right gene pool and gene expression conditions are considered. The presentation will review among others different challenges as the efforts directed to the exploitation of microbial resources for NPs d rug discovery, the impact of genomics to untap silent biosynthetic pathways, the use of culture -based approaches to address underexploited silent genes, the epigenetic remodeling of fungal biosynthetic systems or the interspecies cross -talk in co-culturing.

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Whereas the main conditions under which a given gene cluster is expressed in a microbial community is still

largely unknown, research in natural products and in synthetic biology are providing fundamental insights into microbial communication and chemical ecology and many of these innovative approaches have already demonstrated the potential of their exploitation in the development of novel compounds. These disciplines are becoming more necessary than ever not only to understand the trends of the NP s production and its influence in other organisms in the same niche, but as well as to stimulate and simulate the ecosystems of the NP producing microorganisms. Future research in this field will continue to develop innovative strategies to stimulate the biosynthesis and to simulate natural microbial habitats. These efforts will require to be always complemented by the extraordinary advances in analytical chemistry with the semi -automated chemical de-convolution and the chemical isolation and structure elucidation of potential novel NPs leads. With no doubts all these advances are establishing the new trends in the discovery and development of future novel NP -based drug candidates.

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Isothermal calorimetry: a response to challenges in modern biomedicine and biotechnology

Tino Krell. Estación Experimental del Zaidín (CSIC), Prof. Albareda 1, Granada 18008 . tino.krell@eez.csic.es; http://krell-laboratory.com/

Life is based on the specific interaction of molecules and the study of molecular recognition is therefore of fundamental importance. A number of different techniques have been developed to characterise binding events. Amongst these is Isothermal Titration Calorimetry (ITC) in which he at changes caused by ligand binding is monitored. ITC is now considered as the gold-standard to characterise binding interactions and is becoming increasingly popular. The advantages of ITC over alternative techniques include the fact that no ligand immobilization or labelling is required and there are little restrictions as to the choice of the buffer system and the analysis temperature. Since heat generation is a generic feature of an interaction, almost all types of ligands can be analysed including small molecules, proteins or ribonucleic acids. However, the major advantages resides in the amount of information obtained in a single experiment which permits the determination of the equilibrium binding

stoichiometry.

In this talk I will present the fundamentals of the method and instrumentation. In a number of case studies the usefulness of a microcalorimetric analysis to tackle issues of different sort in biomedicine and biotechnology will be illustrated. Particularly, the usefulness of a microcalorimetric analysis in the optimisation of lead drug compounds will be discussed. In addition practical guidelines will be provided for the generation of high -quality ITC data and trouble shooting. We will i llustrate on several examples how ITC can be successfully used to study signal transduction mechanisms in different bacteria.

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Exopolysaccharides from bacteria inhabiting hypersaline environments. Emilia Quesada Arroquia. Department of Microbiology, School of Pharmacy and the Biotechnology, the University of Granada, Spain. equesada@ugr.es; http://www.ugr.es/~eps/es/index.html

About Exopolysaccharides (EPSs) are often found outside the prokaryotic or eukaryotic microbial cells. EPSs exhibit a large variety of unique and complex chemical structures and enable free -living bacteria to adhere to and colonize solid surfaces forming biofilms. In addition they protect microorganisms against physic -chemical adverse conditions, phagocitosis and antimicrobial agents.

Nowadays increasing attention is being paid to exopolysaccharides because of their bioactive role and the wide range of potential applications in industry, pharmacy, agriculture, and various other areas. During the past decades a number of microbial EPS’s have been introduced into the market, yet few of these remain commercially available in commonly sold products. The major obstacl e to commercialization of any of them lies in finding novel or superior properties to products already on the market.

An alternative approach to finding novel products is to investigate new environments such as hypersaline habitats. Regarding the extreme nature of the hypersaline environment the presence of unusual microorganisms of biotechnological interest could be expected. During recent years we have carried out wide research programmes looking among microorganisms from hypersaline habitats in an attem pt to find new EPS’s, possibly with different characteristics. As a result we have discovered that polymers produced by halophilic bacteria have differential and useful characteristics that other polysaccharides do not have. They differ one to another depe nding on the strain in question; however, all of them have three common characteristics from which derive their potential applications: a protein fraction intimately attached to the polysaccharide, high sulphate content, and a distinctly anionic or acid character due to their sulphate, phosphate and pyruvate residues, and to the uronic acids. Some of our polymers also have a high content of the sugar fucose that is of great interest in cosmetics and medicine.

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Round table 5 June 9th, 9:30-12:00

Advanced Chemistry for Biomedicine Chairman: Fernando Hernández Mateo (Department of Organic Chemistry, School of Sciences, University of Granada, Spain)

Development of nanostructured biological biomaterials for peripheral nerve tissue engineering. Miguel Alaminos (Tissue Engineering Group, Department of Histology, School of Medicine, University of Granada, Spain)

Cyclodextrins: Simple Molecules for Smart Drug Transport and Vectorization Fernando Hernández Mateo (Department of Organic Chemistry, School of Sciences, University of Granada, Spain)

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Development of nanostructured biological biomaterials for peripheral nerve tissue engineering

Miguel Alaminos, Víctor Carriel. Tissue Engineering Group, Department of Histology, School of Medicine, University of Granada, Spain. Peripheral nerves are complex structures mainly composed of several fascicles of neuron axons with or without a Schwann cell-synthesized myelin sheath, and a complex and well -organized extracellular matrix (ECM). Integrity and proper function of peripheral nerves is crucial for the maintenance and physiology of these organs and to prevent muscle atrophy. However, peripheral nerves can be affected by several diseases, including traumatic injury (i.e. traffic accidents), neurotoxicity, neuritis, autoimmune diseases, and many others. The large extension of these nerves and their location in superficial areas of the body make them vulnerable to damage. Once damaged, the human nerve has very limited regeneration capability, and patients with peripheral nerve damage are very difficult to treat, causing high economic and social costs for the National Health Systems. Once the nerve is severed, several processes are activated. In the distal nerve segment, Wallerian degeneration occurs, with the complete removal of the damaged distal axon by macrophages. In the proximal segment, axon sprouts grow distally into the bands of Büngner, become myelinated and reach the target organs. The only effective treatment for patients with peripheral nerve damage is restoration of the nerve continuity. Unfortunately, this is not possible in cases with long nerve gaps between both nerve ends, and interposition of a connecting device -a neural conduit or a bioengineered artificial nerve substitute - is necessary to restore nerve integrity. In this context, the recent development of tissue engineering methods allows the generation of bioengineered artifi cial nerve substitutes (ANS) in the laboratory. By using tissue engineering techniques, different researchers have developed efficient tissue substitutes for therapeutic use, such as the human skin, cornea, oral mucosa, bone and blood vessels, but very few efficient models of bioengineered ANS have been developed to the date. The main challenge is probably the generation of a complex multifasciculated nerve. Recent advances in this field include development of novel biofabrication methods combining the use of mesenchymal stem cells, growth factors and biocompatible biomaterials for the generation of ANS. One of the biomaterials showing very promising results is a combination of human fibrin and agarose, which allowed the construction of several tissue mo dels, including the peripheral nerve. However, the biomechanical properties of these models are very poor, and surgical implantation of these ANS is difficult. For this reason, we have optimized a fabrication method allowing us to permanently modify the biomechanical properties of fibrin-agarose biomaterials by inducing the formation of 3D bonds among the fibers of the biomaterials subjected to plastic compression. This method -nanostructuration- resulted in a change of the spatial structure of the biomaterial at the nanometric scale and permitted us to engineer a multifasciculated ANS whose structure was analogue to the native nerve. The combination of a dehydration method with control pressure and temperature resulted in a biomimetic nerve substitute for use in tissue engineering. Ex vivo and in vivo analyses confirmed the usefulness of these nanostructured ANS at the biomechanical, histological and gene expression levels, suggesting that these bioengineered nerves could have potential clinical application in the future.

