Annual Report 2011

Institut Pasteur de Montevideo Annual Report 2011

TECHNOLOGICAL UNITS

Unit: Head:

Cell Biology Unit Mariela Bollati, PhD

Members:

Soledad Astrada (Master in Sciences) Perelmuter Karen (Master in Biotechnology) Josefina Louge (Biochemist) Valentina Porro (Medical Doctor) Sofa Ramirez (Bachelor in Sciences) Ins Tiscornia (Master in Sciences)

Research Our focus of research deals with three main topics: 1. CELL CULTURE TECHNOLOGY In this respect, the main area of research focuses on different aspects of cell culture technology with special emphasis in the optimization of recombinant protein production in mammalian cell cultures (employing transient or stable expression, culture media optimization, as well as metabolic engineering of animal cells, as a platform to improve cell productivity of recombinant proteins). 2. TYPE I IFNS Interferons (IFNs) are potent biologically active proteins synthesized and secreted by somatic cells of all mammalian species. Type I IFN (IFN-alpha and IFNbeta) is secreted by virus-infected cells while type II, immune or IFN-gamma is mainly secreted by T cells, natural killer cells and macrophages. Historically, IFN antiviral activity assays were the first type of bioassays developed to measure the relative activity or potency of IFN preparations. Recently, we report a new virus free, cell-based assay to quantify murine type I IFN, which basically consists of an indicator cell line. At the present we are using such a cell line for the screening of components, which are able to induce or interfere murine type I IFN signaling. 3. INFLAMMATION AND PROBIOTICS Inflammation and inflammatory pathways are key regulators of a wide range of processes, from innate- and adaptive-immunity, to the amplification of external signals (antigens), tissue homeostasis or disease. Lactic Acid Bacteria (LAB) are commonly employed as probiotics because of its immunmodulatory properties. Our aim is to characterize the in vitro effect of bacterial strains, which could present anti-inflammatory properties, on human intestinal epithelial and immune cells.

Services 1. Culture of different cell lines, cell banking. 2. Detection of mycoplasma contamination in cell culture by PCR. 3. Citotoxicity and proliferation assays. 4. Adaptation of different cell lines to the suspension growth mode and to serum free medium. 5. Generation of reporter cell lines, with broader applications. 6. Flow cytometry analysis: immunophenotyping, DNA content and cell cycle analysis, fluorescent proteins detection, apoptosis, etc. 7. Sorting of heterogeneous cell populations into homogeneous populations: sterile sorting, single cell deposition, 4 way sorting. 8. Imaging using Confocal Microscopy.

Publications 1. Brgi, M.; Prieto, C.; Oggero, M.; Bollati-Fogoln, M.; Etcheverrigaray, M.; Kratje, R. New reporter cell clones to determine the biological activity of human type I interferons. BMC Proceedings 2011, 5(Suppl 8):P4. Pils, M; Bleich, A; Fasnacht, N; Bollati-Fogolin, M; Schippers, A; Rozell, B; Mller, W Commensal gut flora reduces susceptibility to experimentally induced colitis via T-cell derived Interleukin-10. Inflammatory Bowel Diseases, 2011, 10: 2038-46.

2.

Grants 1. Desarrollo de un nuevo proceso de produccin de vacunas virales para uso veterinario mediante el empleo de cultivos de alta densidad Responsible: Mariela Bollati. ANII- Alianza para la Innovacin (Uruguay), 2010-2011 (1 year), USD 55,000. Status: On going. 2. Evaluacin de una vacuna peptdica basada en el oncogen HER-2/Neu utilizando un modelo de cncer de mama murino que sobre-expresa HER-2/Neu de origen humano Responsible: Mariela Bollati. Comisin Honoraria de Lucha contra el Cncer (Uruguay), 2009-2011 (2 years), USD 10,000. Status: On going. 3. Compuestos naturales y sintticos que modulen la actividad biolgica de los interferones humanos de tipo I: Anlisis mediante una nueva herramienta biolgica Director: Marcos Oggero, Co/director: Mariela Bollati. PICT 2007Tipo III A Cooperacin internacional. Mincyt-Foncyt (Argentina) 2010-2013 (3 years), USD 71,500. Status: On going.

Other activities TRAINING COURSES LOCAL, REGIONAL 1. 2. 3. Theoretical and practical intramural course: Introduction to Flow cytometry and its applications. Hand on training on CyAn - June 2011. Basic aspects of Cell Culture. PEDECIBA BIOLOGY, Montevideo, Uruguay. The Cell Biology Unit participated in the Theoretical and practical activities. August, 2011. Recombinant Protein Expression Course. PEDECIBA - BIOLOGY. Montevideo Uruguay, July 2011. Mariela Bollati was invited lecturer.

VISIT OF SCIENTISTS AND TRAINING OF STUDENTS Renzo Martino. PhD student from Universidad Nacional de San Luis, Laboratorio de Inmunologa, San Luis, Argentina. Andrea Comba. PhD student from Facultad de Ciencias Mdicas Universidad Nacional de Crdoba, Argentina. AMSUD-Pasteur Fellowship. Javier Eliabe. PhD student from Universidad Nacional de San Luis, Laboratorio de Inmunologa, San Luis, Argentina. AMSUD-Pasteur Fellowship. Ivanna Rolny. PhD student from Centro de Investigacin y Desarrollo en Criotecnologa de Alimentos, La Plata, Argentina CONICET.

Unit: Head:

Molecular Biology Unit Carlos Robello, PhD

Members:

Adriana Parodi-Talice (PhD - Senior, Facultad de Ciencias) Dolores Pieyro (PhD - Postdoc, Facultad de Medicina) Ma. Laura Chiribao (MSc Student, Facultad de Medicina) Talia Arcari (MSc Student) Gabriela Libisch (MSc Student) Gonzalo Greif (Technological activities) Cecilia Portela (Tecnician, Facultad de Ciencias) Pilar Zorrilla (Technological activities) Lorena Gil (Technological activities) Andrea Trochine (Postdoctoral position, CONICET-Argentina)

Research In the last year, our main focus of research has been centered on molecular parasitology with emphasis in the identification of virulence factors and drug targets. More recently we started to use next generation sequencing in our parasite models. 1. MOLECULAR PARASITOLOGY A. Nucleobase derivatives as drugs against trypanosomal diseases The main objective is the identification of new purine and pyrimidine derivatives for the treatment of the leishmaniases and trypanosomiases. A two-pronged approach is proposed to discover new leads for the treatment of leishmaniasis and trypanosomiasis targeting nucleoside/ nucleotide metabolism. 1) The phenotypic approach exploring the potential of large collections of novel nucleobase derivatives against trypanosomal diseases. 2) The target-based approach specifically centered on the development of inhibitors of the enzyme deoxyuridine triphosphate nucleotidohydrolase. The trypanosomal enzyme shows structural and functional characteristics which differ profoundly from the mammalian counterpart. The aim is to identify potent inhibitors that are active against parasitic protozoa, active in rodent models of infection and have drug-like properties. This project is financed by the Framework Program 7th (European Union) and the Molecular Biology Unit is participating by evaluating mode of action of compounds through proteomic and functional genomics approaches. Information about the project and the consortium can be found in http://www.ipb.csic.es/trypobase/ B. Oxidative stress in T. Cruzi Cytosolic and mitochondrial Trypanosoma cruzi tryparedoxin peroxidases belong to the family of 2-Cys peroxiredoxins. These enzymes play an essential role as antioxidants by their peroxidase and peroxynitrite reductase activities. TXNPx are key components of

the trypanosomatid peroxide detoxification pathways. Our group solved the structure of the cytosolic TXNPx. We found peroxiredoxins as relevant proteins in survival, replication and differentiation of T. cruzi, which could constitute virulence factors. Moreover, their expression in the infective forms of the life cycle and their low intracellular concentration make them good candidates to become targets for drug design. Currently we are studying interactions between peroxiredoxins and tryparedoxins from T. cruzi, both at the structural and functional level. C. Metacyclogenesis in T. Cruzi Metacyclogenesis is a process that takes place in the invertebrate host, comprising morphogenetic transformation from a noninfective form to an infective form, such that parasites acquire the ability to invade human cells. We analyzed the metacyclogenesis process by 2D electrophoresis coupled to MALDI-TOF MS. A large proportion of unique proteins expressed during metacyclogenesis were observed, and 50% of the spots were found to differ between epimastigotes and trypomastigotes. Several isoforms for a number of proteins were detected, some displaying differential expression during metacyclogenesis. The results indicate that posttranslational modifications constitute a fundamental part of the parasites strategy for regulating gene expression during differentiation. This study contributes to the identification of relevant proteins involved in the metacyclogenesis process. The identification and molecular characterization of these proteins will render vital information about the steps of the parasite differentiation into the infective form. D. Trypanosoma vivax transcriptome Phylogenic analysis locates T. vivax as the earliest branching species in African trypanosomes. Therefore the identification and evolutionary analysis of VSG genes in this species will shed light on the origin of one of the most fascinating mechanisms of immune evasion. Moreover, deep sequencing of transcriptome will give clues about transcription in these parasites. We have sequenced the transcriptome of T. vivax by 454 technology, assembly has been performed, and the analysis of results in going on. 2. TECHNOLOGICAL PROJECTS Although one of the aims of UBM is to provide services (see below Services), some of the proposals in functional genomics were considered as collaborations since our team participates actively in the design and the analysis of results. These projects are related to Uruguayan livestock production problems, and are being done according to necessities generated by the Faculty of Agronomy, the Faculty of Veterinary, and the INIA (National Institute of Research in Agriculture and Livestock). A. Pigmented fibres in Corriedale sheep: study of their genetic variability through quantitative and molecular approaches The presence of pigmented fibres (PF) in Uruguayan wool diminishes its value. The aim of this project is the genetic characterization of the presence of dark spots through quantitative and molecular approaches. Gene expression on dark spots (with and

without the presence of PF) and normal skin have been studied by microarrays with a bovine commercial chip (Agilent). The three kinds of skin samples were hybridized against each other in a two-color microarray design. Subsequent molecular characterization of the genes expressed only in spots will be done. B. Analysis of the gene expression of Solanum commersonii in an incompatible interaction with Ralstonia solanacearum The aim of this project is to gain insight about the characteristics of the resistance of Solanum commersonii to the bacteria Ralstonia solanacearum through the analysis of the transcriptional expression. Some resistant clones of S. commersonii were inoculated with R. solanacearum, and samplings were studied in microarray two-color experiments. This project obtained some interesting results over different time points that are being validated by Real-Time PCR, in order to contribute to the knowledge of the mechanisms involved in the resistance response of S. commeronii facing the infection of R. solanacearum. C. Study of genes and metabolic pathways involved in the bovine gestation-lactancy period with the Microarray technique The main objectives of the experiment were: To evaluate temporal changes in the hepatic gene expression profile and their relation with the most important metabolic pathways associated with energetic changes. To identify these changes in the different experimental animal models (pure and cross breeds) evaluated in high and low offer of natural countryside fodder. The general aim is to improve the reproductive efficiency in Uruguayan livestock.

Services

1. DNA sequencing (Sanger methodology) 2. Real Time PCR 3. Microarrays 4. Bioanalyzer 5. Next Generation Sequencing (Illumina)
Publications

1.

Schijman AG, Bisio M, Orellana L, Sued M, Duffy T, Mejia Jaramillo AM, Cura C, Auter F, Veron V, Qvarnstrom Y, Deborggraeve S, Hijar G, Zulantay I, Lucero RH, Velazquez E, Tellez T, Sanchez Leon Z, Galvo L, Nolder D, Monje Rumi M, Levi JE, Ramirez JD, Zorrilla P, Flores M, Jercic MI, Crisante G, Aez N, De Castro AM, Gonzalez CI, Acosta Viana K, Yachelini P,

Torrico F, Robello C, Diosque P, Triana Chavez O, Aznar C, Russomando G, Bscher P, Assal A, Guhl F, Sosa Estani S, DaSilva A, Britto C, Luquetti A, Ladzins. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. J. PLoS Negl Trop Dis. 2011 Jan 11;5(1):e931.

2.

Pieyro MD, Arcari T, Robello C, Radi R, Trujillo M. Tryparedoxin peroxidases from Trypanosoma cruzi: high efficiency in the catalytic elimination of hydrogen peroxide and peroxynitrite. Arch Biochem Biophys. 2011 Mar 15;507(2):287-95. Pieyro MD, Parodi-Talice A, Portela M, Arias DG, Guerrero SA, Robello C. Molecular characterization and interactome analysis of Trypanosoma cruzi Tryparedoxin 1. J Proteomics. 2011 74(9):1683-92 De Figueiredo Peloso E, Vitor SC, Ribeiro LH, Pieyro MD, Robello C, Gadelha FR. Role of Trypanosoma cruzi peroxiredoxins in mitochondrial bioenergetics. J Bioenerg Biomembr. 2011 Jul 6. 43(4):419-24. F. Peagaricano, P. Zorrilla, H. Naya, C. Robello, and J.I. Urioste. Gene expression analysis identifies new candidate genes associated with the development of black skin spots in Corriedale sheep. J Appl Genet. 2012 Feb;53(1):99-106. Epub 2011 Sep 28.

3.

4.

5.

Grants

1. Nucleobase derivatives as drugs against trypanosomal diseases Carlos Robello FP7 UE

2009 (3 years) -

2. Trypanosoma vivax: un modelo para comprender el origen de la variacin antignica en

tripanosomas Carlos Robello ANII 2009 (2007 Call) (2 years) Amount Granted USD 34.000.

3. Caracterizacin de la propuesta de T. Cruzi al perdixo de hodrgeno: rol de las

peroxirredoxinas Mnica Gardner ANII 2011 (2009 Call) (2 years) Amount Granted USD 13.000.

4. Impact of temperature on detoxifying enzyme expression and activity, and its

consequences on Aedex Aegypti insecticide resistance- Carlos Robello ACIP 2010 (2 years) Amount Granted Euros 17.100.

Other activities TRAINING COURSES 1. Regional workshop Massive Sequence ant their application 13th April, 2011 Institut Pasteur de Montevideo 2. Basic course of technologies of massive sequence and biology of small ARNs, 29th Aug 2nd Sept 2011, Institut Pasteur de Montevideo co organization Carlos Robello, Alfonso Cayota 3. International Course: Second Edition. Functional genomics and its applications in biomedicine. Institut Pasteur de Montevideo November 21st - December 2nd, 2011

Unit: Head: Members:

Recombinant Proteins Unit Pablo Oppezzo, Ph.D Cecilia Abreu (Doctoral student-staff Research Assistant) Florencia Palacios (Doctoral student-staff Research Assistant) Agustn Correa (Doctoral student-staff Research Assistant) Mariana Pegazzano (Staff Research Assistant) Pilar Moreno (Ph.D)

Research Our major scientific interest is focused on B lymphocyte development and the tumoral lymphoproliferative disorders of this cell type. In this context we chose a leukemic model as Chronic Lymphocytic Leukemia (CLL) and we directed on different target proteins-pathways in order to understand the basis of tumoral development in B-cells. Our work lies on the interface between biochemistry, molecular and cellular biology in combination with crystallography and biophysics protein constituting the core of our experimental methods. Three major lines of research are being pursued: 1. Functional role of the mutagenic B-cell specific enzyme, Activation-Induced Cytidine Deaminase (AID) in the tumoral process 2. Genomic and proteomic characterization of a proliferating CLL B-cells subset over-expressing AID enzyme with a typically profile to an aggressive tumoral disease. In these two items special interest is given to understand the molecular mechanisms underlying the tumoral process in those lymphoproliferative disorders that over-express AID. Some key questions involving AID enzyme and tumoral B-cells development are raised: How AID enzyme is regulated during development of B lymphocyte? How AID contributes to lymphomagenesis? Which are the partners of AID involved in the deamination process? How transcribed non Ig-genes escape to deamination? 3. Effects of constant regions in the antigen binding site and their applications in the recombinant antibodies used in cancer immunotherapy. 1. FUNCTIONAL ROLE OF THE MUTAGENIC B-CELL SPECIFIC ENZYME, ACTIVATION-INDUCED CYTIDINE DEAMINASE (AID) IN THE TUMORAL PROCESS At this moment we need to learn more about how, Ig gene diversification occurs, and how it is regulated in order to prevent the mutagenic action of AID enzyme. The different haematopoietic B-cell malignancies are excellent models to dissect and understand the different pathways involved in the deamination event and in the tumoral origins. In this context our laboratory has established a successful interaction with the Hematological Service of Maciel Hospital and put forward two main diagnostic methods used as predictors in the CLL progression. This collaboration is coordinated with the national bank of cancer in Uruguay allowing the establishment of a CLL cellular bank and the continuation of different scientific projects on this leukemia.