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Cyclodextrins: Simple Molecules for Smart Drug Transport and Vectorization

Fernando Hernandez-Mateo. Department of Organic Chemistry, Faculty of Sciences, University of Granada, Spain. Email: fhmateo@ugr.es Cyclodextrins (CDs)1 are a family of simple cyclic molecules compounds made up by glucose molecules bound together in a ring (cyclic oligosaccharides) that were discovered more that 100 years ago. This structure confers to those compounds with a hydrophilic outer surface and a lipophilic central cavity. The most common natural CDs

consist of six and eight (α-1,4-)-linked-α-D-glucopyranose units (α-, β- and γ-CDs, respectively). CDs have

found a wide panel of applications owed to their ability to host compound in their lipophil ic cavity forming inclusion

complexes (host–guest complexes) with solid, liquid and gaseous compounds.

On the basis of their excellent biocompatibility as well as their powerful chemical functionalization capacity, CDs have a wide range of applications in different areas2 including drug transport and delivery owed particularly to their capability to enhance the solubility, stability, safety and bioavailability of drug molecules. 3 More recently, CDs and their derivatives have been engineered with the aim to prepare novel functional materials to be used in vectorization of drugs (targeted drug delivery) 4 and gene delivery and transfer.5

The objective of the present lecture is to review the pharmaceutical applications of CDs on drug transport and vectorization with an emphasis on the fundaments that govern the formation of the CD -drug complexes.

Bibliography

(1) Kurkov, S. V.; Loftsson, T. Int. J. Pharm. 2012.DOI: 10.1016/j.ijpharm.2012.06.055

(2) Valle, E. M. M. D. Process Biochemistry 2004, 39, 1033.

(3) (a) Zhang, J.; Ma, P. X. Adv Drug Deliv Rev 2013 DOI: 10.1016/j.addr.2013.05.001; (b) Rao; Palem; Chinna, R.; Chopparapu; Karthik Siva, C.; P, V. R. S.; Subrahmanyam; Yamsani; Madhusudan Curr. Trends Biotechnol. Pharm. 2012, 6, 255. (c) Loftsson, T.; Brewster, M. E. J. Pharm. Pharmacol. 2010, 62, 1607. (c) Devi, N. K. D.; Rani, A. P.; Javed, M. M.; Kumar, K. S.; Kaushik, J.; Sowjanya, V. J. Global Pharm. 2010, 3, 155; (d) Rasheed, A. Scientia

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Pharmaceutica 2008, 76, 567; (d) Loftsson, T.; Duchene, D. Int. J. Pharm. 2007, 329; (e) Loftsson, T.; Jarho, P.;

Másson, M.; Järvinen, T. Expert. Opin. Drug. Deliv. 2005, 2, 335.

(4) Salmaso, S.; Sonvico, F.; In "Cyclodextrins in Pharmaceutics, Costemics and Biomedicine", John Wiley & Sons, Inc.: 2011, p 251.

(5) Mellet, C. O.; Fernández, J. M. G.; Benito, J. M. Chem. Soc. Rev. 2011, 40, 1586.

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Round table 6 June 11th, 9:30-11:00

Employment and Research opportunities Chairman: Ignacio Molina Pineda de las Infantas (Vice-rector, Parque Tecnológico Ciencias de la Salud, University of Granada, Granada, Spain)

Employment And Health Oportunities. The Granada Health Sciences Technology Park. Ignacio Molina Pineda de las Infantas (Vice-rector, Parque Tecnológico Ciencias de la Salud, University of Granada, Granada, Spain)

Round tab le 6

Round Tables

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EMPLOYMENT AND HEALTH OPORTUNITIES. The Granada Health Sciences Technology Park.

Ignacio Molina Pineda de las Infantas. Vice-rector, Parque Tecnológico Ciencias de la Salud, University of Granada, Granada, Spain. The Granada Health Sciences and Technology Park is a reference area for the creation, establishment and expansion of institutions and companies that turn knowledge into economic and social development, particularly in the Pharmaceutical, Bio-health, Healthcare and Food Industry sectors, which makes it the first in Spain and one of only a few in the world specialising in Health. This unique Health Sciences Technology Park (HTP) model includes: Teaching. Research. Healthcare. Business development. The following institutions are to be found in the Technology Park: The Universidad de Granada: Faculties of Medicine, and Health Sciences (whose construction, together with a

Central Services Building, is about to be finished). The new Faculties of Pharmacy and Dentistry will be built in a second phase.The Centre for Biomedical Research houses the university’s research institutes of Biopathology and Regenerative Medicine; Neurosciences; Nutrition and Food Technology as well as the Biobank of the Andalusian Public Health System. Another building houses the Joint University Institute for Sports and Health.

San Cecilio Clinical University Hospital. The Spanish National Research Council (CSIC), with the “López Neyra” Institute of Parasitology and Biomedicine

(IPB). Two public-private initiatives: The regional Ministries of Innovation and Science and Health, and the University

of Granada, have developed the Foundation Center of Excellence for Innovative Medicines in Andalucia (MEDINA) together with the pharmaceutical company Merck, Sharp&Dhome de España; and the Pfizer -Universidad de Granada-Junta de Andalucía Center for Genomics and Oncological Research, a joint project with Pfizer.

Institutions belonging to the regional Ministries of Innovation, Science and Business, Health, Justice, and Employment at the Junta de Andalucía.

Technology-based, pharmaceutical and bio-health companies.

A total of 25 buildings are planned on a surface occupying 625.000 m2. As of today, over € 600M have been invested, which allowed to fin ish the construction of 15 buildings (11 of them are already fully operative) and 9 more are under construction. The Granada HTP is a model for transferring basic clinical Research and Technology in the areas of health and biomedicine that contributes to economic development through: Promotion of international interdisciplinary research in biomedicine in order to make advances in health

through understanding, diagnosis, treatment, cure and prevention of diseases. Protection and transfer of knowledge, particularly in the areas of health and biomedicine. Consolidation of a technology-based bio-health business sector serving clinical practice. Being a centre of excellence for healthcare, responding to the health nee ds of the patient.

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Round table 7 June 13th, 9:30-14:00

Frontiers in Biomedicine Chairman: María Dolores Girón González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes Rafael Salto González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

Frontiers In Biomedicine: Gene Therapy for Primary Immunodeficiencies. Theraphy in Monogenic Disorders. Ignacio Molina Pineda de las Infantas (Department of Biochemistry and Molecular Biology II I & Immunology, School of Medicine, University of Granada, Spain)

Regulation of LINE-1 retrotransposition José Luis García Pérez (Pfizer Center-Junta de Andalucia Center for Genomics and Oncological Research (Genyo), University of Granada, Granada, Spain)

Round tab le 7

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Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes Rafael Salto González, Dámaso Víchez and María Dolores Giron. Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain Skeletal muscle mass depends on the net balance between the rates of protein synthesis and degradation. It is generally accepted that these two events are regulated by PI3K/Akt signaling. Here we focus on the role of tungstate-induced ERK activation on protein turnover in rat L6 myotubes and dissect the molecular mechanisms involved in this process. Sodium tungstate activated ERK, but not PI3K/Akt, and induced an increase in protein synthesis as a result of the activation of mTOR through the phosphorylation of Tuberous Sclerosis Factor 2 (TSC2). Moreover, tungstate prevented protein degradation i nduced by dexamethasone. This effect was caused by inhibition of FoxO3a-mediated transcription, which resulted in the normalization of downstream targets of FoxO: ubiquitin promoter transcription and the expression of ubiquitin ligases, MuRF1 and Atrogin -1. Moreover, tungstate reduced dexamethasone-induced autophagy. In summary, activation of ERK by sodium tungstate in skeletal muscle suffices for the observed increase in protein synthesis and decrease protein degradation. On the basis of our results, we propose that sodium tungstate be considered an alternative to IGF -I and its analogs in the prevention of skeletal muscle atrophy.

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The FRONTIERS IN BIOMEDICINE: Gene Therapy for Primary Immunodeficiencies.

Ignacio Molina Pineda de las Infantas. Vice-rector, Parque Tecnológico Ciencias de la Salud, University of Granada, Granada, Spain. Primary immunodeficiencies are a group of severe diseases arising from mutations in non -redundant genes that play critical roles in the development of the immune response. Due to the inability to properly recognize and/or destroy the target pathogen, patients suffering from these conditions experience frequent life -threatening infections. Curative treatment involves a transplant of bone marrow progenitors from a co mpatible donor. However, many patients lack such donors and therefore the therapeutic intervention is confined to palliative care. In this, the administration of intravenous immunoglobulins is a key element. In the absence of a bone marrow reconstitution, the prognosis for almost all of these patients is fatal. Therefore, clinical trials based on gene therapy have been carried out in an attempt to find alternative curative approaches. Gene therapy involves the introduction of an intact gene into a target cell with the aim to modify or repair the defective functions. The ideal target for primary immunodeficiencies is the bone marrow stem cell, as the molecular reconstitution of this cell will result in functional correction of all lineages derived from it. To integrate a curative gene into the host’s genome, the transgene has to be transferred in a delivery system called gene therapy vector. Gene therapy vectors use virus-modified sequences that produce viral particles containing the gene of interest. A number of bio-safety features are incorporated into the constructs to ensure that the virus is self -inactivating, and therefore, capable of only one cycle of integration -replication and thus preventing uncontrolled viral replication. Depending upon the target cell or organ, therapeutic transgenes are packaged into vectors derived from adenoviruses, onco-retroviral vectors or lentiviruses, which are based on the HIV. Over 100 patients suffering from several types of primary immunodefi ciencies have been treated so far, such as patients with X-Linked Severe Combined Immunodeficiency, Adenosin -Deaminase deficiency, X-linked Chronic granulomatous disease and the Wiskott-Aldrich syndrome. A clinical benefit or even cure has been achieved i n over 80% of treated patients, which represents a great success. The appearance, however, of undesired side effects resulting in lymphoproliferation prompted researchers to find new strategies that increase biosafety and reduce risks for the patients. In spite of initial difficulties, gene therapy now faces an exciting future to offer an effective therapy to patients who otherwise would face a certain death.

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Regulation of LINE-1 retrotransposition.

Jose L. Garcia-Perez. GENYO. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government. Av. de la Ilustración, 114 18016 GRANADA, Spain

More than 40% of the human genome is composed of repeated DNA, and some types can be mob ilized within the genome (e.g., Transposable Elements (TE)). Long Interspersed Element -1 (LINE-1 or L1) is the only autonomous retrotransposon (a class of TE) in the human genome, with more than 600,000 copies per genome (~17% of the genome). Furthermore, L1 is able to mobilize other non-autonomous TEs (Alu, SVA, U6) and cellular mRNAs (giving rise to processed pseudogenes). Overall, L1 is responsible for a third of the human genome.

Although most L1 elements in the genome are inactive, each human genome c ontains ~100 active L1 retrotransposons. Its mobility has resulted in a variety of human diseases, and its activity has shaped the human genome during evolution. L1 insertions within genes can alter their function, can modulate their expression at both splicing and poly-adenylation steps, and can provide regulatory sequences to genes. In addition, L1 insertions can be accompanied by genomic instability processes (deletions, translocations, etc), adding to the myriad of ways they shape our genome. Despite their abundance, little is known about human cell types that can accommodate the mobility of such repeated DNA sequences or its regulation.

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Workshops

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Workshop 1

June 2nd-June 6th

School of Pharmacy, University of Granada

Quorum Sensing and Quorum Quenching: Extraction and analysis of quorum sensing molecules. Detection of AHL -degrading bacteria

Inmaculada Llamas Company (Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain)

Steve Atkinson (Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom)

Stephan Heeb (Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom)

Cecilia Arraiano (Control of Gene expression laboratory, ITQB -Institute of Technological Chemical and Biological Technology, University of Lisboa, Portugal)

José Carlos Reina Cabello (Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain)

David Castro (Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain)

Miguel Cámara (Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom)

Workshop 1

Workshops

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Workshop 1: Quorum Sensing and Quorum Quenching

Detection and analysis of quorum sensing molecules in Gram-negative Proteobacteria by a diffusion agar-plate assays and Thin-layer chromatography. Identification of AHL-degrading bacteria.

Inmaculada Llamas1, Steve Atkinson2, Stephan Heeb2, Cecilia Arraiano3, José Carlos García1, David Castro1, Miguel Cámara2 1Department of Microbiology, Faculty of Pharmacy, University of Granada, 18071 Granada, Sp ain 2Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, United Kingdom 3Control of Gene expression laboratory, ITQB-Institute of Technological Chemical and Biological Technology, University of Lisboa, Portugal Bacteria have evolved sophisticated mechanisms to co -ordinate gene expression, such as quorum sensing (QS), which involves the production of signal molecules known as autoinducers. This system controls numerous bacterial cell functions such as the expressi on of virulence factors and exoenzymes, conjugal DNA transfer, control of plasmid-copy number, production of and susceptibility to antibiotics, biofilm formation and exopolysaccharide production (Parker and Sperandio, 2009; Williams, 2007). Within the Gram-negative Proteobacteria, the most studied autoinducer signals are N -acylhomoserine lactones (AHLs) which differ in the length and substitution of their respective acyl side chains, conferring their signal specificity (Fuqua et al. 1995). Other QS signal molecules are 2-alkyl-4-quinolones (AQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) described in Pseudomonas aeruginosa (Dubern and Diggle, 2008). On the other hand, it has been reported that some microorganisms have the p otential to disrupt QS by different mechanisms. One strategy reported in bacteria, known as quorum quenching (QQ), is the enzymatic inactivation of AHLs by the production of acylases or lactonases (Natrah et al., 2011). The aims of this practical will be firstly to extract AHLs and AQs quorum sensing signal molecules from some Gram-negative bacteria and detect the presence of these molecules using a combination of TLC separation and specific QS biosensors. Secondly, the AHL-degrading activity will be detected in Gram-negative bacteria using a diffusion agar-plate assay. References

Dubern, J. F., Diggle, S. P. 2008. Quorum sensing by 2 -alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species. Mol. Biosyst. 4: 882-888.

Fuqua, C., Burbea, M., Winans, S.C. 1995. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator

TraR is inhibited by the product of the traM gene. J Bacteriol 177:1367–1373.

Parker, C.T., Sperandio, V. 2009. Cell-to-cell signalling during pathogenesis. Cell. Microbiol. 11: 363-369.

Williams, P. Quorum sensing, communication and cross -kingdom signalling in the bacterial

world. Microbiology 2007, 153, 3923-3938.

Natrah, F.M.I., Defoirdt, T., Sorgeloos, P., Bossier, P. 2011. Disruption of bacterial cell -to-cell communication by marine organisms and its relevance to aquaculture. Mar. Biotechnol.13:109 -126.

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Workshop 2 June 4th

School of Sciences, University of Granada, Room 04

Bioinformatic identification of proteins involved in signal transduction

José Muñoz Dorado (Department of Microbiology, School of Sciences, University of Granada, Spain)

Juana Pérez Torres (Department of Microbiology, School of Sciences, University of Granada, Spain)

Francisco Javier Marcos Torres (Department of Microbiology, School of Sciences, University of Granada, Spain)

Workshop 2

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Workshop 2: Bioinformatic identification of proteins involved in signal transduction

José Muñoz-Dorado, Francisco-Javier Marcos-Torres and Juana Pérez. Department of Microbiology, School of Sciences, University of Granada, Granada, Spain . Phone +34 958243183 and +34958249830. Fax. +34 958249486. E-mail: jdorado@ugr.es, fjmarcos@ugr.es, jptorres@ugr.es

Many bioinformatics tools are currently available to predict the function of the proteins encoded by genes. In this workshop we will focus on the identification of genes involved in bacterial signal transduction. As these

mechanisms are classified into four different groups (one-component systems, two-component systems, ECF σ

factors, and eukaryotic-like protein kinases and phosphatases), the students will analyze several gene products in order to identify in which kind of signal-transduction mechanism these proteins can be included. Furthermore, as

many of these proteins exhibit a modular organization, the students will have the opportunity to use practical examples to gain knowledge about the most common architectures found in these proteins and their rele vance.

Finally, the students will visit a webpage, MiST2, which is devoted to analyze signal -transduction mechanisms in

sequenced prokaryotic genomes. The will have the opportunity to select different strains to know how many genes in their genomes encode proteins involved in the different types of signal -transduction mechanisms.