We have made progress most notably in the functional characterization (1) and differential expression of AID enzyme in CLL (2). Despite of Somatic Hypermutation (SHM) and Class Switch Recombination (CSR) are physiologically triggered in B-lymphocyte by AID, undesirable actions such as lymphomagenesis has been associated to its over-expression (3) (4). More recently an interesting role for AID in the epigenetic reprogramming including demethylation of DNA was demonstrated (5) (6). Thus, in addition to AID over-expression and lymphomas development, this works highlighted the putative importance of this enzyme in the tumoral origins. In this context we have started a collaborative research with Dr. J Di Noia (Institut de Recherches Cliniques de Montral), concerning the characterization of CLL B-cells and another lymphomas over-expressing AID. In this line recombinant AID protein has being produced in order to characterize putative cofactors of this enzyme that could be implicated in the specificity of deamination process. Different solubilization methods have been optimized in order to obtain functional AID enzyme in collaboration with Dr. Vincentelli in the laboratory of Cloning, Expression & Protein purification from CNRS, Universits Aix-Marseille. Despite of known problems to obtain soluble AID, the results from this high-throughput analysis in this collaboration have been very successful. At the present two constructions were selected from 250 conditions test and preliminary functional Biacore analysis are being carried out with AID enzyme and different single and double strand DNA molecules.

2. GENOMIC AND PROTEOMIC CHARACTERIZATION OF A PROLIFERATING CLL B-CELLS SUBSET OVER-EXPRESSING AID ENZYME WITH A TYPICALLY PROFILE TO AN AGGRESSIVE TUMORAL DISEASE To understand how AID over-expression could be responsible in the tumoral development of CLL B-cell, we isolated a particular proliferative subset of CLL B-cells with active CSR described in (2). Since AID expression results from interaction with activated tissue microenvironment, we speculated that the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in peripheral blood (PB) of 50 CLL patients and we characterized the expression of different molecules related to microenvironment interaction. Our results show that among unmutated (UM) patients: 1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; 2) this small subset expresses high levels of proliferation, anti-apoptotic and progression CLL markers (Ki-67, c-myc, Bcl-2, CD49d and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment. This research line has resulted in the first high impact article made in our laboratory, including most of the work performed at the IP Montevideo (7). At present, new collaborations are being developed in this field with the laboratory of Dr. Giordano at the Academy of Medicine in Bs As (Argentina).

3. EFFECTS OF CONSTANT REGIONS IN THE ANTIGEN BINDING SITE AND THEIR APPLICATIONS IN THE RECOMBINANT ANTIBODIES USED IN CANCER IMMUNOTHERAPY Although the CSR process is frequently associated with affinity maturation, one of the immunological dogmas is that the constant region does not assume any role in the binding with the antigen. However in the last decade several works suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form. (8, 9). Although these data support clear evidence for this hypothesis, no structural proofs are available at this time. In the context of structural platform of IP Montevideo (Protein Crystallographic Unit, Biophysics Protein Unit and Recombinant Protein Unit), our group decided to provide crystallographic and biophysical data in order to confirm whether the constant regions of the Ig play a role in the recognition of the antigen. The three-dimensional structure of Igs has provided essential information about antigen recognition. Despite the fact that there are over 300 crystal structures of human IgG Fabs currently available, there are no crystal structures of a human IgA1 fab fragment. The major difficulty in obtaining crystal structures from human IgA1 is related to the presence of several heterogeneous O-linked glycosylation sites in the hinge region of IgA1. To solve this we produced Fab fragments (FabA) by cleavage with a recombinant protease from Clostridium ramosum which specifically cuts the IgA1 molecule at the very end of the hinge region, releasing a Fab fragment without any glycan residues. With this FabA, we were able to solve two different crystal structures at high resolution (1.5 and 2.3 ), resulting in the first crystal structure of a human IgA1 Fab fragment. (10) (Manuscript in preparation). Additionally to this we have also solved the Fab from a monoclonal IgG1 with identical variable region of the IgA described above. Comparison and crystallographic studies from these two structures with identical variable regions but with different CH1 domain might finally support clear evidence on the relevance of CH1 fragment in the antigenic binding site. At present, we are carrying out BiaCore analysis in order to characterize the kinetic constants for both Igs and a second manuscript is envisaged this year. Part of this work corresponds to the degree thesis of Correa Agustin (MSc student staff Research Assistant) which has been defended in May 2011. RESEARCH LINES PERSPECTIVES Tasks for the next future involves the consolidation of the research lines 1. and 2. mentioned above. Two doctoral fellowships for the two doctoral theses involved in these research lines has been obtained in June 2011 and the project entitled Genomic characterization of a proliferating B-cell subset in CLL: Could AID expression be implicated in the development and progression of this disease? has been financed by Lady TATA Foundation, England 2011. Additionally, in collaboration with Dr. Giordano from Immunologic Department in the Academy of Medicine of Bs As, Argentina we applied for funding (PICT 2011, Argentine under revision) with the project entitled Origins and clinical evolution in CLL: Role of auto antigen stimulation

Furthermore, a project in the CLL field developing a recombinant antibody in order to generate a new prognostic marker entitled Lipoprotein Lipase expression in Chronic Lymphocytic Leukemia (CLL): Towards the development of a new prognostic marker has been approved by Comision Honoraria de Lucha contra el Cancer in May 2011. Finally, in order to consolidate a CLL regional group we have presented the project entitled "Red-iberoamericana de Leucemia Linfoide Crnica: hacia el desarrollo de nuevos marcadores pronsticos". at the international convocatory of IBERO-AMERICAN PROGRAMME FOR SCIENCE, TECHNOLOGY AND DEVELOPMENT (CYTED). Regarding point 3. of the research lines, it will probably become a more general project including other IP Montevideo laboratories. In this line we expect the publication of the manuscripts above mentioned and we will subsequently focus on the antibodies engineered design for the therapeutic tumoral area. We believe that we have the knowledge and the necessary equipment for the development of this area in order to provide these tools to the academic area. (see publication number 6). In this context a collaborative project with Dr. Osinaga, Dr. Pritsch at IPMont and Dr Amingorena at Institut Marie Curie, France entitled Pharmacokinetic study of Chi-Tn", has been obtained.

Services SERVICES THAT ARE CURRENTLY BEING PROVIDED 1. Protein expression in prokaryotic and eukaryotic systems: a. E. coli expression b. Baculovirus system c. Mammalian cells expression 2. Manual solubilization system allowing test of 74 different culture conditions in order to optimize the protein expression. 3. Manual and robotic purification system.

UPR progress 2007-2011

As shown in the figure the number of expressed and purified proteins in the UPR is on rise since 2007 to date. This increase is related to several variables such as consolidation of the laboratory staff, optimization of different expression systems and acquisition of new equipment.

FUTURES SERVICES LINES Acquirement and installation of a prokaryotic fermentor and eukaryotic bioreactor, both with 5 lts capacity each one. Installation of prokaryotic fermentator has been performed in July 2011 and the eukaryotic bioreactor will be carried out in the next months. This technology will increase the service lines offered by the UPR. Special interest is given to the development of eukaryotic bioreactor cells in order to improve the possibilities to obtain greater quantities of those proteins which are difficult to express. In this context the research assistant Abreu C. has been trained at the USA New Brunswick platform. The second perspective for coming years in the UPR service line is to develop a highthroughput platform dedicated to solubilization and crystal optimization of recombinant proteins. We think that it is a central element for this laboratory to provide a better and original system for solubilization and crystallization high-throughput screenings in the regional recombinant protein area. This technology is being performed through a doctoral thesis work carried out by the research assistant Correa A. in collaboration with Dr Alzari in the Structural Biochemistry Unit at the Pasteur Institut in Paris.

Expected

Publications

1.

CCR4 expression in a case of cutaneous Richter's transformation of chronic lymphocytic leukaemia (CLL) to diffuse large B cell lymphoma (DLBCL) and in CLL patients with no skin manifestations. Nannini P., Borge M, Mikolaitis V, Abreu C, Morande Pablo E., Zanetti S, Oppezzo P., Palacios F., Ledesma I, Bezares R., Giordano M, Gamberale R. Eur J Haematol. 2011 Jul; 87 (1):806. doi:10.1111. PMID:21443542 Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: Advantages of high-throughput screening. Correa A, Oppezzo P. Biotechnol J. 2011 Jun;6(6):715-30. doi: 10.1002/biot.201100025. Epu 2011 May 12. PMID:21567962 Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: the carbohydrate binding mechanism involves residual sialidase activity. Oppezzo P, Obal G, Baraibar MA, Pritsch O, Alzari PM, Buschiazzo A. Biochim Biophys Acta. 2011 Sep;1814 (9):1154-61. Epub 2011 Apr 30.PMID: 21570497 Antibody-dependent cell cytotoxicity synapses form in mice during tumor-specific antibody immunotherapy. Hubert P, Heitzmann A, Viel S, Nicolas A, Sastre-Garau X, Oppezzo P, Pritsch O, Osinaga E, Amigorena S. Cancer Res. 2011 Aug 1;71(15):5134-43. Epub 2011 Jun 22. PMID: 21697279

2.

3.

4.

Financed ongoing projects 1. "Responsible mechanisms for the association between autoimmune hemolytic anemia and Chronic Lymphocytic Leukemia: role of erythrocyte protein band 3 Mirta Giordano and Pablo Oppezzo (Principal Investigators) Programa de Ciencia y Tecnologia (PICT)Argentina 2008 Amount Granted USD 40.000 (4 years). Characterisation of the proliferating pool in CLL. Is AID expression a marker of this subpopulation? Pablo Oppezzo - Lady Tata Foundation (2009 - 2011) Amount Granted 50.000 Sterlin Pounds Estudio de la expresin de gen LPL en la progresin de pacientes con Leucemia Linfoide Crnica Pablo Oppezzo Comisin Honoraria de Lucha contra el Cncer. CHLCC Uruguay Amount Granted 10.000 USD (1 year)

2.

3.

Other activities 1. 2. 3. 4. 5. 6. 7. Training of three PhD degree students. Training of one Master degree student. Correa A. - Doctoral training trip, under the supervision of Dr Pedro Alzari, Biochemistry Structural Unit, Institut Pasteur, Paris France (six months, 2011) Oppezzo P. - Editor of book of Chronic Lymphocytic Leukaemia. InTech publisher Oppezzo P. - Reviewer of >10 peer-reviewed journals, including Blood, Leukemia, Haematologica, Interntaional Journal of Hematology and others. Oppezzo P. - Associate professor - Department of Immunobiology. School of Medicine Universidad de la Repblica, Uruguay International Workshop in Chronic Lymphocytic Leukaemia, 3 posters presentation of, 2011, Houston-TEXAS-USA. Young Investigator Meeting in CLL, Oppezzo P. invited speaker, 2011 Houston-TEXAS-USA.

Unit: Head:

Protein Biophysics Unit Otto Pritsch, PhD

Members:

Gonzalo Obal (MSc student, Technical Assistant) Sergio Bianchi (MD, MSc, PhD student) Gonzalo Moratorio (MSc, PhD student) Lorena Tom (MSc, PhD student) Federico Carrin (Technical Assistant) Gonzalo Rama (MSc student)

Research Our scientific activity in the context of the Institut Pasteur de Montevideo is focused in two principal projects: 1. SEARCHING OF A VIRAL ETIOLOGY FOR HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA In this project, we aim to search for the presence of a virus in human CLL, by using subtractive techniques employed to detect the last two viruses implicated in human cancer: the hepatitis C virus and the HHV-8. As it was the case for these last two viruses, if there is a virus implicated in CLL, it should be a new virus. In our laboratory at the Pasteur Institute of Montevideo, we have studied the putative presence of viral DNA in the genome of CLL B cells by combining high-throughput sequencing technology (454 Life Sciences and Illumina). Despite having analyzed more than 100 million sequences and reached enough transcriptomic depth, we failed to identify any putative viral candidate of the disease (Search for an etiological virus candidate in chronic lymphocytic leukemia by extensive transcriptome analysis. Rego N, Bianchi S, Moreno P, Persson H, Kvist A, Pena A, Oppezzo P, Naya H, Rovira C, Dighiero G, Pritsch O. (submitted to Haematologica 2011) 2. STUDY OF AN ANIMAL MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA INDUCED BY VIRUS. Bovine leukaemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus of the family Retroviridae. BLV infects cattle worldwide and is the causative agent of Enzootic Bovine Leukosis (EBL). In Uruguay, more than 50% of dairy cattle is seropositive for BLV, and this situation involves important economical concerns in our economy. The main objective of our work is to integrate a multidisciplinary national team to study the epidemiology and pathophysiology of the enzootic bovine leukosis, its causative agent and the oncogenic process triggered by BLV. The specific aims are: a. to analyze the genetic variability of BLV in Uruguay; Enzootic Bovine Leukosis can be divided into three clinical forms: a) Asymptomatic (AS), b) Persistent Lymphocytosis (PL), characterized by a malignant polyclonal expansion of CD5+ B cells and c) Lymphosarcoma (LS), characterized by the formation

of tumors. The general objective of this work is to contribute to knowledge on Bovine Leukemia, by molecular characterization and analysis of viral strains present in infected cattle with different clinical manifestations of the disease. We obtained for first time the complete genomic sequence of a BLV strain from a lymphosarcoma. The amplified BLV genome was cloned, sequenced, and bioinformatically studied with other BLV full-length sequences from other manifestations of the disease including PL and asymptomatic ones. b. to develop new methods for diagnosis of BLV infection: we have developed a rapid and sensitive real time PCR assay using SYBR green chemistry to detect and quantify BLV proviral DNA by amplifying gp51 gene from bovine peripheral blood. A comparative analysis with validated diagnostic tests (AGID, ELISA and direct nested PCR) was performed in 45 dairy cattle samples. In summary, our results reveal that real-time PCR is comparable to nested PCR, and confirm an increased sensitivity of the PCR (realtime and nested) over the ELISA and AGID tests respectively. Overall, our results show that this SYBR Green -based PCR assay may be a useful, simple, and rapid tool to detect BLV infection in dairy cattle samples that could be adapted to high-throughput diagnostic procedures c. to analyze at the structural level the principal viral proteins: we have characterized in vitro self-assembly of Bovine Leukemia Virus capsid protein. In common with other retroviruses, formation of a functional core structure during morphogenesis of BLV viral particles is essential for infectivity. In this process, thousands of capsid (CA) molecules self-assemble to form a shell which encases the genome. The assembly mechanism of BLV-CA protein to form mature-type core in vivo and in vitro are unknown. We recently started the characterization of in vitro BLV-CA protein assembly and developed a turbidimetry-based assembly assay using the purified recombinant BLV-CA protein. Specifically, the effect on self-assembly triggering and kinetics was analysed for protein concentration, pH, ionic strength, temperature, phosphate and polyphosphates. The influence of both independent N-terminal and C-terminal domains on oligomerization kinetics was also assessed. In parallel, we performed electron microscopic analysis of assembly products in order to evaluate the physical characteristics of the material formed under these conditions. This work provides the first description of the BLV capsid protein assembly properties, which may also be of relevance for understanding other Deltaretrovirus assembly characteristics. d. To identify and characterize the proteome expressed in BLV virions. This includes the proteins encoded by the viral genome and their post-translational modifications; and also the proteins encoded by the genome of the host infected cell. For this we have purified viral particles from culture supernatants of an persistently infected cell line; separated virion proteins by two-dimensional electrophoresis and identified them by mass spectrometry. Services 1. Thermodynamic analysis of protein-protein and protein-ligand interaction through determination by isothermal titration microcalorimetry (ITC) of binding constants (KB), reaction stoichiometry (n), enthalpy (H) and entropy (S).