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Workshop 3 June 9th-June 11th

School of Pharmacy, University of Granada

Site directed mutagenesis as a tool for the analysis of the structure-function in proteins

José Dámaso Vílchez Rienda (Department of Biochemistry and Molecular Biology II , School of Pharmacy, University of Granada, Spain)

Elena Cabrera Cazorla (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

María Dolores Girón González (Department of Biochemistry and Molecular Biology II, School of Pharmacy , University of Granada, Spain)

Rafael Salto González(Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of

Granada, Spain)

Workshop 3

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Workshop 3: Site directed mutagenesis as a tool for the analysis of the structure-function in proteins

Dámaso Vílchez Rienda, Elena Cabrera Cazorla, María Dolores Girón González and Rafael Salto González.Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Granada, Spain.

Structure–function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that

allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. Here, we have implemented a workshop based on the site -directed mutagenesis of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The GFP is ideal because the students are able to correlate the changes introduced into the

structure of the protein with the observable modification of its fluorescence properties. By using noncommercial kits, we set up a non PCR-thermocycling reaction using mutagenic primers, followed by removal of the original plasmid template by DpnI digestion. By introducing only one (Y66H) or two mutations (Y66H/Y145F) in the ‘ ‘cycle 3’’

variant of GFP (F99S, M153T, and V163A) or GFPuv, students are able to analyze the changes from green to blue in the fluorescence emission of the mutated proteins and to correlate these differences in fluorescence with the structural changes using three-dimensional structure visualization software. This inexpensive laboratory course familiarizes the students with the design of mutagenic oligonucleotides, site -directed mutagenesis, bacterial transformation, restriction analysis of the mutated plasmids, and protein characte rization by SDS-PAGE and fluorescence spectroscopy.

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Workshop 4 June 12th-June 13rd

School of Sciences, University of Granada

Synthesis and Characterization of Neoglycoproteins by Click Chemistry and the Chemistry of Vinyl Sulphones

Fernando Hernández Mateo (Department of Organic Chemistry, School of Sciences, University of Granada, Granada, Spain)

María Dolores Girón González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

Rafael Salto González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

Workshop 4

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Workshop 4: Synthesis And Characterization Of Neoglycoproteins Trought Vinyl Sulfone Chemistry

Fernando Hernández Mateo1, Rafael Salto González2 and María Dolores Girón González2

1Department of Organic Chemistry, School of Sciences, University of Granada, Granada, Spain.

2 Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain.

Day 1: Reactivity of the vinyl sulfone group toward biomolecules

The purpose of the experiment is to provide a ge neral outlook on the reactivity of the vinyl sulfone chemistry towards proteins. Vinyl sulfones readily form covalent adducts with many nucleophiles (“hard” and “soft”) via a Michael-type 1,4-addition. In biomolecules, both amino and thiol groups are nucleophiles that can react with vinyl sulfone derivatized reagents (tags, labels, solid supports...) 1.

Three characteristics are especially attractive in the context of life sciences: 1) the possibility to perform the se reactions in physiological conditions (aqueous media, slightly alkaline pH and room temperature) that preser ve the biological function of the proteins, 2) the absence of catalysts and by-products and 3) the formation of a covalent bond. In addition, the introduction of the vinyl sulfone is not a difficult task and the resulting functionalized reagents or intermediates are stable.

In order to study the reactivity of biomolecules toward s the vinyl sulfone group, two proteins (BSA and lysozyme) will react with a vinyl sulfone derivatized model compound (VS -Remazol blue) at two temperatures (room temperature and 37º C) as a function of time. Samples will be analyzed by SDS -PAGE to detect the blue color resulting from the coupling of the remazol blue to the protein. Try to correlate the in tensity of the blue color with 1) pH, 2) temperature, 3) protein (the larger the number of reactive groups the stronger the intensity of the blue color), and 4) time.

1 Morales-Sanfrutos et al. Vinyl sulfone: a versatile function for simple bioconjugation and immobilization. Org. Biomol Chem. 2010,

8: 667- 675

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Materials and Methods

Materials

Proteins

Protein MW (kDa) Iso. point His + Lys Cysa Solution 5 mN

Lysozyme 14.3 11 1 + 9 8 (4 S-S bonds) 10.2 mg/ml

BSA 66.5 5.6 59 + 17 35 (17 SS bonds) 4.32 mg/ml

aCys involved in S-S bonds are not reactive

bNormality = Molarity x (His+Lys+reactive Cys)

Buffers

0.5 M HEPES pH 8

0.5 M Acetate pH 5

Label

20 mN VS-Remazol blue in water

Methods

Reaction

Buffer (0.5 M) 10 µ l (=50 mM)

Protein (5 mN) 40 µ l (=200 neq)

VS Remazol Blue (20 mN) 40 µ l (=800 neq)

H20 10 µ l

Take aliquots (20 µ l) at the time suggested by the instructor and transfer them to Eppendorf tubes with 40 µ l of

loading buffer.

SDS-PAGE.

Heat samples at 92º C for 4 minutes then load 10 µ l of sample per well and run the electrophoresis at 150 V (constant) for 30 minutes.

VS-Remazol Blue

MW= 506

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Day 2: Synthesis of neoglycoconjugates

In the context of Glycomics, new functionalizations to efficiently promote the reaction of oligosaccharides with solid supports and yield their immobilization or the glycosylation of proteins are desirable. The purpose of the experiments is to provide a general outlook on the application of the vinyl sulfone derivatized saccharides to

explore protein− carbohydrate interactions 2.

Two proteins (BSA and lysozyme) will be reacted with a vinyl sulfone derivatized mannose at the best condition found in Day 1 to yield neoglucoconjugates. Samples will be analyzed by ELLA (Enzyme-Linked-Lectin-Assay). Concanavalin A (ConA) is a lectin that interacts with mannose. The glycosylation of lysozyme and BSA with VS -mannose yields neoglycoconjugates that can be recognized by ConA. The interaction can be visualized if ConA conjugated to horseradish peroxidase (ConA -HRP) is used instead of ConA. The lectin-neoglyconjugate can be quantified if the neoglyconcojugate is adsorbed on a well and is first incubated with ConA-HRP and finally with a substrate for the peroxidase that produces colour upon transformation into the product.

Materials

Proteins

Protein MW (kDa) Iso. point His + Lys Cysa Solution 5 mN

Lysozyme 14.3 11 1 + 9 8 (4 S-S bonds) 10.2 mg/ml

BSA 66.5 5.6 59 + 17 35 (17 SS bonds) 4.32 mg/ml

aCys involved in S-S bonds are not reactive

bNormality = Molarity x (His+Lys+reactive Cys)

Buffers

0.5 M HEPES pH 8

100 mM Carbonate pH 9

VS derivatized sugar

10 mN VS-mannose

2 Lopez-Jaramillo et al. Vinyl sulfone functionalization: a feasible approach for the study of the lectin-carbohydrate interactions. Bioconjugate Chem.

2012, 23, 846-855

VS-mannose

MW= 314

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Methods

Reaction

0.5 M HEPES pH 8 10 µ l (=50 mM)

Protein (5 mN) 40 µ l (=200 neq)

VS Remazol Blue (10 mN) 40 µ l (=400 neq)

H20 10 µ l

At the time suggested, add 5 µ l of β-mercaptoethanol and continue the incubation while preparing the plate for the

ELLA assay

ELLA assay.

Add 200 µ l of the carbonate buffer to each well. Please take care to ensure that you add 200 µ l and avoid the

formation of bubbles. The distribution of your samples will be:

Lines A and B: Lysozyme (control)

Lines C and D: glycosylated lysozyme

Line E and F: BSA (control)

Line G and H: glycosylated BSA

Fill each row as described below:

To well 1, add 50 µ l of sample, mix well and then transfer 50 µ l to well 2 and repeat the procedure up to well 12.

Do not forget to remove 50 µ l from well 12.

Incubate the plate at 4 º C overnight.

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Visits

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VISITS TO RESEARCH COMPANIES AND LABORATORIES

Visit 1: Bio-Iliberis R & D June 5th, 9:30-14:00

Amalia Roca Hernández. Departamento de Agronomía- I+D+i, Bio-Iliberis R&D, Poligono Industrial Juncaril, Peligros, Granada, Spain.

Visit 2: Abbott Laboratories June 10th, 9:30-12:00

José María López Pedrosa. Department I + D, Abbott Laboratories, Granada, Spain.

Visit 3: Fundación Medina June 12th, 9:30-12:00

Olga Genilloud. Scientific Director, Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, Spain.

Visit 4: Neuron Bio June 12th, 12:00-14:00

Elena Requena Rodríguez. Neuron Bio, P. T. S. Granada, Granada, Spain.

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Visit 2: Abbott Laboratories

ROLE OF FEEDING WITH SLOW DIGESTING CARBOHYDRATES DURING PREGNANCY ON IMPROVING METABOLIC HEALTH IN THE OFFSPRING: MECHANISTIC INSIGHTS (NIGOHealth study) Manzano M1, Martin MJ1, Bueno P1, Andujar E2, Perez M2, Girón MD3, Salto R3, Vílchez JD3, Cabrera E3, Rueda R1, López-Pedrosa JM1 1Discovery Strategic Research ANR&D Granada Spain 2Cabimer-CSIC. Genomics Unit, Sevilla, Spain. 3 Dpt of Biochemistry and Molecular Biology, School of Pharmacy, University of Granada, Spain Background and objectives: Maternal obesity prior to and through pregnancy program offspring to a broad spectrum of metabolic and physiological alterations later in life. This work summarizes preclinical results obtained up to date on NIGOHealth study. The main goal for this study is to evaluate the effects of feeding with slow digesting carbohydrates (SDC) during pregnancy on programming adiposity and muscle development in the offspring from obese mothers. Methods: Rats were assigned to one of three experimental groups: Control dams fed a standard rodent diet before mating and throughout pregnancy; dams fed a high fat for 6 weeks before mating and then fed a HF diet containing either SDC or high digesting carbohydrates throughout pregnancy. Offspring’s body composition and plasma biochemical markers were analysed by using MRI and bio-analyzer, respectively. Western blot was used to analyse signalling pathways. Muscle transcriptome, pathway and biofunction were analysed using the Agilent microarray and Ingenuity software. Results: Offspring from pregnant rats fed with SDC showed changes on adipose tissue glucose transporters and insulin signalling, that were consistent with reduced adiposity at adolescence. Reduction on adiposity was associated to reduce levels of plasma glucose, triacylglycerides and cholesterol. Feeding with SDC during pregnancy also enhanced skeletal muscle development in the offspring. Conclusions: Results from this study point out the importance of nutrition during critical periods of development and show the role of carbohydrate profile on maternal diet influencing key outcomes related to adipogenesis and muscle development in the offspring. This influence may translate into prevention of metabolic diseases and other alterations later in life. Key words: Early programming, dietary carbohydrate

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Students communications

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Students Communication Session 1 June 9th, 12:00-14:00

Chairman: Fernando Hernández Mateo (Department of Organic Chemistry, School of Sciences, University of Granada, Spain)

Susana Barahona (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Anabela Vieira (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Lidia Delgado Calvo-Flores (University of Granada, Granada, Spain)

Christian Arenas López (University of Nottingham, Nottingham, UK)

Natalie Barratt (University of Nottingham, Nottingham, UK)

Session 1

Students communications

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Unravelling the role of Escherichia coli BolA, a pleiotropic protein involved in cell survival

Susana Barahona Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. The Escherichia coli bolA is promptly induced in harmful situations, conferring protection to the cells. Its expression is tightly regulated at transcriptional and post -transcriptional levels involving different players and mechanisms. Its overexpression is known to induce sp herical morphology due to its recently described role as a transcription factor and regulator of a complex network, including penicillin binding proteins and MreB. Additionally, in E. coli, BolA was shown to influence biofilm formation. In nature, most bac teria live in community attached to surfaces as biofilms and these communities are of extreme relevance in biotechnology and health -related issues. We were interested in the characterization of the role of this protein in cell motility and adhesion mechani sms. Transcriptomic analyzes were performed to obtain a global overview of BolA influence in E. coli mRNA expression. We show that this protein is modulating genes with consequences in motility. Different metabolism pathways were observed to be affected, including those that are involved in the composition of the extra -cellular matrix, important to promote biofilm formation. Evidences provided establish BolA as a possible key player in the control of cell adhesion and/or motility and shows how it can modula te a network of genes involved in cell growth and survival.

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Microbial activity assessment in biological nutrient removal from wastewater

Anabela Vieira Microbiologyof Man-Made EnvironmentsLaboratory,iBET- Instituto de Biologia Experimental e Tecnológica, Av. República, Qta. do Marquês, 2780-157 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa (UNL), Qta do Mar quês, 2780-157 Oeiras, Portugal. The biological nutrient removal (BNR) processes are the most economic and environmentally friendly way to remove nutrients, such as nitrogen, from wastewater. Nitrogen removal is normally comprised of a sequence of nitrification and denitrification steps, carried out by different groups of organisms. Moreov er, specific enzymes are involved in each of the nitrification/denitrification steps, which, when unbalanced, can lead to accumulation of intermediates and incomplete nitrogen removal. In order to optimise the performance of the nutrient removal process, it is important to improve our knowledge on the (micro)organisms present in wastewater treatment plants at different operating conditions, as well as their enzymatic potential. In this study, genomic real-time polymerase chain reaction (qPCR) was optimised to quantify the number of gene copies of functional genes of the denitrification pathway, such as nitrate reductase ( narG), nitrite reductase (nirS and nirK) and nitrous oxide reductase (nosZ). The effect of various carbon sources (acetate, ethanol and methanol) on denitrification rate were evaluated in batch tests experiments in different biomass samples, collected from the same reactor in different time periods. Simultaneously, the relative abundance of denitrifying genes was assessed in the same samples. The abundance of nirS was higher than the abundance of nirK, nosZ and narG genes for all experiments. The results were generally in agreement with the nitrate, nitrite and nitrous oxide redu ction rates. However, some variations were observed, which are likely related to differences in expression of these genes at different operating conditions, such as different carbon sources. Future work will be carried out to quantify the mRNA of each of the target genes, in order to assess their expression in different conditions.

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Study of metabolomics biomarkers of obesity in children born from obese and diabetic women Lidia Delgado Calvo-Flores. University of Granada, Spain. In recent years, urine samples are commonly used in metabolomic studies because they can identify changes in the metabolic profile of diseased subjects, represents a more accessible substrate and are useful for population studies. My thesis will have as a main topic the characte rization of the metabolome of urine samples collected of the participants in the PREOBE project. The PREOBE project is a longitudinal observational study to analyze the effect on medium -term of maternal obesity and diabetes during pregnancy on the growth and development of their children, deepen on the study of the factors and mechanisms involved in early programming of childhood obesity and the impact on neurodevelopment. The PREOBE cohort was established with 345 pregnant women recruited between 12 and 20 weeks of pregnancy, from them 302 completed the follow-up which finally formed the actual cohort based on 4 groups of "mother -children pairs". The inclusion criteria for pregnant women were as follow according to their health status: 1)

gestational diabetes (n:61), 2) overweight (25≥BMI<30) (n:55), 3) obesity (BMI>30) (n:57) or 4) healthy and norm

weight (18.5>BMI<25) (n:129), aged between 18 and 45 years, single and non -complicated pregnancies. Pregnant women were supervised throughout gestation up to d elivery, and then up to 18 months postpartum. Standardized fetal anthropometry including abdominal thickness was performed after delivery and the infants were followed -up until 3 years old. Important information has been obtained, such as maternal body com position during pregnancy (weight gain) and weight retention after delivery, dietary intake by the mother and offspring during the first years of life; lifestyle including physical activity; lipid and immunological biomarkers in the mother and umbilical co rd, etc. In the project presented here, the aim is to characterize the metabolome of urine samples collected in PREOBE mothers at 34 weeks of gestation and their children at 3 years; this study will provide us with important information about possible changes in nutrient utilization in children born to obese and diabetic mothers compared to normal. The metabolome represents the ultimate expression of the genome, transcriptome and proteome, accounting for the interrelation between the different levels of information in biology. Therefore, direct measurements of expression of proteins and metabolites are essential to study biological processes in both states; normal and disease. The objective of developing a global metabolic profile in urine is monitoring important nutrients metabolites related with neurodevelopment and dietary intake and is expected to reflect differences due the pro-inflammatory state in obese and diabetic mothers and in their children, as a result of the prenatal metabolic imprinting. Besides to obtain information about methylmalonic acid and total homocysteine, which are biomarkers of folate and cobalamin deficiency related to neuropsychiatric disorders and mental performance, we can obtain valuable information on some other metabolites of interest, such as amino acids, fatty acids, carboxylic acids, alcohols, sugars, hormones, etc. which allow us to obtain a complete picture of the samples studied and the effect of metabolic imprinting suffered on the child.