2. Thermodynamic analysis of conformational changes of proteins including assessment of stability and folding of recombinant proteins through differential scanning microcalorimetry (DSC) to study a wide range of thermal transitions in biological systems, to determine melting temperatures as well as thermodynamic parameters associated to these changes. 3. Kinetic analysis of protein - ligand interaction through Surface Plasmon Resonance (SPR) measurements, determination of kinetic association (kass) and dissociation (kdiss) constants. 4. Determination of the hydrodynamic radius of macromolecules or particles through dynamic light scattering measurements coupled to size exclusion chromatography SEC-HPLC. Publications

1. Lima A, Durn R, Schujman GE, Marchissio MJ, Portela MM, Obal G, Pritsch O, de Mendoza D, Cerveansky C. Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: Biochemical characterization and identification of interacting partners through proteomic approaches. J Proteomics. 2011 Aug 24;74(9):1720-34. 2. Manta B, Obal G, Ricciardi A, Pritsch O, Denicola A. Tools to evaluate the conformation of protein products. Biotechnol J. 2011 Jun;6(6):731-41. 3. Oppezzo P, Obal G, Baraibar MA, Pritsch O, Alzari PM, Buschiazzo A. Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: The carbohydrate binding mechanism involves residual sialidase activity. Biochim Biophys Acta. 2011 Sep; 1814(9):1154-61. 4. Hubert PM, Heitzmann A, Viel S, Nicolas A, Sastre-Garau X, Oppezzo P, Pritsch O, Osinaga E, Amigorena S. Antibody-dependent cell cytotoxicity synapses form in mice during tumorspecific antibody immunotherapy. Cancer Res. 2011 Aug 1;71(15):5134-43. 5. C Tabares-da Rosa S, Rossotti M, Carleiza C, Carrin F, Pritsch O, Ahn KC, Last JA, Hammock BD, Gonzlez-Sapienza G. Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response. Anal Chem. 2011 Sep 15;83(18):7213-7220.

Unit: Head:

Protein Crystallography Alejandro Buschiazzo, PhD

Members:

Horacio Botti, MD/PhD (staff Research Scientist) Sofa Horjales, MSc (PhD student) Nicole Larrieux (staff Technician) Natalia Ruetalo, MSc (Research Assistant) Felipe Trajtenberg, PhD (staff Research Scientist)

Research Within our scientific interests, as a first general goal, we wish to understand how signaling and subsequent cell regulation are involved in microbial pathogenesis. In a second line, structural studies of glycosyltransferases in pathogens are also pursued as potential drug targets. To these ends our efforts mainly, although not exclusively, involve the study of two different biological models: Leptospira spp. (prokaryotic Spirochetes), and the Trypanosomatid parasites Leishmania spp. and Trypanosoma spp. (eukaryotic Protozoa). In terms of the scientific approach, we intend to understand protein function at the molecular level. This is why we believe it is essential to explore a diverse set of organisms, with interest in eventually extending our studies to other clinically relevant bacterial species. Our work lies on the interface between biology, chemistry and physics: protein crystallography in combination with biochemistry, biophysics and molecular biology constitute the core of our experimental approaches. 1. SIGNALING AND REGULATION IN PATHOGENIC MICROORGANISMS Bacterial two-component systems (TCSs) and mitogen activated protein kinases (MAPKs) in eukaryotes, constitute the main protein systems that we work on. The common theme is how cells use proteins to sense extra- and intra-cellular signals in order to regulate specific functions. Signaling through TCSs in bacteria To understand the molecular means by which bacteria transduce signals, adapting to a changing environment, during the last few years we have been using a non-pathogenic model (Bacillus subtilis) focusing our efforts in elucidating the molecular mechanisms of signaling and regulation of lipid synthesis in Gram+ bacteria. Our main contribution concerns the structural studies of DesK, a trans-membrane histidine kinase that, together with its cognate response regulator DesR, constitutes a TCS, ultimately regulating the membranes fluidity in response to cold shock in B. subtilis. The 3D structure of the entire cytoplasmic region of DesK from B. subtilis (Albanesi et al., Proc Natl Acad Sci U S A. 2009. 106(38):16185-90), determined in different functional configurations along the regulation cycle, allowed us to propose a mechanistic model that appears to be general for histidine kinase-mediated signal transduction. The high resolution 3D structure of the ATP-binding domain of DesK (Trajtenberg et al., J Biol Chem. 2010 285(32):24892-903) led us to

identify conserved regions suspected to be relevant in the autophosphorylation mechanism. Subsequent structure-based mutagenesis, lead to the kinetic trapping of a putative transient state of autophosphorylating DesK, confirming the structural hypotheses. More recently, we have been able to solve the structures of the response regulator DesR, in its inactive state as well as in the activated conformation (using beryllofluoride to mimic the phosphorylated state). Novel features of the activation mechanism are being analyzed (Trajtenberg et al., manuscript in preparation). This project has been supported by the French Research Agency (ANR) and the Uruguayan National Research Agency (ANII). It has been embedded in a collaborative work with the teams of P Alzari and Michael Nilges (Units de Biochimie Structurale and Bioinformatique Structurale, Institut Pasteur, Paris) and Prof. D de Mendoza (Dept of Microbiology, Instituto de Biologia Molecular y Celular de Rosario IBR, Rosario, Argentina). During the last year, with the aim of studying how TCS-mediated signaling eventually regulates pathogenesis, we have started to work with Leptospira spp., spirochetal bacteria that cause leptospirosis. This disease is the most widespread zoonosis in the world, reemerging as a major health problem. In Uruguay its prevalence as a veterinary issue is very significant, identified as the second most serious problem after brucellosis (Plan Nacional de Investigacion en Salud Animal http://www.fvet.edu.uy/planisa). A collaborative partnership has been established with Albert Kos lab (Yale Univ) and Mathieu Picardeaus (Institut Pasteur). Combining genetics and structural biology, we have identified TCS-related virulence attenuation in L. interrogans (hamster model), particularly involving intra-cellular hybrid histidine-kinases. Heme biosynthesis, critical for Leptospira survival (at difference with other Spiroche tes) is also controlled via a TCS, through a poorly understood pathway that we are now investigating. Finally, we are studying proteins that structure the leptospiral motility apparatus, which in spirochetes displays important differences compared to similar Gram-negative bacteria. This pilot project has received support from the Institut Pasteur, through ACIP (Actions Concertes Inter-Pasteuriennes) and P T R ( Projet Transversal de Recherche) grants, currently underway. This line of research is also extended towards the generation of chimeric sensor histidinekinases, within a Synthetic Biology approach, combining structural modules of better characterized proteins from bacteria such as E. coli and B. subtilis. This project is being supported by the Uruguayan National Research Agency (ANII). Signaling through MAPKs in Trypansomatid parasites With regards to eukaryotic MAPKs, we are studying their mode of action and regulation in Leishmania major, a trypanosomatid protozoa that causes human leishmaniasis. Trypanosomatids cause severe human diseases such as sleeping sickness, Chagas disease, and leishmaniasis. Together these diseases provoke millions of deaths in endemic areas around the world and represent a major burden for the socio-economic development of affected countries. No vaccine exists for any of these diseases. Current treatment options have serious side -effects and are increasingly threatened by the spread of drug-resistant parasite strains. We have solved the crystal structure of LmaMPK10, the first MAPK 3D structure to be disclosed from a Trypanosomatid species (Horjales et al., manuscript under review). The crystal structures of LmaMPK10 have been determined both alone (at 1.9 resolution) and in complex with the p38specific inhibitor SB203580 (2.6 ), revealing parasite-specific features and a novel autoinhibitory mechanism that involves its C-terminal end. LmaMPK10 participates in the regulation of the promastigote to amastigote differentiation process. A strong collaboration with Gerald Spaeths group at Institut Pasteur has been established. This project has been supported by the

multicentric program Targeting the Leishmania kinome for the development of novel antiparasitic strategies, funded by the European Union (FP7). 2. STRUCTURAL BIOLOGY OF GLYCOSYLTRANSFERASES We have a long-standing background in the structural biology of sialidases and trans- sialidases (TS) from trypanosomal parasites. The trans-sialidase from Trypanosoma cruzi, the etiologic agent of Chagas disease, is a virulence factor directly involved in pathogenesis within the vertebrate host. It is also a unique enzyme, not present in humans, able to efficiently transfer terminal sialic acids from the host sialoglycoconjugates, onto the parasites own glycoproteins and glycolipids. Using our structural information on parasite sialidases, we have been concentrating in the rational design of T. cruzi TS inhibitors that could eventually evolve towards specific compounds for biochemical and cell biology approaches in understanding T. cruzi pathogenesis linked to sialic acid biology. In the end, these efforts may prove useful in a strategy to develop therapeutic applications, badly needed to combat this neglected disease. In this line, collaborative research is being actively pursued with Dr O Campetella (Universidad de San Martin, Argentina), concerning the use of neutralizing antibodies able to inhibit the trans-sialidase activity from T. cruzi. The crystal structure of TcTS in complex with a subnanomolar affinity monoclonal antibody has been recently solved in our lab (Buschiazzo et al. PLoS Pathogens 2012). We have also contributed to the understanding of molecular mechanisms linked to a sialic acid-specific lectin function of catalytically inactive members of the TS family in T. cruzi (Oppezzo et al. Biochim Biophys Acta. 2011 1814(9):1154-61), among other recent work in the field. Services The Protein Crystallography Facility (PXF) has been consolidated; its visibility in Uruguay and the region has been strengthened. The web page has been updated, and for the last 3 years our facility has become fully operational to receive and process all the service requests (mainly from IPMontevideo, from the Uruguayan community and several users from Argentina). The number of users has been steadily growing : 5 in 2010, 10 in 2011, figures that are anticipated to keep rising in 2012. Our staff has received permanent training in high quality centers worldwide, and we have been able to improve sample management and tracking within a fully digitalized database (XTRACK). Services currently provided 1. Robotic and manual protein crystallization screenings 2. Follow-up and optimization of initial crystallization hits 3. X ray Diffraction Testing & Crystal Characterization 4. X ray Diffraction single crystal data collection 5. Crystal Structure Determination & Refinement

Progress 2011 1. Over 10 structures solved in our facility during the reported period (among >50 since the facility is up and running on the first semester of 2007). Notably including glucose-6Pdehydrogenase from Trypanosoma cruzi (2: apo 4E9I & in complex with Glc-6-P 4EM5); mitochondrial iron superoxide dismutase from T. cruzi (4DVH); the metallo-beta-lactamase GOB from 2. Six (6) of these structures have been deposited in 2011 in the Protein Data Bank: human IgA Fab (2: 3QNX, 3QNY) and IgG Fab (3: 3QNZ, 3QO0, 3QO1); MAP kinase LmaMPK10C from Leishmania major in complex w/inhibitor (3UIB, still unreleased). 3. Based on information from the Protein Data Bank, we are becoming one of the most productive centers in the region. Our figures in terms of deposited crystal structures solved and refined at the facility: 1 deposit in 2007; 4 in 2008; 8 in 2009; 6 in 2010; 5 (plus 1 not yet released) in 2011; 1 (plus 4 not yet released) so far in 2012. This does not include over 12 structures that have been solved and are currently being refined. As a comparative reference, in 2010, considering all depositions from Argentina added up to 11 structures, in Brazil 48, in Finland 27, in the Netherlands 26, in Ireland 2, in the USA 3364, among a few selected countries (PDB statistics 2011). 4. We have accepted short traineeship students from abroad : 3 facility users to collect X ray diffraction data (Sebastian Klinke, Jimena Rinaldi, Melissa Jacobs, FILeloir, Arg), with complementary support from CeBEM.

Publications 1. Musumeci MA, Botti H, Buschiazzo A, Ceccarelli EA. Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases. Biochemistry. 2011 Mar 29;50(12):2111-22. 2. Oppezzo P, Obal G, Baraibar MA, Pritsch O, Alzari PM, Buschiazzo A. Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: The carbohydrate binding mechanism involves residual sialidase activity. Biochim Biophys Acta. 2011 Sep;1814(9):1154-61. 3. Ortiz C, Larrieux N, Medeiros A, Botti H, Comini M, Buschiazzo A. Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Nov 1;67(Pt 11):1457-61. Epub 2011 Oct 27. Ongoing Grants 1. "Signal transduction in Leptospira virulence regulation: a multidisciplinary approach" - A Buschiazzo, Principal Investigator - Institut Pasteur, Paris, Actions Concertes Intra Pasteuriennes ACIP - 2011 (2 years).

2. Cell signaling in bacterial pathogenesis: iron metabolism regulation in Leptospira as a working model A Buschiazzo, Principal Investigator Institut Pasteur, Paris, Projet Transversal de Recherche PTR 2011 (2 years) 3. "Structural biology of the membrane sensor kinase from bacteria: a rotation mechanism of the alpha-helices" - F Trajtenberg (finishing PhD grant) Agencia Nacional de Investigacion e Innovacion ANII, Uruguay - 2010 (2 years) 4. Targeting the Leishmania kinome for the development of novel anti-parasitic strategies A Buschiazzo, collaborative partner (PI: Gerald Spaeth) European Union, Framework Program 7 2009 (3 years) 5. Virulence factors in the pathogenesis of Chagas Disease A Buschiazzo, Associate Researcher (PI: Oscar Campetella) National Institutes of Health NIH R01 2008 (4 years)

Other activities 1. Active contribution of our group to the consolidation of the Center for Structural Biology of the Mercosur (Centro de Biologia Estructural del Mercosur, CeBEM). www.cebem.org.ar The network now includes 9 nodes in Argentina, Brazil, Paraguay and Uruguay. CeBEM gathers one the most important group of laboratories engaged in Structural Biology in Latin America. 2. Organization of the III CeBEM Meeting, Institut Pasteur de Montevideo, Sep 6-7, 2011 3. PhD Thesis defense of our first graduate student in protein crystallography: Felipe Trajtenberg (May 30, 2011): "Allosteric mechanisms in the functional regulation of a bacterial thermosensor. The Jury included one of the world specialists in the field (P rof Alberto Marina, Valencia, Spain), delivering the highest academic grade. 4. F Trajtenberg postdoctoral training trip, under the supervision of Dr A Ko, School of Public Health, Yale University, New Haven USA (three months, 2011) 5. H Botti post-doctoral training trip, under the supervision of Dr M Picardeau, Biology of Spirochetes Unit, Institut Pasteur, Paris France (three months, 2011) 6. A Buschiazzo - Reviewer of >5 peer-reviewed journals, including PNAS, JACS, Glycobiology, J Med Chem, PLoS ONE, among others. 7. A Buschiazzo Associate Editor of the journal PLoS Neglected Tropical Diseases. 9. Our staff has attended to several national and international meetings and scientific activities, including as recent activities: cluster meeting of the consortium LEISHDRUG, European Union FP7 project (AB invited speaker; 2011 Rome, Italy); AB lecturer at the Depto Quimica Inorganica, Analitica y Quimica Fisica, Universidad de Buenos Aires (invited speaker; 2011 Buenos Aires, Argentina); XXII International Congress and General Assembly of the Internation Union of Crystallography (2011 Madrid, Spain); Grand Colloque Biologie & Sant, National Research Agency (ANR) (AB invited speaker; 2011 - Lyon, France); among others.