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Cupriavidus necator: A new approach to the sustainable production of biofuels and chemical commodities

Christian Arenas López. Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, UK

The sustainable production of advanced biofuels and chemical commodities requires efficient release of sugars and other fermentable compounds from lignocellulosic biomass. However, the recalcitrance of this material to deconstruction poses a major problem. In particular the lignin component remains inaccessible, as it cannot be efficiently degraded by physical, chemical, or enzymatic treatment.

A possible alternative is therefore the gasification of biomass to yield syngas, a gas mixture consisting of carb on monoxide and hydrogen. In the absence of oxygen, syngas can be utilised by several strictly anaerobic bacteria as the sole source of carbon and energy and this ability is currently being commercially exploited for the production

of ethanol from industrial off- gases. Unfortunately, however, the described anaerobic fermentation process cannot drive the production of more complex and energy -demanding compounds. We will therefore investigate whether aerobic gas-fermenting organisms, in particular the bacter ia Cupriavidus necator, can be engineered to

produce desirable chemicals and fuels. Initially, the resistance of the organism to a range of commercially interesting chemicals will be assessed. Metabolic routes to those tolerated at high concentrations will be evaluated and one chosen for implementation using state -of-the-art metabolic engineering and synthetic biology approaches.

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The role of quorum sensing and type three secretion in the regulation of virulence determinants in Yersinia spp

Natalie Barratt. Centre for Biomolecular Science, School of Life Sciences, The University of Nottingham, Nottingham, United Kingdom. Quorum sensing (QS) is the term used to refer to the regulation of gene expression in a population density manner. This involves the secretion of chemical signal molecules, autoinducers, which accumulate with increasing cell number. Once the signal concentration reaches a threshold level the population will respond with changes in gene expression, thus enabling coordinated group behaviour. In the zoonotic gastric pathogen Y. pseudotuberculosis, the autoinducers are N-acylhomoserine lactones (AHLs). There are two pairs of regulators and synthases of the AHLs, namely YpsRI and YtbRI which produce and transduce the signal molecu les (Atkinson et al., 2008).

In Y. pseudotuberculosis the yps/ytb quorum sensing system controls a number of virulence related traits including: type three secretion (T3S), motility and biofilm production on surface of the nematode worm Caenorhabditis elegans.

The plasmid-encoded Ysc T3S system is a method o f protein secretion through a needle -like projection across the bacterial membrane directly into the host cell, allowing the bacterium to evade the host’s innate immune defences. These proteins are either regulatory and effector proteins; the effector prot eins are the secreted proteins that have the cytopathologic effects acting on the target cell (Cornelis, 2002).

Y. pseudotuberculosis is able to form an extracellular biofilm, this can aid in the evasion of host defences and resistance to antimicrobials. B iofilm production is an important virulence factor and is also regulated by AHL -mediated quorum sensing (Atkinson et al., 2011).

It has previously been shown that quorum sensing, the T3S system and biofilm production are interlinked at the

molecular level since quorum sensing mutants show reduced biofilm production and increased expression of the T3S system (Atkinson et al., 1999) (Atkinson et al., 2011). However the same mutation in a strain without pYV did not effect biofilm production.

In order to further investigate the relationship between the quorum sensing system and virulence in Y. pseudotuberculosis this project aims to examine the role of the structural protein responsible for the T3S needle, YscF, in biofilm formation and the regulatory links to the T3S system.

References:

Atkinson S, Throup JP, Stewart GS, Williams P. 1999. A hierarchical quorum -sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping. Mol. Microbiol. 33:1267–77

Atkinson S, Chang CY, Patrick HL, Buckley CM, Wang Y, Sockett RE, Cámara M, Williams P. 2008. Functional interplay between the Yersinia pseudotuberculosis YpsRI and YtbRI quorum sensing systems modulates swimming motility by controlling expression of flhDC and fliA. Mol Microb iol. 69(1):137-151

Atkinson S, Goldstone RJ, Joshua GW, Chang CY, Patrick HL, Cámara M, Wren BW, Williams P. 2011. Biofilm development on Caenorhabditis elegans by Yersinia is facilitated by quorum sensing -dependent repression of type I I I secretion. PLoS P athog. 7(1):e1001250

Cornelis GR. (2002) The Yersinia Ysc–Yop 'Type II I ' weaponry. Nat. Rev. Mol. Cell Biol. 3:742 -754

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Students Communication Session 2 June 10th, 12:00-14:00

Chairwoman: María Dolores Girón González (Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Spain)

Ricardo F. dos Santos (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Catia M. Morgado Pérez (Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa)

Francisco Reche Pérez (University of Granada, Granada, Spain)

Francisco Ismael Román Moreno (University of Granada, Granada, Spain)

Amy Slater (University of Nottingham, Nottingham, UK)

Session 2

Students communications

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A novel protein complex involved in ribosome biogenesis and rRNA quality control Ricardo F. dos Santos. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB/UNL), Oeiras, Portugal The ribosome is the macromolecular decoder responsible for protein synthesis. It is build of three ribosomal RNAs (5S, 16S and 23S rRNA) and several ribosomal proteins. The maturation and assembly of all these components is a tightly regulated process, leading to the formation of the functional 70S ribosomal particle. In this work we describe a new quality control pathway involved in the degradation of defective rRNA and surveillance of ribosome biogenesis that requires the RNA-binding proteins Hfq and RNase R. The RNA chaperone Hfq and the 3’-5’ exoribonuclease R interact and this novel protein complex is critical for the elimination of aberrant rRNA fragments and processing of rRNA precursors. Moreover, the ribosomal profile of the Δhfq Δrnr strain is significantly altered. In the absence of Hfq and RNase R the amount of immature 30S and 50S subunits increase and the levels of functional 70S ribosomes are drastically decreased, which is consistent with the existence of severe defects in ribosome assembly. This is the first demonstration that the well -conserved Hfq and RNase R proteins interact in vivo, unraveling an unknown mechanism of RNA -quality control. This cooperation adds a new layer of regulation for the multi-stepped process of ribosome biogenesis.

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Characterization of probiotic strains of epiphytic microbiota of table olives Cátia M. Morgado Peres. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, P- 2780-157 Oeiras, Portugal. Consumption of functional probiotic foods has increased over recent decades, alongside with increasing consumer and researcher awareness of their health-promoting effects. This has prompted an interest toward development of novel functional food formulations. Despite fermented dairy products still remaining the most common vectors for delivery of probiotics to humans, such other food matrices as fruits and vegetables offer a promising performance as sources (of wild) and carriers of probiotic strains. Hence , these types of matrices were thoroughly reviewed – with table olives being subjected to comprehensive discussion as case study, owing to their suitable microstructure, and unique sensory and nutritional features. Adventitious strains from olive fermentation belong to Lactobacillus genus – and exhibit a remarkable capacity to tolerate the high acidity and salt content of fermented vegetables, as well as an ability to adhere to the colon wall and survive transit through the gastrointestinal tract where they will exert antimicrobial roles against local pathogens, further to exhibiting antitumoral features. Because of the health relevance of bioactive components from olives, e.g. phenolic compounds, coupled with the capacity of probiotics to adhere to intestinal cells, the relationship between probiotics, phenolic compounds and intestinal microbiota has been under scrutiny in order to provide new insights into their beneficial interactions, chiefly mechanisms of action against pathogens involving exocelular polymer production by probiotics. Capsular polysaccharides can indeed promote adherence of bacteria to biological surfaces, thereby facilitating colonization of various ecological niches. In the context of probiotic f oods, the synthesis of polymers aimed at surviving the adverse conditions prevailing in the upper part of the gastrointestinal tract after food ingestion is worthy studying, as they also provide a longer residence time in the gastrointestinal tract. Therefore, this project attempts to address this new kind of probiotic food, aiming at a better understanding of its potential toward improvement of health – while consubstantiating a case study of a novel plant -based functional food.