Unit: Head:

Transgenic and Experimental Animal Unit Martina Crispo, DVM

Members:

Geraldine Schlapp, MSc (Full time technician) Luca Goyeneche, Bach (Technician) Ana Paula Arvalo, Bach (Technician) Gabriel Fernndez, Bach (Technician, animal caretaker) Sergio Anchetta (animal caretaker) Ana Paula Mulet , MSc (research project member) Herjan Koopman, MSc (research project member) Veronica Gutierrez, MSc (Internship)

Research Our research team has concentrated his area of interest in the production of in vivo and in vitro embryos, embryo micromanipulation and the production of transgenic animals. During the last 10 years we have generated and applied this knowledge both in laboratory animals and in species of productive interest. The technologies that we have developed have interest in the field of biomedicine, pharmaceutical industry and animal production in the farming area. We have initiated a new research area related to the culture of murine embryonic stem cells and the effect of their microenvironment in the phenotype modulation of tumor cells. This research area is developed in collaboration with the Mouse Functional Genetic Unit of Institut Pasteur de Paris, the Cell Biology Unit and the Cancer Program of Institut Pasteur de Montevideo. Another research area is the generation of diverse transgenic murine models with the aim to study the function of genes linked to cancer, neurodegenerative or immune diseases, as well as the generation of transgenic sheep for the production of recombinant proteins. This last research area is developed in collaboration with Instituto de Reproduccin Animal de Uruguay (IRAUy) and the laboratory of Jean Paul Renard at INRA, Jouy en Josas, France. These activities represent an important support for the scientific structure of the region, since the Transgenic and Experimental Animal Unit provides genetically modified animals to different groups in the regional countries. Projects 2010 2012 2011 - 2013 2011 - 2013 2009 2011 Generation, characterization and posible aplication of a murine transgenic line reporter of quimioquine CXCL2. PI. Transgenic model development by nuclear transference of ovine somatic cells. Co - PI New alternatives of gamete criopreservation of female ruminants. Co-PI Development of a murine model of breast cancer from a tumoer cell line expressing HER-2/NEU. Co - PI

2011 - 2013

Differential genic expression during meiosis: identification and characterization of specific products in male meiotic prophase in rodents. Collaboration. Rol of circulating endothelial progenitors from bone marrow in the tumoral neovascularization of NHL. Murine model. Collaboration. Strenghtening of murine models plattforms in controlled environment for use in reserach, drug control and diagnostic. Collaboration. Creation of an International Consortium for preclinical evaluation of probiotic strains for diary food prototypes. Collaboration. Oxitocine and variability in parental behaviour. Collaboration. Trypanothione biosynthesis as a basis for drugs against trypanosomes. Collaboration. EMU: Adquisition of multi-users equipment for cryopreservation, transgenesis and education in the field of Laboratory Animal Science. Collaboration. Rol of IL-17 in immunoprotection of respiratory airways mucosae. Collaboration. Colon anticancer immunotherapy using Trypanosoma cruzi antigens. Collaboration.

2011 2013 2009 - 2012 2010 - 2012 2010 2012 2009 2011 2009 - 2011 2008 2011 2008 2011

Services 1. Generation of transgenic mice by pronuclear microinjection (3 projects finished - mCXCL2luc, mCXCL1-luc and OTR). Two projects waiting for financial support. 2. Generation of transgenic mice by homologous recombination in embryonic stem cells (2 projects finished - mgc-1203). Two projects waiting for DNA construction (A. Geisinger, G. Grompone). 3. Embryo and sperm cryopreservation (2 projects ended + 2 waiting). 4. Rederivation of mouse lines (3 project finished- Oct4 GFP, Tx/Ty, CCL20 + 1 project waiting). 5. Breeding and housing of SPF and conventional mice (C57BL/6J, BALB/cJ, DBA/2J, SWISS, SJL/J, Nude and 16 different transgenic lines). Actual production: aprox. 500/month. 6. Trials of biological activity for recombinant eritropoyetin (Lab. Clausen). (12 per month). The animal facility was certified by the Ministry of Health. 7. Trials of toxicity for biotechnological products (EPO, Filgen, Interferon) for Lab. Clausen (8 per month).

Publications Full article in peer review journals: 1. Roldn A, Comini MA, Crispo M, Krauth-Siegel RL. Lipoamide dehydrogenase is essential for both bloodstream and procyclic Trypanosoma brucei. Mol Microbiol. 2011 Aug;81(3):62339. Abstracts in peer review journals: 1. Schlapp G; L. Goyeneche; G. Fernndez; A Menchaca; M. Crispo Use of tolfenamic acid in recipient mice given at the moment of surgical embryo transfer. Transgenic research, 2011 30:11391189 2. M. Vilario; M. Crispo; A Pinczak; A Menchaca Association between sperm morphology and in vitro embryo production in mice. Reproduction, Fertility and Development 23(1) 217218.

Grants 1. Generacion, caracterizacion y posible aplicacin de una linea trasngenica murina reportera para la quimioquina CXCL2 Martina Crispo (PI) PRONIBIO (2009-2012). U$S 20.000. 2. Fortalecimiento de plataformas de produccin de biomodelos murinos en ambiente controlado para uso en investigacin, control de medicamentos y diagnostico Enrique Pochintesta (PI) and Martina Crispo (co-participant) ANII (2009-2012) USD 376.440. 3. Evaluacin de una vacuna peptdica basada en el oncogen HER-2/neu utilizando un modelo de cncer de mama murino que sobreexpresa HER-2/neu de origen humano Mariela Bollati (PI) and Martina Crispo (co-responsible). CHLCC (2009-2011). USD 10.000. 4. Desarrollo de modelos transgenicos mediante transferencia nuclear de celulas somaticas en ovinos Alejo Menchaca (PI) and Martina Crispo (Co-responsible). ANNI INNOVAGRO (2011 2013) U$S 60.000. 5. Nuevas alternativas para la criopreservacin de gametos en hembras rumiantes. Coresponsible. ANNI Maria Vias (2011-2013). U$S 20.000.

Other activities

Human resources formation Pedro Claudino dos Santos Neto - Transgnesis en ovinos. MSc Thesis (2011-2013), Facultad de Veterinaria - UDeLaR. (Co-Tutor) Robert Wijma Cabrera - Efecto de la vitrificacin de ovocitos y estadio de desarrollo al momento de la transferencia sobre la sobrevivencia de embriones ovinos producidos in vitro. Initiation ANII (2011-2012). Instituto de Reproduccin Animal de Uruguay. (Co-Tutor) Vernica Gutirrez Castro, DVM (Microsules Laboratory). Animal House Management (2011). Institut Pasteur de Montevideo. (Tutor) Herjan Koopman, MSc (University of Utrecht). Generacion, caracterizacion y posible aplicacin de una linea trasngenica murina reportera para la quimioquina CXCL2. Institut Pasteur de Montevideo. (Tutor) Teaching Course "Genetics of Laboratory Rodents" December 5th- 14th December 2011, Institut Pasteur de Montevideo. Seven lectures in postgraduate national and regional courses. Three presentations in international meetings. Other Member of Comite de Etica en el Uso de Animales (CEUA). Institut Pasteur de Montevideo (2009 -) Member of Comite de Etica en el Uso de Animales (CEUA). Facultad de Ciencias, UdelaR (2011- ) Member of Consejo de Instituto, Institut Pasteur de Montevideo (2011). Member of Scientific Comitee of Centro Multidisciplinario para Investigao Biolgica (CEMIB) Universidad de Campinas (2010 -). Member of Comisin Nacional de Experimentacin Animal (CNEA) (2010-2014). Member of Comisin de Evaluacin del Riesgo en Bioseguridad, MGAP (2009 -).

Unit: Head: Members:

Bioinformatics Unit Hugo Naya, PhD Martn Graa (PhD, AI) Natalia Rego (TA, MSc student in Zoology) Luca Spangenberg (contract, MSc in Bioinformatics) lvaro Pena (MSc student in Bioinformatics) Agustn Gonzlez (MSc student in Bioinformatics) Tamara Fernandez (BSc in biology, Electrical Engineering student) Sebastin Valenzuela (undergraduate student in Biology)

Research Our current research activities begin to acquire the profile suggested in our initial proposal. On the functional genomics side our efforts will be centered in three axes: a) analysis of expression data originated through microarray experiments, altogether with the UBM, and Next Generation Sequencing; b) analysis of microRNAs as well as of existing algorithms for target prediction; and c) the use of SNPs in Genome Wide Association Studies (GWAS). In what concerns structure-function relationships, our group is entering this field, with its longstanding conceptual and methodological (still open) questions. A central topic here is the role played by structural information in genome annotation and re-annotation, as seen from lab members' previous publications in Structural Genomics. Other directions are being launched which, despite incipient, are key to IP Montevideot in what concerns developing in-house scientific capacities in Structural Biology/Bioinformatics. The recent creation of a five years group working in molecular simulation of proteins and DNA, as well as the know-how linked to the crystallographic Unit, makes it feasible to tackle scientific questions from complementary points of views. For example, we are planning to provide Sergio Pantanos group with a list of potential microRNAs and their sequence targets, in order to perform calculations and evaluate theoretical affinities between them. Our hope is to gain insight into the 'ranking problem', as microRNAs and their targets do not follow strict Watson-Crick pairing rules and the algorithms are plagued with false positives and negatives. More generally, we hope that the structural eye recently integrated to our Unit will provide a balancing view to ours of what bioinformatics is and what are its limits. Within the Institute, the iterative process solved protein crystal structure; comparative and functional analyses through bioinformatics; molecular mechanics and dynamics simulations, seems a natural one. Given the appropriate question, the potential to give interesting answers seems high. Finally, an important part of our research will be centered in methodological developments in relevant areas for our international projects, more specifically, the application of NLP to biological research and data mining in information systems and software development for High Throughput Flow Cytometry.

Services 1. NCBI, EMBOSS, eBi suites locally installed. 2. Sequence alignment and phylogenetic inference software. 3. Sequence analysis software. 4. 3D molecular modeling software. 5. Database hosting and querying. 6. Tools for complex systems analysis. 7. Basic biostatistics and use of specific software advice. 8. Software development.

Publications 1. Persson H, Kvist A, Rego N, Staaf J, Vallon-Christersson J, Luts L, Loman N, Jonsson G, Naya H, Hoglund M, Borg A, Rovira C. Identification of new microRNAs in paired normal and tumor breast tissue suggests a dual role for the ERBB2/Her2 gene. (2011). Cancer Res. 71(1):78-86. Peagaricano F, Urioste JI, Naya H, de los Campos G, Gianola D. Assessment of Poisson, probit and linear models for genetic analysis of presence and number of black spots in Corriedale sheep. (2011). J Anim Breed Genet 128(2):105-13. Olivera-Couto A, Graa M, Harispe L, Aguilar PS. The eisosome core is composed of BAR domain proteins. Mol Biol Cell. 2011 Jul;22(13):2360-72. Spangenberg L, Battke F, Graa M, Nieselt K, Naya H. Identifying associations between amino acid changes and meta information in alignments. Bioinformatics. 2011 Oct 15;27(20):2782-9.

2.

3.

4.

5. Duhagon MA, Smircich P, Forteza D, Naya H, Williams N, Garat B. Comparative genomic analysis of dinucleotide repeats in Tritryps. Gene. 2011 Nov 1;487 (1):29-37. 6. F. Peagaricano, P. Zorrilla, H. Naya, C. Robello, and J.I. Urioste. Gene expression analysis identifies new candidate genes associated with the development of black skin spots in Corriedale sheep" acpetado en la revista: Journal of Applied Genetics 2012 Feb;53(1):99106. Epub 2011 Sep 28.

Grants

1. Bsqueda de un agente etiolgico viral de la leucemia linfoide crnica - Fondo Clemente Estable ANII Responsable: Guillermo Dighiero - 2010 - 18 Months Amount Granted USD 50.000 Research Associates: Hugo Naya and Pablo Oppezzo. 2. Genome-wide analysis of chromatin modifactions and gene experession profiles in human adult stem cells Fiocruz-Pasteur Mission - Hugo Naya 2011 24 months Amount Granted 10000 Euros Research Associate: Samuel Goldenberg.

Other activities We are currently involved in several teaching activities, mainly on bioinformatics-related topics. The recently created MSc in Bioinformatics is currently highly demanding, courses design and impartment being in charge of the Faculty of Sciences, School of Engineering, and our group at Pasteur. We also have punctual participations in several PEDECIBA courses, including topics in bioinformatics and quantitative genetics. Human resources are clearly needed in this somewhat new research domain; this calls for our effort in such teaching activities, as well as for maximizing the number of graduate and undergraduate students in our lab (eight persons at the moment).

Unit: Head:

Analytical Biochemistry and Proteomics Unit Carlos Cerveansky, PhD (IIBCE IP Montevideo)

Members:

Rosario Durn, PhD (Associate Investigator, IIBCE IP Montevideo) Carlos Batthyny, MD, PhD (Associate Investigator, Interim Head) Magdalena Portela (Technical Assistant, School of Sciences-IP Montevideo) Anala Lima, MSc (Technical Assistant, PhD student, IP Montevideo) Magdalena Gil (Technical Assistant, post-graduate student, IIBCE-IP Montevideo) Florencia Festari (Technical Assistant, IP Montevideo) Associate members: Mara Noel Alvarez, PhD (Associate Investigator, Adjunct Professor of Biochemistry, School of Medicine, Universidad de la Repblica, Uruguay)

The mission of the Analytical Biochemistry and Proteomics Unit (UByPA) at the Institut Pasteur de Montevideo is: 1. to perform and support mass spectrometry (MS)-based proteomic research, 2. to provide researchers with training, scientific assistance and access to MS and proteomic related technologies, 3. to improve available MS techniques for biomedical research, and 4. to contribute to local and regional training and education programs. Our specific goals are: provide open access to mass spectrometry and proteomics technologies to local and regional researchers, pursue biomedical research projects based on mass spectrometry and proteomic, facilitate collaborative scientific projects with other national and international research groups, supporting a join-effort to seek funding, provide state-of-the-art laboratory for education of graduate students and researchers in mass spectrometry and proteomics related technologies. Research In the past years, members of our research group have been involved in different areas of biological/biochemical research. A major contribution made by the UByPAs scientist was the incorporation of modern mass spectrometry (MS) and 2D-electrophoresis to our local academy, bringing totally new analytical capabilities to perform comprehensive protein studies, including posttranslational modifications of proteins and the ability to decode cell signaling networks. Proteomic approaches are contributing in full to understand protein function as expressed in normal and disease states. In this sense, our near future challenge is to improve our local capabilities to perform comparative-quantitative proteomics studies.

Now days, we are involved in two main areas of research concerning protein-mediated cell signalling events: 1. Proteomic profiling of host-pathogen interactions: A. Effects of Mycobacterium tuberculosis Ser/Thr kinase PknG on the macrophage. PR_FCE_2009_1_2479, PIs: Rosario Duran, Carlos Batthyany, 2011-2012. B. Characterization of the phosphoproteome and acetylome of phagosomes during the maturation process: effects of PKnG. PhD Thesis Project MSc. Anala Lima Raimondo, Pro.In.Bio., UdelaR, 2011-2013. 2. Oxidative stress-induced protein modifications: A. Nitration of Tyr residues: i. Detection and Characterization of Alternative Conformations of Citochrome c in Biological Systems. PR_FCE_2009_1_2486, PI: Dr. Rafael Radi, 2011-2012. ii. Nitroxidative and Functional Modifications of Prx2 from Human Red Blood Cells. Lia Randall Graduate Thesis Project. PI: Ana Denicola B. Electrophilic/Nitrated-fatty acids mediated protein modifications: i. Inhibition of PKnG of Mycobacterium Tuberculosis by Nitrated Fatty Acids. PIs: Rosario Durn & Carlos Batthyany. ii. Inhibition of PtpB of Mycobacterium Tuberculosis by Nitrated Fatty Acids. PIs: Carlos Batthyany & Andrea Villario C. Effect of Redox Environment on Rubredoxin-Domain Mediated PKnG Activity Regulation. Magdalena Gil Graduate Thesis Project, PIs: Rosario Duran, Ana Denicola; PEDECIBA Qumica, UdelaR; 2010-2012. Services Our Unit received in January 2007 a MALDI TOF-TOF MS instrument (AB-SCIEX, Framingham, USA) and, in December 2009, completed the MS platform with the arrival of a nano-electrospray/ion trap LTQ Velos instrument (Thermo, USA). Both instruments together complement each other and will expand the quality and type of mass analysis we offer to local and regional research groups through the Service, in fulfillment of a main goal of our technological Unit. Two main modes to get access to the unit are available: 1. Routine Service For routine analysis, users are welcome to access the UByPA as a fee for service facility supported by the Institut Pasteur de Montevideo. The facility offers this kind of service to researchers in the region, with priority given to users from the Institute and local academy. The analysis will be performed by members of our technical staff and will be done following standard protocols. The routine service includes analysis and interpretation of raw data based on routine practices only. Routine analysis includes: 2-D gel electrophoresis; Protein sample preparation for MS analysis: in-gel digestion, in-solution digestion, desalting; Molecular mass determination for peptides and small proteins by MS; Protein identification by MALDI-TOF/TOF MS (peptide mass fingerprinting, MS/MS ion search) and database search; Protein identification by nanoflow LC ESI tandem MS (nano LC-MS/MS) and database search.