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Tamoxifen effects killing MCF-7 cells Francisco Reche Pérez. School of Pharmacy, University of Granada, Granada, Spain. Estrogens, in particular 17β-estradiol, are involved in many physiological processes; however they are also involved in development of breast cancer. The functions of these hormones are mediated by nuclear receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Approximately 70% of human breast cancers express ERα. In contrast, ERβ function is related with antiproliferative properties. Using selective estrogen receptors modulators (SERMs) we can suppress estrogen signaling and kill breast cancer cells. Tamoxifen is the SERMs of first choice treatment for breast cancer. It is ER antagonist, acting as agonist under particular conditions. However is proved that Tamoxifen reduce breast cancer by approximately 40 -50%.Tamoxifen inhibits proliferation and induces apoptosis by its interaction with the ERα. Tamoxifen activates ERK 1/2, a downstream member of the mitogen -activated protein kinases (MAPK). In serum-free culture conditions, estradiol was found to protect MCF -7 cells from Tamoxifen induced death. Albeit the activation of ERK is a possible mechanism by which estradiol opposes the effects of Tamoxifen, we have found that treatment with 5 μm Tamoxifen led to phosphorylation of ERK in MCF -7 cells. The result suggests that ERK is involved in the death response induced by Tamoxifen, related with an increased expression of caspase3, which activates its expression in the mitochondrion and produces apoptosis.

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Method And Product For Protecting And Reinforcing Construction And Ornamental Material Francisco Ismael Román Moreno, University of Granada, Spain. The invention relates to a method and product for protecting and reinforcing construction and ornamental materials, which activate the natural microbiota present in construction materials, such as to induce the formation of calcium carbonate. The application of the product generate a new calcium carbonate cement which i s compatible with the original material, respects the porosity thereof and improves the degree of reinforcement of same. Inventors: Gonzalez Muñoz, Maria, Teresa Rodriguez Navarro, Carlos Jiménez Lopez, Concepcion Rodríguez Gallego, Manuel

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Uncovering the regulatory mechanisms underpinning cell -to-cell signalling and type three secretion in Yersinia spp. Amy Slater. Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, UK. The genus Yersinia presents three human pathogens: enterocolitica, pseudotuberculosis and pestis encompassing a broad spectrum of diseases ranging from gastric infections through to the legendary causative agent of plague. Many Gram negative bacteria are known to regulate population-wide communication via AHL mediated quorum sensing (QS), a system comprised of diffusible signal molecules and transducers. In response to a threshold population density, the signal concentration is sufficient to promote coordinated gene express ion. Quorum sensing regulatory systems are composed of both a synthase gene and a regulatory gene of which discreet systems exist for specific AHL molecules. Many virulence genes are under the control of this bacterial ‘social networking’ including those located on a virulence plasmid known as pYV which is present in all pathogenic Yersinia. pYV codes for components of the type three secretion system (T3S) which is analogous to a hypodermic needle. The T3S system encodes an array of Yersinina Outer Proteins known as Yops. The Yop proteins are divided into two groups, firstly the regulatory Yops which encode both the proteins involved in the T3S apparatus and the associated regulators and secondly, the virulence effector proteins that are injected into the host. Virulence is energetically expensive therefore the T3S system is tightly regulated by two higher level regulator proteins known as VirF and YmoA. VirF is a temperature-dependent transcriptional activator which operates as a dimer. Upon host entry the rise in temperature to 37oC induces characteristic ‘melting’ of DNA at specific sites on pYV. This melting permits the access of transcriptional machinery and subsequent gene expression. As a regulatory Yop, VirF is expressed at this increased temperature and contributes to the phenotypic shift. Alongside VirF, a histone-like protein YmoA adds a secondary level of regulation. YmoA is chromosomally encoded an d is responsible for inhibiting Yop expression below 37 oC. We have recently revealed that quorum sensing regulates T3S secretion but the regulatory mechanism which underpin these observations are yet to be investigated. This project aims to examine whic h key regulators form part of the QS-T3S pathway.

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Students Communication Session 3 June 11th, 11:00-14:00

Chairwoman: Inmaculada Llamas Company (Department of Microbiology, School of Pharmacy, University of Granada, Spain)

Teresa Pinto (Universidade Nova de Lisboa, Lisboa, Portugal)

Rebeka Krebesova (University of Granada, Granada, Spain)

Carlos Peris Torres (University of Granada, Granada, Spain)

Daniella Spencer (University of Nottingham, Nottingham, UK)

Carmen Tong (University of Nottingham, Nottingham, UK)

Session 3

Students communications

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The RNase R from Campylobacter jejuni has unique features and is involved in the first steps of infection

Teresa Pinto . Universidade Nova de Lisboa, Avª da República, 2780 -157 Oeiras, Portugal.

Bacterial pathogens require a fast adaptation to different environmental stimuli. Ribonucleases (RNases) control RNA levels and contribute to the rapid adjustment to the new condit ions. It was already demonstrated that exoribonuclease polynucleotide phosphorylase (PNPase) facilitates survival of C. jejuni in low temperatures, and favors swimming, chick colonization and cell adhesion/invasion. However, little is known about the mechanism of action of other ribonucleases in this microorganism. Members of the RNB-family of enzymes shown to be involved in virulence of several pathogens. We have searched C. jejuni genome for homologues and found one candidate which displayed properties m ore similar to RNase R (Cj-RNR). We show that Cj-RNR is important for the first steps of infection, since it is involved in adhesion and invasion of C. jejuni to eukaryotic cells. Moreover, Cj-RNR proved to be active in a wide range of conditions, which m ay be crucial for the ability of C. jejuni to adapt to different environments during the infection process. The results obtained lead us to conclude that Cj-RNR has an important role in the biology of this foodborne pathogen.

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Optimization of a method for extracting total proteins from olive oil valid for electrophoretic and other downstream applications Rebeka Krebesova. University of Granada, Granada, Spain. The main compounds of olive oil are fatty acids. Besides, low amounts of proteins (0.05 -2.4 mg/kg) are also detectable in the olive virgin extra oil. These proteins may be bioactive molecules and have a potential allergenic impact on consumers, so its identification and characterization is of great importance for human health. Protein extraction is a critical step in the comprehensive study of olive oil proteins. During the olive oil extraction process, contaminating compounds migrate from the fruit/seed into the oil. The significant low concentration of proteins, together with these undesirable compounds and the lack of extraction protocols make the study of these proteins difficult. The aim of my master thesis was to develop a method suitable for extracting total proteins from olive oil and seed tissues for downstream applications such as SDS -PAGE, capillary electrophoresis, and further protein identification by mass spectrometry analysis. For this purpose, we have assayed different protein extraction methods including several aqueous buffer/organic solvent (i.e. acetone) mixtures and aqueous SDS -containing buffers. Our data indicate that acetone extraction followed by TCA/acetone precipitation and centrifugal filtration through a 3 kDa cut-off cellulose membrane renders the best results in terms of protein quantity and purity as demonstrated in electrophoretic experiments. Protein bands were picked form gels and are currently being analyzed by mass spectrometry. In conclusion, this method is a first good approach to obtain significant amounts of proteins from olive oil but further studies are nece ssary to validate it.

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Role of the matrix metalloprotease ADAMTS1 in melanoma: Tumor angiogenesis

and plasticity

Carlos Peris. University of Granada, Granada, Spain

The extracellular microenvironment is very dynamic, due to the constant modifications of their components in response to very different stimuli. The extracellular matrix (ECM) is a tridimensional net between every organ, tissue and cell of the organism. It composition is very diverse, with proteoglycans and polysaccharides with different physical and biological properties. The reorganization of the extracellular matrix during development is under a strict control of the enzyme production like extracellular proteases (MMPs, ADAMs and ADAMTSs). This control could be lost with aging or under pa thologies conditions, like cancer. Extracellular proteases are

responsible of important post‐translational protein modifications to allow the reorganization of the extracellular

space for proper cell migration and to modulate the activation of signaling mo lecules.

Angiogenesis (formation of new vessels) plays a fundamental role on the establishment and development of a

tumor. Nowadays, the research on this field is focused against this formation of new vessels. Blocking the angiogenesis process will stop the increment of the tumor mass, and this point could be the start of a different type of therapy. In this study we focus our attention in the first member of the ADAMTSs family of metalloproteases, ADAMTS1 (a disintegrin and metalloproteinase with thromposp ondin repeats 1). For this protease, its main actions as an angiogenesis inhibitor have been described, although its contribution for cancer progression is still controversial.