In 2011 we have performed more than 4000 routine analysis from 750 different samples. Approximately 50% of the analyzed samples were from our local academy and the other 50% from regional academy (Argentine, Brazil, Chile, Colombia, and Venezuela). 37 different research groups from local academy and 28 different research groups from the region were users of our routine service during 2011. 2. Non-Routine Service The facility welcomes collaborative research on projects beyond routine services. In this case, members of the Unit are expected to significantly contribute to the conception, design of experiments and custom-design protocols, original ideas as well as data analysis and interpretation beyond routine practice. Non routine analysis includes: Custom sample preparation, Post-translational modification analysis, 2-D gel electrophoresis based proteomics, "shotgun" based proteomics, Quantitation, De novo peptide sequencing In any case, a great flexibility is pursued in order to agree on a case-by-case basis on the best way to use the facility. In 2011 we participated in several collaborative research projects mainly in the area of posttranslational modification analysis and shotgun proteomics. Main collaborations with local and regional academic partners are list below: Structural Study of PK10, a MAPK from Leishmania major. PI: A. Buschiazzo, Protein Crystallography Unit, IPMon. Proteomic Analysis of Bovine Leukemia Virus Particles. PI: O. Pritsch, Protein Biophysic Unit, IPMon. Functional and structural analysis of antigen B from Echinococcus granulosus. PI: Dra. A. Ferreira, Facultad de Ciencias, UdelaR, Uruguay. Phagocyte-specific S100 proteins in the local response to the Echinococcus granulosus larva. PI: Dr. A. Diaz, Facultad de Qumica, UdelaR, Uruguay. Exploring the Structural Details of Cu(I) Binding to -Synuclein. PI: Dr. C. Fernandez, IBR, Rosario, Argentina. Exploiting the cAMP pathway in Chagas Disease Therapy, Fogerty-NIH grant, PIs: Dr. D. Altschuler, University of Pittsburgh, PA, USA & Dr. M. Edreira, Universidad de Buenos Aires, Buenos Aires, Argentina. Post-translational modifications in Dengue Virus Capsid Protein. PI: Dr. A. Gamarnik. Molecular Virology Laboratory, Instituto Leloir, Buenos Aires, Argentina. Identification of specific antigens from Mycobacterium bovis whit potential application in diagnosis improvement. PI: Dra. M.I. Romano, Instituto de Biotecnologa, INTA, Argentina. Identification of phosphorylation sites in protein kinases from Streptococcus pneumoniae. PI: Dr. J. Echenique, Dpto. de Bioqumica Clnica CIBICI-CONICET, Universidad Nacional de Crdoba, Argentina.

Identification of acetylation sites in ppGalNAcT2. Dr. F. Irazoqui, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba, Argentina.

Publications

1. Exploring the Structural Details of Cu(I) Binding to -Synuclein by NMR Spectroscopy. Binolfi A, Valiente-Gabioud AA, Durn R, Zweckstetter M, Griesinger C, Fernandez CO. J Am Chem Soc. 133, 194196 (2011) 2. Proteomic survey of the cestode Mesocestoides corti during the first 24 hours of strobilar development. Laschuk A, Monteiro KM, Vidal NM, Pinto PM, Durn R, Cerveansky C, Zaha A, Ferreira HB. Parasitol Res. 108, 645-56 (2011). 3. Electrophilic Fatty acids regulate matrix metalloproteinase activity and expression. Bonacci G, Schopfer FJ, Batthyany CI, et al. J Biol Chem 286, 16074-81 (2011). 4. Adenosine triphosphate-dependent calcium signaling during ventilator-induced lung injury is amplified by hypercapnia. Briva A, Santos C, Malacrida L, Rocchiccioli F, Soto J, Angulo M, Batthyany C, Cairoli E, Piriz H. Exp Lung Res. 37, 471-81 (2011). 5. Pieyro MD, Parodi-Talice A, Portela M, Arias DG, Guerrero SA, Robello C. Molecular characterization and interactome analysis of Trypanosoma cruzi Tryparedoxin 1. J Proteomics. 74, 1683-92 (2011). 6. Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo versus in vitro conditions. D Alessandro B; Lery L; Von Kruger V; Lima, A.; Piccini C; Zunino P. FEMS. Imm Med Microbiol 63, 174-82 (2011). 7. Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: Biochemical characterization and identification of interacting partners through proteomic approaches. Lima A, Durn R, Schujman G, Marchissio MJ, Portela MM, Obal G, Pritsch O, de Mendoza D, Cerveansky C. J Proteomics 74, 1720-34 (2011).

Grants In Door Grants: 1. "Efectos de la Ser/Thr quinasa PknG de Mycobacterium tuberculosis en el macrfago: protemica de la interaccin husped-patgeno Fondo Clemente Estable- ANII (Uruguay) Rosario Durn/Carlos Batthyny 2011 (2 years).

Other Grants (scientist of the UByPA are participating as investigators):

2.

Deteccin y Caracterizacin de Conformaciones Alternativas del Citocromo c en Sistemas Biolgicos Fondo Clemente Estable- ANII (Uruguay) PI: Dr. Rafael Radi, Facultad de Medicina, UdelaR, 2011 (Call 2009) (2 years). PR_FCE_2009_1_2486. Estructura y funcin del antgeno B del parsito Echinococcus granulosus, Proyecto I+D Comisin Sectorial de Investigacin Cientfica (CSIC), PI: Dra. Ana Ferreira, Facultad de Ciencias, UdelaR, 2010-2012.

3.

Other activities 1. Training of three post-graduate students. 2. In-house Thermo training course: LTQ with ETD Operations (Dr. Detlef Schumann, Thermo Fisher Scientific, USA). One week internal training course (IPMont) (August 2011) 3. Post-graduate course Protein production, purification and characterization (PEDECIBA) Co-Organizers (May August 2011). 4. Magdalena Gil, training stay at the Unit de Microbiologie Structurale, Institut Pasteur, Paris (Dr. Pedro Alzari) (November 2011 January 2012). 5. Analia Lima, RIIP Traineeship Grant for three months stay at G5 Virulence Parasitaire, Departament of Parasitology and Mycology, Institut Pasteur, Paris (Dr. Gerald Spaeth). Training on 2D-DIGE technology (September-December 2011). 6. Magdalena Portela, training stay at Facultad de Biologa, Universidad de la Habana, Cuba (Dr. Luis Javier Gonzlez). Training in peptide de novo sequencing by Mass Spectrometry (December 2011). 7. Magdalena Portela, training course: Proteomics at Universidad de la Habana, Cuba (December 2011)

RESIDENT LABORATORIES

Cancer Program Institut Pasteur de Montevideo

Cancer Program
Laboratory 1: Functional Genomics - Research Program in Molecular Oncology Head: Alfonso Cayota, MD, PhD

Staff:

M Catalina Gida (Postdoctoral Fellow) M Rosa Silva (Doctoral Fellow) Juan Pablo Tosar (MSc Student) Braulio Bonilla (MSc Student) Florencia Cabrera (Undergraduate Student) Fernanda Bangueses (Undergraduate Student) Julia Sanguinetti (Undergraduate Student)

Research In the last 3 years, our main focus of research has been centered on the molecular and cellular events determining the initiation and progression of human cancer with special emphasis in chronic lymphocytic leukemia. Ongoing work deal with three main research areas: 1. ROLE OF MICRORNAs IN THE INITIATION AND PROGRESSION OF CHRONIC LYMPHOCYTIC LEUKEMIA Recently, a novel class of small noncoding RNAs dubbed microRNAs has been identified in plants and animals. Mature microRNAs are 19-22 nt in length and regulate target gene expression post-transcriptionally through base pairing with the 3 untranslated regions of the target messenger RNA inducing either degradation or translation inhibition. They participate in the regulation of developmental timing, cell death, cell proliferation and differentiation, fat metabolism and haematopoiesis. The finding that more than half of the known human microRNAs are located at cancer-associated regions of the genome suggests that microRNAs might play a broad role in cancer pathogenesis. In order to gain insight into the role of microRNAs in the initiation and progression of Chronic Lymphocytic Leukemia we have recently performed a cloning-based analysis of small RNAs in leukemic B cells from affected patients. Our results showed an almost complete loss of expression levels of several relevant microRNAs associated to the finding of new microRNAs candidates associated to the malignant phenotype. Currently we analyze the role of several cancer associated microRNAs in the malignant phenotype of leukemic B cells with eukaryotic expression vectors specially designed to express microRNAs. This project can afford emerging and relevant new mechanisms in cancer biology with a great potential in actual knowledge of B-CLL biology.

2.

MicroRNA-DEPENDENT CHROMOHELICASES AS A NOVEL TUMORIGENIC PATHWAY IN HUMAN CANCER In previous work, we succeeded to identify five novel microRNAs. In this proposal we intend to identify a novel tumorigenic pathway in human cancer in which these microRNAs appear to play an important role. In silico predictions showed that a significant proportion of mRNA targets of these novel microRNA mapped within a small region in the short arm of human chromosome 1 (1p311p36) which is frequently deleted in neuroblastoma and other neural-related malignancies. We speculate that these novel miRNAs could use as an effector arm the p19Arf/Mdm2/p53 pathway. The link between both molecular circuitries is represented by the recently identified Chromohelicase 5 (CHD5) which was demonstrated to act as a tumor suppressor via transcriptional activation of the INK4a-ARF locus. Surprisingly, CHD5 is a target shared by three out of these 5 novel oncogenic microRNA reported in this proposal. The validation of this tumorigenic pathway could provide new insights into the molecular mechanisms predisposing to human cancer and the identification of new therapeutic targets. We aim to identify a novel tumorigenic pathway which could be activated through a microRNAinduced deficiency of the chromatin-remodeling protein CHD5 expression. This could predispose to malignancy by crippling tumor suppressive pathways involving p16lnk4a, p19Arf and p53. We will try to establish an unrecognized role of hCHD5 in both facilitating transcriptional programs providing tumor suppression and as an alternative regulating pathway for the p19Arf/Mdm2/p53 axis. SMALL RNA PATHWAYS IN THE PROTOZOAN PARASITE TRYPANOSOMA CRUZI Over the last years an expanding family of small RNAs (i.e. microRNAs, siRNAs and piRNAs) was recognized as key players in diverse forms of gene silencing and chromatin organization. Effectors functions of these small RNAs are achieved through ribonucleoprotein (RNP) complexes containing at their center an Argonaute/Piwi protein. Although these proteins and their small RNA-associated machinery can be traced back to the common ancestor of eukaryotes, this machinery seems to be entirely lost or extensively simplified in some unicellular organisms including Trypanosoma cruzi, which are unable to trigger RNAi related phenomena. Speculating about the presence of alternate small RNA-mediated pathways in these organisms, we constructed and analyzed a size-fractionated cDNA library (2035 nt) from epimastigotes forms of T. cruzi. Our results showed the production of an abundant class of tRNA-derived small RNAs preferentially restricted to specific isoacceptors and whose production was more accentuated under nutritional stress. These small tRNAs derived preferentially from the 5 halves of mature tRNAs and were recruited to distinctive cytoplasmic granules. Our data favor the idea that tRNA cleavage is unlikely to be the consequence of non-specific degradation but a controlled process, whose biological significance remains to be elucidated. Detailed phylogenetic analysis of the small RNA-related machinery components revealed that they can be traced back to the common ancestor of eukaryotes. However, this machinery seems to be lost or excessively simplified in some unicellular organisms such as

3.

Saccharomyces cerevisiae, Trypanosoma cruzi, Leishmania major and Plasmodium falciparum which are unable to utilize dsRNA to trigger degradation of target RNAs. We recently reported a unique ORF encoding for an AGO/PIWI protein in T. cruzi which was expressed in all stages of its life cycle at the transcript as well as the protein level. Database search for remote homologues, revealed the presence of a divergent PAZ domain adjacent to the well supported PIWI domain. Our results strongly suggested that this unique AGO/PIWI protein from T. cruzi is a canonical Argonaute in terms of its domain architecture. We proposed to reclassify all Argonaute members from trypanosomatids as a distinctive phylogenetic group representing a new subfamily of Argonaute proteins and propose the generic designation of AGO/PIWI-tryp to identify them. Inside the Trypanosomatid-specific node, AGO/PIWI-tryps were clearly segregated into two paralog groups designated as AGO-tryp and PIWI-tryp according to the presence or absence of a functional link with RNAi-related phenomena, respectively.

Publications 1. Rovira, C., Guida, M.C. and Cayota, A. (2011) MicroRNAs and other small silencing RNAs in cancer. IUBMB Life 62, 859-68.

Grants 2. Expersin y rol funcional de microRNAs en el epitelio tmico en la regulacin de la maduracin linfocitaria - Bilateral Cooperation Agreement (Uruguay-Brasil) IP Montevideo (Alfonso Cayota)/Fiocruz (Wilson Savino) Amount Granted USD 15.000

Other activities 1. ORGANIZATION OF EDUCATIONAL AND TRAINING ACTIVITIES June 22 - 24, 2011 United States-Latin America Cancer Research Network Microarray Training Workshop August 29 September 2, 2011 Basic course of technologies of massive sequence and biology of small ARNs, Institut Pasteur de Montevideo co organization Carlos Robello, Alfonso Cayota

Laboratory 2: Tumor Immunology and Glycobiology Laboratory - Research Program in Molecular Oncology Head: Eduardo Osinaga, MD, PhD

Members:

Nora Berois (MD, PhD, Associate Investigator) Edgardo Berriel (MD, MSc, PhD student) Mara Florencia Festari (PhD student) Sabina Victoria (PhD student) Patricia Moerzinger (MSc, student) Diego Touy (MSc, student) Claudia Schvartzman (undergraduate student) Sofa Russo (undergraduate student)

Research 1. STRUCTURAL, FUNCTIONAL AND BIOLOGICAL STUDIES ON O-GLYCOSYLATED ANTIGENS AND GLYCOSYLTRANSFERASES ABNORMALLY EXPRESSED IN CANCER CELLS The most abundant form of O-linked glycosylation in higher eukaryotes, termed mucintype, is characterized by the covalent linkage of an -N-acetylgalactosamine residue (GalNAc) to the hydroxyl group of Ser/Thr residues. Mucin core O-glycosylation is catalyzed by a group of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases (ppGalNAc-Ts) (EC. 2.4.1.41). Subsequent elongation of O-linked sugar chains is achieved by the transfer of additional saccharide units, catalyzed by specific glycosyltransferases. Malignant transformation of epithelial cells is commonly associated with changes in the expression level and/or glycosylation pattern of mucins, including exposure of simple mucin-type carbohydrates, such as Tn, sialyl-Tn and TF antigens. These determinants contribute to the phenotype and biology of cancer cells and are involved in their metastatic activity. Moreover, they are considered among the most specific cancer-associated structures, and are thus being evaluated as promising targets for tumor immunotherapy. We have recently identified some apomucins and glycosyltransferases, which are abnormally expressed in certain cancer cells. One of these enzymes, ppGalNAc-T13, is probably associated to the aggressiveness of some tumors. In this project we investigate the molecular mechanisms underlying the regulation of the initial steps of mucin-type O-glycosylation in human cancer, and evaluate how this abnormal process influences malignant cell behavior. 2. CROSS-REACTIVE INTERACTIONS BETWEEN CANCER AND PARASITES: THE POSSIBLE INVOLVEMENT OF SIMPLE MUCIN-TYPE O-GLYCOSYLATED ANTIGENS In the last years, different groups independently reported that certain parasite infections reduced the incidence of cancer. For instance, in patients with hydatid disease the prevalence of cancer was significantly lower than in normal subjects. Furthermore, a lower level of colon cancer induced by 1,2-dimethylhydrazine has been reported in rats

chronically infected with Trypanosoma cruzi, compared to a non-infected control group. In the same direction, Balb/C mice infected with T. cruzi CH4 strain developed anti-tumor activity which inhibited the growth and metastasis of a subsequently transplanted solid L5178Y-R lymphoma. Presumably, tumor cells and these parasites expose similar antigenic determinants, and hence, some parasite antigens induced an effective cross-reacting immune response against cancer cells. This is in agreement with data reported by our laboratory regarding the expression of simple mucin-type antigens by some parasites. In particular, we have shown Tn and sialyl-Tn antigen expression in different parasites, raising the interesting possibility that these antigens act by altering the host susceptibility to cancer. On the basis of this hypothesis, we investigate whether these parasitic antigens are able to induce an effective immune response capable of inhibit the malignant tumors growth in animal models. 3. GLYCOPROTEIN ENGINEERING TOWARDS THE DESIGN OF ANTI-TUMOR VACCINES This project aims to develop a new approach for the production of glycosylated hemisynthetic vaccines for anti-tumor immunotherapy. Our strategy will be based on the directed enzymatic glycosylation of a mucin-like protein from parasitic origin. The proof of concept of this strategy will be obtained using the tumor-associated Tn antigen. Tn-based vaccines will be prepared by in vitro glycosylation of the mucin-type parasite C317 protein using several recombinant ppGalNAc-Ts, including those specifically expressed by cancer cells. The fact that C317 comes from an evolutionary distant organism should overcome tolerance issues encountered with human-mucin based cancer therapeutic approaches. If successful, this innovative procedure could have broad applications in the development of anti-tumoral and anti-parasite glycoprotein-based vaccines.

Publications 1. Hubert PM, Heitzmann A, Viel S, Nicolas A, Sastre-Garau X, Oppezzo P, Pritsch O, Osinaga E, Amigorena S. Antibody-dependent cell cytotoxicity synapses form in mice during tumorspecific antibody immunotherapy. Cancer Res. Cancer Research 71:5134-43 (2011). Patents 1. "An antibody specific for the tn antigen for the treatment of cancer" Authors: HUBERT PASCALE; AMIGORENA SEBASTIAN; SASTRE XAVIER; OSINAGA EDUARDO; PRITSCH OTTO; OPPEZZO PABLO; PEREZ FRANCK; MOUTEL SANDRINE. Facultad de Medicina (UdelaR), Uruguay. Institut Marie Curie, Francia. Date: 2010-01-15 International Patent number: A61K39/395; A61P35/00; C07K16/28; C07K16/30;G01N33/577. International European number: C07K16/30S

Grants 1. Design of anti-tumor vaccine by glycoprotein engineering, ICGEB CRP (Italy): Euros 45.000, 2009-2011. 2. Gnotypage des papillomavirus humains dans le cadre du dpistage organis du cancer du col de lutrus en Uruguay et en Cambodge. ACI RIIP. Euros 15.000. 2010-2011 3. Contribucin al desarrollo de la glicoprotemica en el Institut Pasteur Montevideo. Proyecto Transversal, Institut Pasteur Montevideo. U$S 20.000. 2010-2011

4. Targeting of the Tn antigen by a specific chimeric antibody in ovarian cancer immunotherapy. ANR France. Euros 15.000, 2011-2012

Other activities Thesis approved 3 - Leticia Monn Master student in Biology, Faculty of Sciences, University of the Republic, Uruguay. Title of the Thesis: Evaluacin de respuestas inmunitarias anti-tumorales inducidas por antgenos de Trypanosoma cruzi". Directors: Eduardo Osinaga and Teresa Freire. 4 - Vernica Noya Master student in Biology, Faculty of Sciences, University of the Republic, Uruguay. Title of the Thesis: Evaluacin de las propiedades anti-tumorales de un pptido tipo mucina de origen parasitario Directors: Teresa Freire and Eduardo Osinaga

Unit: Head:

Neurodegeneration Laboratory Luis Barbeito, MD

Members:

Hugo Peluffo (PhD, patternship with Faculty of Medicine, UR) Natalia Lago (PhD) Natalia Puig (Student) Andrs de Len (Student) Luciana Negro (Student, patternship with Faculty of Medicine, UR) Past members: Andrea Cragnolini (PhD, fellow from CONICET, Argentina) (until 2010) Javier Ganz (Student) Until 2009

Research 1. NEW NEUROTROPHIN SPECIES: THEIR ROLE IN NEURODEGENERATIVE DISEASES, AGING AND PAIN The neurotrophins are growth factors required by discrete cell types for survival and maintenance, with a broad range of activities in the nervous system and beyond. In 2006 we have described new species of NGF resulting from posttranslational modification of the mature neurotrophin resulting from nitration of tyrosine residues. Importantly, nitrated NGF form high molecular weight oligomers and display a gain-of-function through interaction with p75NTR. We propose nitrated NGF is formed during inflammation, playing a specific activity in nociception. The aims of the present research are to identify the specific activity of nitrated neurotrophins in different models including inflammatory pain and cornea wound healing and to develop neutralizing antibodies with potential therapeutic application. In collaboration with other groups within the IPM, we also attempt to determine the structural and physicochemical properties of nitrated NGF and characterize the binding to receptors. 2. CHARACTERIZATION OF ABA CELLS AND THEIR PATHOGENIC MECHANISMS AbA cells (from aberrant astrocytes) are a new type of glial cells recently isolated by our group from degenerating spinal cord from SOD1G93A rats and mice. These cells display high proliferative potential, inflammatory features and markers of un-differentiated astrocytes. Notably, AbA cells are highly and specifically neurotoxic to neurons, being pathogenic candidates for the progression of neurodegenerative diseases. The biology of AbA is being studied in the context of a collaborative study involving other local institutions. The aim of our research at the IP Montevideo is to characterize the transcriptome and secretome of AbA cells, using microarrays and mass spectrometry technologies respectively. Also, we attempt to identify markers to label these cells in different models of neurodegenerative diseases and to test whether or not these cells exert a pathogenic role.

3. DEVELOPMENT OF NEW VECTORS FOR GENE THERAPY TARGETING NEUROTROPHIN RECEPTORS The increasing knowledge in molecular pathology made possible the design of therapeutic strategies based on DNA or RNA delivery. These approaches must be biologically safe and guaranty a phenotypical impact. The most conventional vehicles, the genetically modified viral vectors, still present biosafety concerns. There is a need to explore new non-viral vehicles with low biological risks. The present project proposes the application of modular protein design through the rational combination of multiple functional domains. In particular, we are currently developing recombinant proteins that include functional domains combining neurotrophins motifs (to allow macroendocytosis in neurons and glial cells), RGD motifs, nuclear location signals, endosomal escape fusogenic peptides and basic domains. This research will be performed within the frame of an international collaborative program (leaded by Dr. Antoni Villaverde, UAB, Spain), that has pioneered the design and validation of this type of vectors. We have previously shown that they are able to transfer therapeutic DNA resulting in a therapeutic impact in vivo, with no acute inflammatory reaction. The resulting vehicles will be characterized quantitatively in in vitro models of nervous system through a range of properties including productivity, purification, selfassembly, stability, specificity, toxicity, endosomal escape, nuclear transport, uncoating and gene expression.

Publications 1. Diaz-Amarilla P, Olivera S., Trias E., Martinez-Palma L., Cragnolini A., Cassina P., Beckman J., Barbeito L. Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited ALS. Proc Natl Acad Sci U S A. 2o11 Nov 1;108(44):18126-31. 2. Peluffo, H; Al-Ruiz, D; Ejarque-Ortz, A; Heras-Alvarez, V; Comas-Casellas, E; MartnezBarriocanal, A; Kamaid, A; Alvarez-Errico, D; Negro, ML; Lago, N; Schwartz S Jr; Villaverde, A; y Says, J. Overexpression oftheimmunoreceptor CD300f has a neuroprotective role in a modelofacute brain injury. Brain Pathology 2011 Sep 22. doi: 10.1111/j.17503639.2011.00537 3. H. Peluffo, Modular Multifunctional Protein Vectors for Gene Therapy (In Press) . In: Nonviral Gene Therapy, Prof. Xubo Yuan Ed., Editorial INTECH, 2011, ISBN: 9789533075389. 4. Olivera-Bravo S, Fernndez A, Sarlabs MN, Rosillo JC, Casanova G, Jimnez M, Barbeito L. Neonatal astrocyte damage is sufficient to trigger progressive striatal degeneration in a rat model of glutaric acidemia-I. PLoS One. 2011;6(6):e20831. 5. Gonzalez P, Peluffo H, Acarin L, Villaverde A, Gonzalez B, Castellano B. Interleukin-10 overexpression does not synergize with the neuroprotective action of RGD-containing vectors after postnatal brain excitotoxicity but modulates the main inflammatory cell responses. J Neurosci Res. 2011 Sep 15. doi: 10.1002/jnr.22741.

6. Domingo-Espn J, Vazquez E, Ganz J, Conchillo O, Garca-Fruits E, Cedano J, Unzueta U, Petegnief V, Gonzalez-Montalbn N, Planas AM, Daura X, Peluffo H, Ferrer-Miralles N, Villaverde A. Nanoparticulate architecture of protein-based artificial viruses is supported by protein-DNA interactions. Nanomedicine (Lond). 2011 Aug;6(6):1047-61.

Grants

1.

Project PEIP-FOCEM-MIEM Neutralization of NGF and pain relief using horse polyclonal antibodies (2009-2011) Desarrollo de nanopartculas modulares recombinantes como vectores de terapia gnica para sistema nervioso lesionado ANII - Uruguay 2009 (2007 Call) 2 years Amount Granted USD 10.000 Project Collaboration Agreement- IP Paris/IP Montevideo/RECALCINE (Chile) Development of Mabs against nitrated NGF USD 300 000

2.

3.

YOUNG GROUP LEADERS LABORATORIES

Unit: Head:

G5 Epigenetics of Cancer and Aging Laboratory Ruben Agrelo, MD

Members:

Natalia Puig Alejandra Garcia Daniela Megrian

Research Our group is interested in the influence of epigenetics on cell proliferation, cell senescence and cell differentiation. We want to understand how epigenetics impacts cell and tissue aging, cancer progression and response to cancer therapies. 1. EPIGENETICS AT THE AGING-CANCER INTERFACE Abnormal epigenetic signaling plays a critical role in tumorigenesis. Epigenetic changes can also be important determinants of cellular senescence and organism aging. The best-defined epigenetic modifications are DNA methylation and histone posttranscriptional modifications. The best known examples are global loss of DNA methylation in aging and cancer and the promoter hypermethylation of genes with a dual role in tumor suppression and progeria, such as WRN and LMNA. In particular we are interested in exploring how epigenetic alterations are accumulated during aging and how these events contribute to the cell transformation process. 2. EPIGENETICS AND NUCLEAR MECHANICS A-type lamins are essential components of the nuclear lamina. Together with B-type lamins, they are the most prominent intermediate filaments forming this network of 10-nM diameter filaments located on the inner side of the nuclear membrane. LMNA has been found to be hypermethylated in hematological tumors. Moreover LMNA is mutated in aging related diseases such as Hutchinson Guilford progeria (HGPS). By the activation of a cryptic donor splice site progerin is expressed in HGPS cells affecting the epigenetic control of facultative and constitutive heterochromatin. We want to elucidate how these epigenetic modifications impact in nuclear mechanics. 3. EPIGENETIC CONTEXT OF CANCER PROGENITORS Using tools derived from the mammalian dosage compensation system are useful for defining the epigenetic context for Xist mediated silencing. This has led to the identification of an epigenetic context that is linked to the potential of Xist to induce chromosome-wide gene silencing in cancer progenitors. The aim of this project is to better characterize this cellular context and its components. As a consequence we expect to gain insight into normal cell biology and identify novel therapeutic targets in cancer.

Publications 1. Ruben Agrelo, X Inactivation and Progenitor Cancer Cells, Cancers 2011, 3, 2169-2175; doi:10.3390/cancers3022169.

Unit: Heads:

G5 - Cellular Membranes Laboratory Arlinet Kierbel, PhD and Pablo S. Aguilar, PhD

Members: Paola Lepanto (M.Sc., full time) Agustina Olivera-Couto (M.Sc. Student, full time) Laura Harispe (Postdoc, part time) Jessica Rossello (Undergraduate Student, part time) Lucilla Pizzo (Intership, part time) Milagros Mailhos (M. Sc. Student, part time) Federico Zancetta (Undergraduate Student, part time) Research Our main goal is to tackle fundamental problems in eukaryotic cell biology and human infectious disease. Focusing on internalization processes, we seek to understand how the cell organizes at its boundaries in order to maintain a continuous exchange of cargo and information. In our lab, we approach these subjects from two different approaches: 1. INTERACTION OF PSEUDOMONAS AERUGINOSA WITH EPITHELIAL CELLS (KIERBEL) Pseudomonas aeruginosa (PA) is a widely distributed environmental bacterium that causes acute infections in immunocompromized individuals and in those who have suffered serious injury. It ranks third among the most common hospital-acquired infections. Chronic infections in cystic fibrosis patients lead to high mortality rates. The formation of biofilms is a factor linked to respiratory infections in cystic fibrosis patients in which airways become blocked. Biofilms are aggregates of bacteria adhering to biotic or abiotic surfaces embedded in an extracellular matrix. This lifestyle is, in fact, predominant in nature and increasing evidence suggests that the formation of these multicellular structures plays a key role in bacterial infections in general. The transition from a planktonic lifestyle to one surface-associated and multicellular, a key step in the development of biofilms, remains poorly understood, specially when involves attachment to biotic surfaces. In our previous studies we observed P. aeruginosa often attached to the apical surface as bacterial aggregates. At the site of bacterial aggregate attachment on the cell surface, an epithelial cell membrane protrusion of considerable size is formed extending from this surface. This protrusion is enriched in the main product of PI3K, i.e., phosphatidylinositol 3,4,5-triphosphate (PIP3), basolateral plasma membrane components and actin. After longer infection periods, bacterial aggregates can be seen inside epithelial cells. We have also shown that the PI3K/AKT pathway is important for both protrusion formation and bacterial internalization (Kierbel et al 2005, 2007).

We focus on: A. How epithelial-associated aggregates are formed and through which mechanism do they enter into epithelial cells? We have shown that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cellcell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)-enriched host cell membrane protrusions. We further show that aggregates can be internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates Pseudomonas dependent PI3K activation, PIP3-enriched protrusion formation and aggregate internalization. We are currently studying the changes in P. aeruginosa signaling network that take place as bacteria face epithelial cells and rapidily transition from a free-swimming, highly-mobile plancktonic state to one sesile, multicellular and cell-attached. A manuscript has been published: Lepanto et. al Cellular Microbiology. Volume 13, Issue 8, pages 1212-1222, August 2011. B. Type III Secretion System (TTSS) regulation in the context of multicellular bacterial structures P. aeruginosa can inhibit its own internalization through the action of TTSS effectors. Such inhibition occurs with logarithmic phase grown P. aeruginosa since expression and secretion of TTSS and its effectors is enhanced during that growth phase. Using confocal microscopy we have measured internalization of both logarithmic and stationary phase grown P. aeruginosa distinguishing between individual and aggregated bacterial cells. Internalization of aggregates, but not individual bacteria, is strongly diminished with log phase grown bacteria. We are currently looking at activation of expression of type three secretion effectors in the context of multicellular bacterial structures. C. Setting up a system to analyze CFTR subcellular localization by fluorescence confocal microscopy (in collaboration with Mnica Marn from Universidad de la Repblica) Cystic Fibrosis (CF) is the most frequent genetic disorder in Caucasians, caused by mutations in CFTR gene, which encodes for the chloride channel Cystic Fibrosis Transmembrane Conductance Regulator. This protein is an ATP-binding Cassette superfamily member and it is normally expressed in the apical membrane of epithelial cells. To date, 1600 mutations have been identified in the gene with an important number with unknown functional effects. Consequently, the interpretation of the phenotypic effect of these changes has become a challenge in genetic counseling. One possible consequence of CFTR mutations is the alteration of its subcellular localization, as has been evidenced for the most frequent CF mutation, pF508del, which is sequestered in the endoplasmic reticulum by its quality control system. Our aim is to design and optimize a cellular system

to assess the subcelullar localization of CFTR mutants with unknown functional effects. For this purpose, GFP fusion constructs will be tested in polarized epithelial cells using fluorescence confocal microscopy. Apical membrane localization will be defined by colocalization with an apical membrane marker and quantified with the image processing program Image J. Two forms of CFTR have been already constructed and assayed: wild type CFTR and pF508del. Two sets of mutants will be tested in this system: those available from patients with atypical CF, and synonymous changes selected from the analysis of CFTR codon usage. 2. EISOSOMES AND PLASMA MEMBRANE ORGANIZATION (AGUILAR) Eisosomes define sites of endocytosis in Saccharmoyces cerevisiae (Nature, 439: 998-1003, 2006). These structures, lay underneath the plasma membrane and are mainly composed by thousands of copies of two paralog proteins Pil1 and Lsp1 (Mol Bio Cell, 20, 809-818, 2009). Eisosomes are plasma membrane domain organizers that concentrate lipids and proteins around them. In doing so, they are thought to help coordinate spatial and temporal execution of diverse plasma membrane functions such as small molecules transport, signaling and bulk endocytosis (EMBO J., 26, 4946-4955, 2007; J Cell Biol. 185:1227-1242, 2009). We focus on: A. What is the physiologcal role of eisosome-generated domains? To reveal functional links between eisosomes and their domains, we generated a quantitative genetic interaction map targeting a large set of genes implicated in plasma membrane function. In collaboration with Nevan Krogan (University of California San Francisco, USA) and Tobias Walther (Max Planck Institute, Martinsried, Germany) we employed the E-MAP (epistatic mini-array profile) approach. We have quantified a total of 57,799 genetic interactions among 374 genes. Using this Plasma Membrane E-MAP, we have linked two new genes (EMP70 and EIS1) to eisosome function and uncovered a link between Rom2-signaling and sphingolipid metabolism. A manuscript has been published: Aguilar et al., Nat Struct Mol Biol, 17: 901-908, 2010. B. How eisosomes organize plasma membrane domains? The list of proteins that encompasses eisosomal components contains no recognizable molecular function. Thus, at first glance, eisosomes are big black boxes. To get an insight in eisosomes molecular function we have executed an exhaustive bioinformatic analysis of all known eisosomal proteins. We found that Pil1 and Lsp1 belong to a superfamiliy of proteins that work as membrane shape modifiers. Site-directed mutagenesis experiments followed by in vivo analysis of eisosomes and plasma membrane organization support a model in which Pil1 and Lsp1 bind to the plasma membrane as filaments that generate regions of negative curvature. In addition, we also found that four proteins which co-localize with eisosomes bear membrane curvature-sensing domains. Overall, our results suggest that bending of the plasma membrane is a mechanism that eisosomes utilize to recruit a subset proteins and therefore to generate domains.