Melanoma is one of the most aggressive tumor types that exist. It has a high in vasive potential and there are still few therapies against it. Blocking the angiogenesis process could be an interesting approach for its eradication, so

it would be very interesting to determine the role of an antiangiogenic element, like ADAMTS1, on mela noma cells. For that reason, in this study we have selected four established melanoma cell lines (MUM2B, A375, G3G1 and SKMEL-28) to evaluate the role of ADAMTS1 in them. We also want to know if these cells are an appropriate model for the study of melanoma and if they have tumorigenic potential, inducing the tumor formation in mice (xenografts).

We are determining by quantitative PCR the gene expression of these cell lines for the genes that we are interested in: stemness markers as Oct4, Nanog, Sox2 or CD 133; and ADAMTS1 and two of its substrates, Nidogen1 and Nidogen2. We are also doing in vivo Matrigel migration and angiogenesis assay, as well as obtaining conditioned media from these cells to determine by Western Blot the presence or absence in it of th e protein of our interest, ADAMTS1, and its substrates. Another point of this study is related with the overexpression of ADAMTS1, by it transfection to these melanoma cells, so that we can study the role of ADAMTS1 and the consequences that its overexpression produces in its substrates.

With all these data we will know the role of ADAMTS1 in melanoma cells and we will have performed a new approach to the anti-cancer therapy based on anti-angiogenic factors.

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Chronic, antibiotic resistant Pseudomonas aeruginosa infections in the Cystic Fibrosis lung could rely on an Arginine-Specific Aminopepdidase AaaA.

Daniella Spencer. Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham,

UK.

The relationship between Cystic Fibrosis and Pseudomonas aeruginosa. Cystic Fibrosis (CF) is genetic disorder arising from the mutation of a single gene coding for an anion channel: the CF transmembrane conductance regulator (CFTR) (Gregory et al 1990). Located on the apical membrane of epithelial cells (Denning et al 1992), this protein maintains an ion gradient permitting optimal hydration of the airways and digestive system. As a result of a mutation in the CFTR gene, mucus lining the airways and digest ive passages of CF patients is dehydrated and difficult to clear (Kreda et al 2012). This causes an increased vulnerability to respiratory infection (Chmiel and Davis 2003).

Pseudomonas aeruginosa is able to establish and maintain a respiratory infection i n CF patients that is difficult to eradicate (Li et al 2005), due to its tendency to form biofilms. A gram -negative, opportunistic, highly adaptable species of bacteria, P.aeruginosa possesses a large genome of 6.3 million bp (Stover et al 2000), encompass ing many metabolic pathways.

The protein. To survive in biofilms, P.aeruginosa needs to metabolise nutrients other than carbon sources and must be able to grow at low oxygen densities (Stewart 2003). One of the alternative nutrient sources available to P.aeruginosa is the amino acid, arginine. This can be metabolised in anaerobic conditions to support cell growth (Mercenier et al 1980). The arginine specific aminopeptidase autotransporter (aaaA) protein cleaves arginine from the ends of peptide chains and could therefore provide the cell with a source of arginine when required (figure 1). Inhibition of this protein could limit P.aeruginosa’s ability to form and maintain a biofilm (Luckett et al 2012).

The aim. This project aims to determine the impact of Aa aA on the survival of P.aeruginosa in the CF airway and to

assess whether the AaaA can be inhibited. There are three main areas of investigation. The first is to determine the presence of AaaA in P.aeruginosa isolated from CF patients and to identify how v ariation in expression corresponds isolate phenotype and patient condition. This will involve the use of an effective RNA extraction procedure and qPCR assay. The second aim is to explore the role AaaA has in biofilms. The selection of an appropriate artificial sputum medium and optimisation of CF -representative conditions will need to be selected in addition to the construction of an inducible P.aeruginosa AaaA mutant. The third aim is to purify a functional AaaA protein which can be used to test the efficacy of inhibitors of its activity in vitro.

References

Chmiel, J. F., & Davis, P. B. (2003). State of the art: why do the lungs of patients with cystic fibrosis become infected and why can’t they clear the infection. Respir Res, 4(8).

Denning, G. M., Ostedgaard, L. S., Cheng, S. H., Smith, A. E., & Welsh, M. J. (1992). Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia. Journal of Clinical Investigation, 89(1), 339.

Gregory, R. J., Cheng, S. H., Rich, D. P., Marshall, J., Paul, S., Hehir, K., ... & Smith, A. E. (1990). Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Kreda, S. M., Davis, C. W., & Rose, M. C. (2012). CFTR, mucins, and mucus obstruction in cystic f ibrosis. Cold Spring Harbor perspectives in medicine, 2(9), a009589.

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Li, Z., Kosorok, M. R., Farrell, P. M., Laxova, A., West, S. E., Green, C. G., ... & Splaingard, M. L. (2005). Longitudinal

development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis. Jama, 293(5), 581-588.

Luckett, J. C., Darch, O., Watters, C., AbuOun, M., Wright, V., Paredes -Osses, E., ... & Hardie, K. R. (2012). A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity. PLoS pathogens, 8(8), e1002854.

Mercenier, A., Simon, J. P., Haas, D., & Stalon, V. (1980). Catabolism of L -arginine by Pseudomonas aeruginosa. Journal of general microbiology, 116(2), 381-389.

Stewart, P. S. (2003). Diffusion in biofilms. Journal of Bacteriology, 185(5), 1485 -1491.

Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., ... & Olson, M. V. (2000). Complete genome sequence of Pseudomonas aerug inosa PAO1, an opportunistic pathogen. Nature, 406(6799), 959 -964.

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Figure 1: A schematic diagram of the SrtA detection assay. After anchoring/ secretion of GLuc, in addition to O2, Coelentrazine substrate is added to the samples in order for bioluminescence to be produced.

Inhibitors of Staphylococcal Sortase A as novel anti-infective agents

Carmen Tong. Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, UK.

Background: Drug-resistant strains of pathogenic bacteria are becoming an increasing issue globally and the need for alternative drug therapies is crucial. In 2014, 80% of S. aureus infections reported in Western Europe were due to Methicillin-resistant Staph. aureus (MRSA) 1. Sortase are a group of bacterial transpeptidases. Their presence has been directly linked to the virulence and the ability of many Gram-positive bacteria to establish infections, including Staphylococcus aureus. Sortase A (SrtA) is responsible for cell wall protein anchoring, through the recognition of a highly conserved LPXTG motif on secreted cell wall proteins 2. In mouse models of infection, S. aureus SrtA mutants displayed a dramatic decrease in virulence3. Consequently, they are of particular interest for alternative antimicrobial drug therapies.

Objective: To develop a novel cell-based assay for the detection of Sortase A activity

Results: We have designed a cell-based assay which allows us to detect the activity of Sortase A using a Gaussia Luciferase photoprotein (GLuc) that has been engineered to possess the LPXTG motif. After the secretion of GLuc in S. aureus, SrtA recognises the LPXTG motif, cleaves and covalently anchors the GLuc onto the cell wall.

When SrtA is inhibited or inactive, GLuc is secreted into the supernatant. By separating the cell lysate from the supernatant we are able to determine SrtA activity based on bioluminescence levels emitted from GLuc proteins; as depicted in figure 1.

Conclusions: In this study we have developed a cell-based assay for the detection of Sortase A. We will use this assay to test previously reported SrtA inhibitors and for High Throughput Screening of chemical compound libraries for potential Sortase A inhibitors. Moreover, we will apply this assay to other Gram positive bacteria including Bacillus spp., Clostridia spp. and Listeria spp. to determine whether potential Sortase A inhibitors are universal to all Gram-positive organisms or specific to Staphylococcus aureus.

1. Health, O. W. Antibiotic Resistance. (2014). at <http://www.who.int/mediacentre/factsheets/fs194/en/>

2. Navarre, W. W. & Schneewind, O. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell

wall envelope. Microbiol. Mol. Biol. Rev. 63, 174–229 (1999).

3. Jonsson, I. M., Mazmanian, S. K., Schneewind, O., Bremell, T. & Tarkowski, A. The role of Staphylococcus aureus sortase A and

sortase B in murine arthritis. Microbes Infect. 5, 775–780 (2003).

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