A manuscript has been published: Olivera-Couto et al., Mol Bio Cell., 22: 2360-2372, 2011.

3. CELL-CELL FUSION (AGUILAR) Eukaryotic cells use intracellular membrane fusion events to move cargo through the various compartments while maintaining compartmental identity. Membrane fusion also occurs between cells, for example when gametes fuse their cell membranes to form zygotes and myoblasts fuse during the development of skeletal muscle, and for virus/cell fusion when enveloped viruses deliver their genome into host cells for infection. All known membrane fusion reactions are catalyzed by proteins collectively referred to as fusases. Remarkably, to this day, only intracellular and viral fusases have been identified and characterized. Cell-cell fusion, despite being investigated for more than 150 years, stands as the field where the fusases still remain elusive. During my postdoctoral work I used yeast mating as a model of cell-cell fusion. We identified new proteins involved in this process and elucidated the role that extracellular calcium plays in cell fusion and cellular lysis (Mol Biol Cell. 18, 547-556, 2007). We also determined the structural role that sterols play during cell polarization and cell fusion (Proc Natl Acad Sci USA., 107: 4170-4175, 2010). Overall, our results support a model in which the still unidentified cell fusion machinery promotes both: fusion and lysis. In collaboration with the Bioinformatics Unit of the IP Montevideo we have designed a battery of genetic and bioinformatic screens to identify cellular fusases. Using biochemical and robotic methods we execute genomic screens aimed to identify genes that mediate cell lysis during mating. Preliminary results validate this approach uncovering genes involved in pheromone-induced calcium uptake as lysis enhancers. -This work is funded by ICGEB-CRP-URU11-01, starting Jan 2012 over 3 years, 57.000 total. 4. DEVELOPMENT OF YEASTS WITH INSECTICIDAL CAPACITY (AGUILAR) Development of new biocontrol agents is a valuable alternative to the vast use of chemical pesticides. In the recent years, the use of different yeasts as biocontrol agents has been widely reported. In collaboration with Celia Carlini (Universidade Federal do Rio Grande do Sul, Brasil) and Enrique Castiglioni (Estacin Experimental Dr. Mario A. Cassinoni, Universidad de la Repblica, Uruguay) we are developing yeast strains that produce the plant-derived entomotoxic peptide Jaburetox. With this project we expect to develop new biocontrol agents that can be used against different insects that affects soybean, cotton and corn production. This work is funded by ANII-FSA-INNOVAGRO 2009, starting Nov 2010 over 2 years, US$ 53.000 total.

Publications 1. Elwell CA, Kierbel A, Engel JN. Species-specific interactions of SRC family tyrosine kinases regulate Chlamydia intracellular growth and trafficking. mBio 2(3):e00082-11. doi:10.1128/mBio.00082-11, 2011.

2. Olivera-Couto A, Graa M, Harispe L, Aguilar PS. The eisosome core is composed of BAR domain proteins. Mol Biol Cell. 2011 Jul;22(13):2360-2372. 3. Lepanto P, Bryant DM, Rossello J, Datta A, Mostov KE, Kierbel A. Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism. Cellular Microbiology. 2011 Volume 13, Issue 8, pages 1212-1222, August 2011. Grants 1. Proteomic approach to identify mediators of PI3K activation by P. aeruginosa. Arlinet Kierbel - N.I.H.- Fogarty International Research Collaboration- Basic Biomedical Sciences Research Award (FIRCA-BB), U.S.A. USD 90.000 - 2008 (3 years). Granted to Arlinet Kierbel. 2. "Desarrollo de levaduras insecticidas". Fondo Sectorial Agropecuario 2009- ANII. 20112012. Granted to P. Aguilar. - USD 53.150. 3. Fondos Clemente Estable ANII Investigacin Bsica, 2010 (24 months) $u 252.000 Paola Lepanto. 4. "Yeast Mating as a model of Cell-Cell Fusion". International Centre for Genetic Engineering and Biotechnology CollaboratIve Research Programme (ICGEB-CRP), 2012-2014. Granted to P. Aguilar. 57.000. 5. Yeast Systems Biology International Course and Symposium November 7th - 22nd 2011 Institut Pasteur de Montevideo, 2011. Institut Pasteur International Network (RIIP), International Union of Biochemistry and Molecular Biology (IUBMB), United Nations University (UNU-Biolac), Ministerio de Ciencia y Tecnologa Argentina (MINCyT), Programa de Ciencias Bsicas (PEDECIBA) Uruguay, Granted to P. Aguilar. Total 70.000 USD. Human Resources Training Paola Lepanto, Masters degree. Ciencias Biolgicas. Opcin Biloga Celular y Molecular. PEDECIBA August 16th, 2011. Advisor: Arlinet Kierbel

Other activities 1. Yeast Systems Biology International Course and Symposium November 7th - 22nd 2011 Institut Pasteur de Montevideo, 2011. (Aguilar, organizer). 2. 14th Bay Area Microbial Pathogenesis Symposium, University of California San Francisco, San Francisco, U.S., March 2011 (Kierbel, speaker) 3. International Course "INTERPLAY BETWEEN PATHOGENS and HOST CELL" organized by Dr. Marisa Colombo. Mendoza, Argentina. August 2011. (Kierbel, speaker)

Unit: Head:

G5 - Molecular and Human Genetics Laboratory Jos Badano, PhD

Members:

Florencia Irigon, PhD (Research associate) Victoria Prieto, PhD (Postdoctoral Fellow) Magdalena Crdenas, MSc (PhD student) Cecilia Gascue, MSc (PhD student) Rossina Novas, Bach (MSc student) Beln Torrado (final laboratory work for her bachelor's degree)

Research The lab is focused in the study of ciliary biology, and more specifically how this organelle and the proteins that compose it modulate cell fate decisions such as whether to proliferate or differentiate. Ciliary dysfunction underlies the pathogenesis of a broad group of human disorders collectively known as ciliopathies that are characterized by a number of phenotypes including retinal degeneration, cystic kidney disease, obesity, and diabetes (1, 2). One example of a ciliopathy, and the model we study in the lab, is Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder for which 16 genes have been cloned to date (Refs. (3-6) and references within). The BBS proteins tested to date localize to centrosomes, basal bodies and in some cases the ciliary compartment and defects in this group of proteins can result in both structural and functional ciliary defects (7-12). Moreover, we have shown that the basal body, and in particular the BBS proteins, participate in the transduction of the Wnt signaling pathway where they modulate the balance between the canonical/ -catenin dependent and non-canonical or planar cell polarity (PCP) pathways (12, 13). However, the exact role of these 16 BBS proteins in the context of the cilium and/or outside of it is still poorly understood. Therefore, we have decided to use BBS as a model to study the biology of cilia and their role in signal transduction to modulate cell fate. Additionally, BBS is an oligogenic disorder whereby mutations at more than one locus collaborate to modulate disease outcome and thus it also represents an opportunity to understand oligogenic inheritance at a cellular and molecular level (14-16). Importantly, we have expanded the spectrum of genetic interactions by showing that they can also involve genes associated with distinct clinical entities. We have shown that mutations in Meckel Gruber (MKS) genes, a perinatal lethal ciliopathy, can either cause BBS or modulate its presentation therefore posing the question of why mutations in a given gene can result in two seemingly distinct disorders (4). Through an in vivo evaluation of the pathogenic potential of different MKS mutations we provided at least a partial answer to this question by showing that the BBS-associated mutations in MKS genes are invariably hypomorphs whereas the mutations in these genes in MKS affected individuals are complete nulls (4). Therefore it is not only the gene mutated but also the type of mutation what appears to determine the final phenotypic outcome. Based on this hypothesis we have collaborated with the group of Dr. Katsanis in an effort to assess the pathogenic effect of an extensive panel of BBS missense mutations that

have been associated with complex inheritance. We have used immunocytochemistry assays to analyze their effect on the cellular localization pattern of the affected proteins, thus complementing Dr. Katsaniss in vivo studies in zebrafish. This collaboration has led to two manuscripts (17, 18). In the lab, we started the characterization of some of the BBS proteins mainly through the identification and characterization of the complex in which they participate. Originally we started focusing our studies on two proteins: BBS7 and MGC1203/CCDC28B (from now on CCDC28B). We selected BBS7 because i) we know that it interacts with other BBS proteins and interactors suggesting that it might be a central component of the complex, ii) it was originally cloned by virtue of its similarity to BBS1 and BBS2 and thus the information gained from this analysis might be extended to these other BBS proteins and iii) there was no information available regarding its function. Using a yeast two-hybrid approach we detected and interaction between BBS7 and a member of the polycomb group (PcG), a protein that is known to regulate transcription through chromatin remodeling. Originally we were extremely interested in this interaction because the activity of polycomb proteins have been indirectly linked to the expression of -catenin, the effector of the canonical Wnt signaling pathway and thus our findings could represent an entry-point to better understand the role of the BBS proteins in Wnt signaling regulation (19, 20). In addition, given that polycomb proteins are nuclear and the vast majority of BBS proteins analyzed up to that moment were reported to localize to centrosomes and basal bodies, it posed the question of first whether this interaction is physiologically relevant and if so, when and where in the cell it takes place. Since that original finding we have now confirmed that the interaction between BBS7 and RNF2 occurs in mammalian cells and it is likely not restricted to BBS7 but involves other BBS proteins. We have shown, as expected, that BBS7 does also localize to basal bodies, centrosomes and at least in some cells, the ciliary axoneme. However, we demonstrate that BBS7, as well as other BBS proteins, is also present in the nucleus. An in silico analysis of the different BBS proteins does not predict any nuclear localization signal but does show the presence of nuclear export signals (NES) in all of them with the possible exception of BBS8. These observations raised the possibility that BBS7 might localize at least transiently to the nucleus. We used a combination cell fractionation studies, immunocytochemistry and site-directed mutagenesis to show that indeed BBS7 is able to enter the nucleus but appears to be actively exported out of it. The next step was to gain insight into the physiological relevance of this interaction. We have been both overexpressing and depleting (siRNA assays) BBS7 to study its effect on the levels of mRNA and protein, the localization patterns and the functionality of the PcG member and vice versa. We have shown that depletion of BBS7 results in increased levels of its interactor, at least at the protein level, and that importantly this effect appears to result in the misregulation of several of its target genes, measured both by real time PCR and a microarray analysis of cells depleted for BBS7. To validate our in vitro results we took advantage of our collaboration with Dr. Katsanis and performed mRNA in situ hybridizations in Danio rerio (zebrafish) embryos that were depleted for BBS7 and other BBS proteins where we confirmed that the level and pattern of expression of different RNF2 target genes are altered in the morphant animals. Importantly, several of the misregulated genes play key roles during development, organogenesis and tissue maintenance, such as the Hox genes and other homeodomain-containing transcription factors (21). Therefore, our studies might provide important insight to understand the cellular basis of

the different phenotypes that characterize BBS and possibly other ciliopathies. In addition, our microarray data indicates that BBS7 might play a broad role in gene regulation, not only through RNF2, thus potentially representing a direct link between ciliary/basal body function and the nucleus. This work has been accepted and is currently in press in the Journal of Cell Science (22). Based on these results with BBS7, we now plan to focus part of our research to understand the mechanisms that regulate the subcellular localization of both BBS and other cilia-associated proteins. The controlled translocation of proteins between cellular compartments is a process used by different cascades during signal transduction and little information is available regarding how the BBSs and ciliary proteins in general are targeted to the cilium or to other compartments. In addition, recent reports have highlighted striking similarities in the mechanims of nuclear and ciliary transport (23). In this context, we are developing an interdisciplinary collaboration with other units at the IPMon (UByPA and UBI) where we plan to use a combination of cell/molecular biology, mass spectrometry and bioinformatics approaches to start tackling an important problem in the field of signal transduction, which is trying to understand how proteins are directed to specific subcellular compartments, in particular exploring the cilia-nucleus connection. Another important research line in the laboratory has been centered on the characterization of MGC1203/CCDC28B (coiled-coil domain containing protein 28b), a novel protein of unknown function that interacts and colocalizes with the BBS proteins at the basal body and centrosome of cells and that we have recently identified as a second site modifier of the BBS phenotype (15). Using a yeast two-hybrid assay we identified several proteins directly involved in the regulation of cell division, and observation that resulted extremely interesting to us given the link between cilia and cilia-dependent signaling and the regulation of cell proliferation and differentiation. Of special interest was an interaction between CCDC28B and a critical player of the mTOR pathway. The mTOR signaling cascade is a central regulator of cellular homeostasis, integrating different extracellular and intracellular stimuli to modulate cell responses that determine growth and proliferation (24). In addition, misregulation of the mTOR pathway has been causally linked to the pathogenesis of cystic kidney disease, a BBS phenotype and a hallmark of the ciliopathies (25). We have designed a two prone approach to assess the function of CCDC28B both in vivo and in vitro. We are generating a mouse model in which Ccdc28b will be ablated by homologous recombination in ES cells. Currently, we are confirming the correct integration of our construct in chimeric animals. Importantly, the targeting construct has been design with a combination of LoxP and Frt sites that will allow us to generate both complete and tissue specific knockout lines as desired. We have also used the zebrafish model through a collaboration with Dr. Phillip Beales at University College London, taking advantage of the use of morpholinos to knockdown protein function. Importantly, we have now completed the installation of a zebrafish facility here at the IPMon that has been financed in part by internal funds (interdisciplinary projects) and a donation from the Ministry of Industry. In vitro, we have been studying the functional relationship between CCDC28B and the mTOR pathway. We have been performing a series of experiments in which we both overexpress and deplete CCDC28B through RNAi in mammalian cell lines to then monitor the effect on expression at the mRNA and protein level of its interactors and downstream targets as well as post-translational modifications that are critical during signal transduction of the mTOR

pathway (i.e. AKT phosphorylation). In this project we have obtained a series of novel and interesting results that we have also validated in vivo. This work is currently being submitted for publication. Bibliography 1. 2. J. L. Badano, N. Mitsuma, P. L. Beales, N. Katsanis, The Ciliopathies: An Emerging Class of Human Genetic Disorders. Annu Rev Genomics Hum Genet 22, 125 (2006). M. Cardenas-Rodriguez, J. L. Badano, Ciliary Biology: Understanding the Cellular and Genetic Basis of Human Ciliopathies. Am J Med Genet Part C Semin Med Genet 151C, 263 (2009). S. K. Kim et al., Planar Cell Polarity Acts Through Septins to Control Collective Cell Movement and Ciliogenesis. Science 329, 1337 (2010). C. C. Leitch et al., Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. Nat Genet 40, 443 (2008). E. A. Otto et al., Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy. Nat Genet 42, 840 (2010). C. Stoetzel et al., Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome. Am J Hum Genet 80, 1 (2007). S. J. Ansley et al., Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome. Nature 425, 628 (2003). O. E. Blacque et al., Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport. Genes Dev 18, 1630 (2004). J. C. Kim et al., The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression. Nat Genet 36, 462 (2004). J. B. Li et al., Comparative genomic identification of conserved flagellar and basal body proteins that includes a novel gene for Bardet-Biedl syndrome. Cell 117, 541 (2004). M. V. Nachury et al., A Core Complex of BBS Proteins Cooperates with the GTPase Rab8 to Promote Ciliary Membrane Biogenesis. Cell 129, 1201 (2007). A. J. Ross et al., Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates. Nat Genet 37, 1135 (2005). J. M. Gerdes et al., Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response. Nat Genet 39, 1350 (2007). J. L. Badano et al., Heterozygous mutations in BBS1, BBS2 and BBS6 have a potential epistatic effect on Bardet-Biedl patients with two mutations at a second BBS locus. Hum Mol Genet 12, 1651 (2003). J. L. Badano et al., Dissection of epistasis in oligogenic Bardet-Biedl syndrome. Nature 439, 326 (2006). N. Katsanis et al., Triallelic inheritance in Bardet-Biedl syndrome, a mendelian recessive disorder. Science 293, 2256 (2001). L. de Pontual et al., Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease. Proc Natl Acad Sci USA 106, 13921 (2009).

3. 4. 5. 6.

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15. 16. 17.

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N. A. Zaghloul et al., Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome. Proc Natl Acad Sci U S A 107, 10602 (2010). A. Tuckfield et al., Binding of the RING polycomb proteins to specific target genes in complex with the grainyhead-like family of developmental transcription factors. Mol Cell Biol 22, 1936 (2002). Y. Xu et al., Intracellular domains of amyloid precursor-like protein 2 interact with CP2 transcription factor in the nucleus and induce glycogen synthase kinase-3beta expression. Cell Death Differ 14, 79 (2007). L. A. Boyer et al., Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349 (2006). C. Gascue et al., A Direct Role of Bardet-Biedl Syndrome Proteins in Transcriptional Regulation. J Cell Sci In Press, (2011). J. F. Dishinger et al., Ciliary entry of the kinesin-2 motor KIF17 is regulated by importinbeta2 and RanGTP. Nat Cell Biol 12, 703 (2010). K. G. Foster, D. C. Fingar, Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem 285, 14071 (2010). J. M. Shillingford et al., The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease. Proc Natl Acad Sci U S A 103, 5466 (2006).

Publications

1. Gascue C, Katsanis N, Badano JL. Cystic diseases of the kidney: ciliary dysfunction and cystogenic mechanisms. Pediatr Nephrol 2011 26:1181-1195. 2. Irigon F, Badano JL. Keeping the balance between cell proliferation and differentiation: the primary cilium. Curr Genomics 2011 12:285-297. Grants 1. The role of MGC1203 a second site modifier of Bardet-Biedl syndrome in cell fate decision Jos Badano 2008 (3 years) - Genzyme - USD 150.000 2. Doctoral Fellowship - Cecilia Gascue 2009 (3 years) ANII 3. Doctoral Fellowship Magdalena Crdenas 2009 (3 years) ANII 4. Master Fellowship - Rossina Novas - 2011 (2 years) - ANII 5. Fondo Clemente Estable - Jos Badano - 2011 (2 years) ANII 6. Research Initiation Fellowship - Beln Torrado 2011 (1 year) - ANII

Other activities As mentioned above, we have installed a zebrafish facility in the IPMon that will not only benefit our own laboratory but will catalyze collaboration with other units at the IPMon and researchers at the School of Science, UdelaR.

Undox Biology of Trypanosomerelo Comini, PhD Unit: Head: Redox Biology of Trypanosomes Laboratory Marcelo Comini, PhD

Members:

Andrea Medeiros (Postdoc) Bruno Manta (PhD Student) Cecilia Ortz (PhD Student) Luca Fiestas (MSc Student) Mariana Curto (MSc Student) Luciana Fleitas (Undergraduate student) Diego Benitez (Technical Assistant)

Research On June 2008 the 5-year-group Redox Biology of Trypanosomes has been established at the IP Montevideo. The research areas of the group encompass basic and more applied aspects of the thiol-dependent redox networks of pathogenic trypanosomes, which are responsible for causing highly disabling and often fatal diseases of human and live-stock (i.e. African sleeping sickness, Chagas disease, black-fever, Oriental Sore, Kala-Azar and Nagana cattle-disease). Unfortunately, there is no prospect for vaccines due to the capacity of the parasites to evade the host immune response and the treatment available suffers from low efficacy, safety and affordability. Therefore, the development of novel drugs and/or therapies is considered a high priority on this field. In this respect, the biochemistry of the trypanosomatids presents some unusual features that can be exploited towards this goal. For instance, in these parasites, the thiol-redox metabolism exclusively depends on a triad absent in mammals: the thiol-polyamine conjugate trypanothione, the flavoenzyme trypanothione reductase and the multipurpose oxidoreductase tryparedoxin. The so called trypanothione system fuels with electrons important cellular functions such as the DNA replication and the antioxidant system. Previous works of the group leader made important contributions to the understanding of the biological significance and biochemical properties of several proteins from the thiol-redox metabolism of trypanosomes (Comini et al. 2004, 2005, 2007, 2008). The main goal of our young group is to deepen the molecular and biological characterization of drug targets associated to this metabolism and, whenever possible, to translate this information into the development or improvement of novel and/or existing therapies. Our studies make focus on proteins involved in the production of important redox cofactors or depend on trypanothione to fulfill essential cellular functions. The chemical and biological validation of compounds/drugs and functional analysis of parasite proteins is carried in an animal model of infection.

Four main subjects constitute the core of our research activities: 1. THE BIOSYNTHESIS AND REGENERATION OF TRYPANOTHIONE We have demonstrated the importance of trypanothione synthetase for the establishment of infection and severity of disease by African trypanosomes in a rodent model (Medeiros et al. unpublished). We are now studying the relevance of a redox active intermediate in the biosynthesis of trypanothione as well as other potential compensatory mechanisms activated under trypanothione-depleted conditions. The biosynthetic pathway operating in the American trypanosome (T. cruzi, etiologic agent of Chagas diseases) is currently being disclosed and we will attempt to validate the indispensability of the enzyme by genetic manipulation (knock-out strategy). Our group has recently (March 2011) become reference lab. of the CM0801 European-COST consortium for the screening of compounds with activity anti-trypanothione synthetase. The studies involve inhibition, cytotoxicity and, eventually, in vivo assays with lead compounds. With these investigations, we expect to facilitate the development and/or optimization of more potent inhibitors against these enzymes. The regeneration of the reduced for of trypanothione requires NADPH, which is mainly supply by the reactions of the oxidative branch from the pentose phosphate pathway. In this respect our studies focus on the first enzyme of this pathway, glucose-6-phosphate dehydrogenase. We have recently completed the biochemical characterization of the T. cruzi enzyme and solved its 3D structure (collaboration with Dr. Buschiazzo, IP Montevideo; Ortz et al., 2011), which represents the first structure reported for an enzyme of a pathogenic organism. Our current research pursues to determine the atomic details that distinguish this enzyme from the human orthologue in order to establish the basis for a rational drug design. 2. THE ROLE OF MONOTHIOL GLUTARREDOXINS IN METAL AND REDOX HOMEOSTASIS By means of a multidisciplinary approach we aim to unravel the molecular and biological properties that distinguish the trypanosomal proteins from classical glutaredoxins and orthologues from other organisms as well as to provide a better understanding of the role these proteins play in iron sulfur cluster biogenesis and/or redox homeostasis. Special interest has been put on the mitochondrial monothiol glutaredoxin 1, a protein that is essential for T. brucei in vivo (animal model). We have recently solved the NMR structure of the protein, the first reported for pathogenic protozoa, in collaboration with Dr. Bellanda (Padova University, Italy) and have identified important structural and mechanistic differences with the human counterpart (Manta et al. in preparation). Based on our findings that altering iron homeostasis is detrimental for parasite survival in a mammalian host we have implemented a new therapeutic strategy with very promising results. Our aim now is to define a therapeutic regime and drug-combined therapy, as well as elucidate the specific role of the protein in vivo. The development of specific inhibitors is under our scope. 3. IN VIVO AND IN VITRO DRUG TARGET VALIDATION Within our program of early-phase drug discovery we are conducting the validation and functional characterization of different drug targets and compounds in a mice infection model of African trypanosomiasis. We have recently demonstrated the essentiality of lipoamide dehydrogenase for T. brucei (Roldn et al. 2011) and have several other

candidates in the validation pipeline: the parasite-specific oxidoreductase tryparedoxin, the mitochondrial glutathione-like trypanothione-dependent peroxidase, glucose-6phosphate dehydrogenase, and a selenocysteine synthase. Moreover, we have conducted therapeutic approaches using a piggy-bag strategy with drugs already approved for use in humans and are currently testing novel candidates provided by our industrial or academic collaborators (Ferrokin-USA, Biochemistry Centre Heidelberg, Germany; Demoro et al. 2011). High-through-put assays are developed in our lab. to screen for compounds for enzyme-inhibitory or anti-trypanosomal activity based on multiwell spectrophotometry or flow-cytometry. 4. MONITORING INTRACELLULAR REDOX CHANGES IN SITU AND ON REAL-TIME We have already initiated in vitro studies with a fluorescence biosensor that allows monitoring in situ and on real-time redox changes in the cells. We will next evaluate the performance of this system to follow the dynamics of the redox changes occurring in T. cruzi and mammalian host cells (fibroblastic, epithelial and macrophagic cell lines) during the invasion process, as well as under different physiological stress conditions. The validation of this technology in trypanosomes will provide a powerful tool for the area of redox cell biology. The biotechnological application of this biosensor is investigated in collaboration with Dr. Bollati (Cell Biology Unit, IP Montevideo) To address some of the issues described above, our group has established new, or continued with precedent, vigorous multidisciplinary collaborations with groups inside and outside the institute. For instance, the group is interacting very actively with most of the technological platforms at the IP Montevideo, and also with regional and interbational renowned laboratories. Publications 1. Roldn A, Comini MA, Crispo M, Krauth-Siegel RL. Lipoamide dehydrogenase is essential for both bloodstream and procyclic Trypanosoma brucei 2011 Mol Microbiol. 81(3):623-639. 2. Ferrer-Sueta G., Manta B., Botti H., Radi R., Trujillo M., Denicola A. Factors affecting protein thiol reactivity and specificity in peroxide reduction. Chem. Res. Toxicol. 2011 24(4): 434450. 3. Ortiz C., Larrieux N., Medeiros A., Botti H., Comini M.A., Buschiazzo, A. Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogenTrypanosoma cruzi in complex with substrate. Acta Crystallographica Section F. 2011 Nov 1;67(Pt 11):1457-61. 4. Comini M. A., Medeiros A., Manta B. (2011) Stress response in the infective stage of Trypanosoma brucei. In: Stress response in microbiology. Ed. J. M. Requena. Horizon Scientific Press, Norwich, UK. In press. 5. Comini M. A., Manta B., Fleitas L. (2011) Iron metabolism in pathogenic Trypanosomes. In: Iron Metabolism. Ed. S. Arora. InTech, Rijeka, Croatia. ISBN 979-953-307-162-5. In press.

6. Demoro B., Sarniguet C., Snchez-Delgado R., Rossi M., Liebowitz D., Caruso F., Olea-Azar C., Kemmerling U., Maya J.D., Guiset H., Moreno V., Pizzo C., Mahler G., Medeiros A., Comini M.A., Otero L., Gambino D., 2011. New organoruthenium complexes with bioactive thiosemicarbazones as co-ligands: potential antitrypanosomal agents. Dalton Transact. 2012 Feb 7;41(5):1534-43. Epub 2011 Dec 2. 7. Manta B., Obal G., Ricciardi A., Pritsch O., Denicola A. Tools to evaluate conformation in protein products. 2011. Biotechnology Journal 6(6):731-41 Other activities 1. Our Lab is associated to the European Cooperation in the Field of Scientific and Technical Research, COST Action CM 0801 "New Drugs for Neglected Diseases". 2. We co-organize the course "Redox chemistry and Biology of Thiols", realized in Uruguay March 21st Ap 1st 2011.

Unit: Head:

G5 - BioMolecular Simulation Laboratory Sergio Pantano, PhD

Members:

Pablo Dans (Staff Member) Leonardo Darre (PhD Student) Matas Machado (PhD Student) Humberto Gonzalez (Msc Student) Astrid Brandner (Udergraduate Student)

Research STRUCTURAL BIOINFORMATICS AND MOLECULAR SIMULATIONS Computational modelling and simulation techniques have become a solid alternative in molecular biology overcoming experimental difficulties and allowing to easily explore different conditions and the integration of apparently uncorrelated biological data. Methodological developments for biomolecular simulations: The central line of research during these years in my group has been the development of simplified or Coarse-Grained (CG) models for simulating biomolecular systems. These CG representations may reduce significantly the computational demands but still capture the physico/chemical essence of the phenomena under examination. Despite the CG model developed by us comports a significant reduction of the molecular components, pseudo atomic information can be recovered at every point in the simulations. The first (and perhaps more challenging) milestone of this project has been the development of a very accurate CG model for the study of DNA. The inclusion of simplified representations for proteins and phospholipids, which is currently in progress, will allow in the short term the study of all the families of biological molecules at the CG level. Our final goal is to provide the tool to follow in silico the temporal evolution of macromolecular ensembles in size and timescales really comparable to those biologically relevant. 1. GENES IN 4D: THE INTEGRATED HIV-1 GENOME The huge amount of data furnished by the latest sequencing technologies provides only a 1D picture of a complex 4D scenario (being time the fourth dimension). Unraveling the molecular basis of gene expression and its connections with human diseases requires study the structure chromatin segments containing entire genes. One of our more ambitious mid-term goals is the use of structural bioinformatics and Coarse Grain methods to model chromatin segments. We aim to build a hierarchical modelling platform for the study of multi Protein/DNA interactions at the level of atoms, molecules, nucleosomes and genes. Our first candidate for structural characterization is the integrated provirus of HIV-1. There are several features that make it an interesting target: its obvious biomedical relevance; the detailed information regarding nucleosomes position; a number of structurally well characterized protein binding motifs within the promoter; the vast documentation about the epigenetic control of its Long Terminal

Repeat; etc This project is carried out in collaboration with Prof. Mauro Giacca at the ICGEB, in Trieste Italy. The group of Prof. Giacca will provide us with experimental data regarding nucleosome positioning and global provirus shape using Chromosome Conformation Capture techniques. 2. SNARE MEDIATED MEMBRANE FUSION. Regulated secretion is an essential process in all eukaryotic cells. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of the socalled SNARE complexes. We have proposed a model according which SNARE complexes assemble together in a rosette-shaped supercomplex. Using a combination of Bioinformatics, site directed mutagenesis and electrophysiology measurements at neuromuscular junctions of Drosophila melanogaster larvae we recently identified putative contact points between SNARE complexes. This information will be used to construct a model of the SNARE supercomplex, which will provide new structural perspectives on the membrane fusion mechanism. This project is carried out in collaboration with the group of Prof. Cesare Montecucco at the University of Padua, Italy. Publications 1. Machado MR, Dans, PD and Pantano S. A hybrid all-atom/coarse grain model for multiscale simulations of DNA. Phys. Chem. Chem. Phys., 2011, Oct 28;13(40):1813444. 2. Perez DI, Palomo V, Prez C, Gil C, Dans PD, Luque FJ, Conde S, Martnez A. Switching reversibility to irreversibility in glycogen synthase kinase 3 inhibitors: clues for specific design of new compounds. J Med Chem. 2011 Jun 23;54(12):4042-56

Other activities 1. Organization of the Hands-on Course: Coarse Grain Methods for Biomolecular Simulations: Institut Pasteur de Montevideo, Uruguay, Sept. 26th- 4th Oct. 2011. 2. Direction of a Degree Thesis in Biochemistry (Fac. of Sciences, Universidad de la Republica, February 2011).