ANNUAL REPORT INBEB. Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem

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1 ANNUAL REPORT INBEB Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem

2 SILVA, JERSON LIMA; MEDEI, EMILIANO; VERJOVSKY, MARINA; TOVAR-MOLL, FERNANDA. ANNUAL REPORT INBEB (INSTITUTO NACIONAL DE CIÊNCIA E TECNOLOGIA DE BIOLOGIA ESTRUTURAL E BIOIMAGEM / SILVA, JERSON LIMA; MEDEI, EMILIANO; VERJOVSKY, MARINA; TOVAR-MOLL, FERNANDA. RIO DE JANEIRO: UFRJ / INBEB, F. : IL. ANNUAL REPORT UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, INSTITUTO NACIONAL DE CIÊNCIA E TECNOLOGIA DE BIOLOGIA ESTRUTURAL E BIOIMAGEM (INBEB), INCT. 2. INBEB. 3. ANNUAL REPORT. 4.MINISTERIO DE C&T. I. SILVA, J. L. II. MEDEI, E. III. VERJOVSKY, M. IV. TOVAR-MOLL, F. V. TÍTULO: ANNUAL REPORT INBEB (INSTITUTO NACIONAL DE CIÊNCIA E TECNOLOGIA DE BIOLOGIA ESTRUTURAL E BIOIMAGEM 2

3 MAIN HEADQUARTERS: UFRJ - Universidade Federal do Rio de Janeiro COORDINATOR: JERSON LIMA SILVA Instituto de Bioquímica Médica, UFRJ VICE-COORDINATOR: WANDERLEY DE SOUZA Instituto de Biofísica Carlos Chagas Filho, UFRJ 3

4 SUMMARY Introduction Science Highlights Cooperative and educational activities Perspectives and future developments

5 1. INTRODUCTION THE NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY FOR STRUCTURAL BIOLOGY AND BIOIMAGING The principal mission of the National Institute of Science and Technology for Structural Biology and Bioimaging is to create and consolidate a technical and scientific infrastructure that facilitates the study of the structure of biological systems (from the macromolecular level to the whole-organism level) while making use of the most advanced analytical techniques and the highest possible resolution images. In addition, our mission is to create conditions in which this infrastructure can be integrated into similar but less complex initiatives in different regions of the country, through the involvement of a large number of institutions. The INBEB emphasizes the use of a multidisciplinary approach to the study of several subjects because we have become increasingly aware of the need to integrate studies on the structure of macromolecules and how they combine to form complex biological structures and macromolecular complexes which in turn are organized into different cell types constituting the different tissues and organs that make up a living being. Understanding the formation of biological structures at their different levels, from the macromolecular to the whole organism level, is the central goal that has led us to assemble a significant number of research groups with proven leadership in biomedical research in Brazil. The INBEB consists of 20 associate laboratories (ALs) at 20 institutions in 7 Brazilian states, as shown on the following map. 5

6 Pará Universidade Federal do Pará (UFPA) Pernambuco Universidade Federal de Pernambuco (UFPE, PE); Centro de Pesquisas Aggeu Magalhães (CPQAG - FIOCRUZ); Centro de Tecnologias Estratégicas do Nordeste (CETENE) Bahia Universidade Federal da Bahia (UFBA, BA) Minas Gerais Universidade Federal do Triangulo Mineiro (UFTM). 5. São Paulo Universidade Estadual de Campinas (Unicamp, SP). 6. Rio de Janiero Universidade Federal do Rio de Janeiro (UFRJ); Universidade Federal Fluminense (UFF); Universidade Estadual do Rio de Janeiro (UERJ); Universidade Estadual do Norte Fluminense (UENF); Universidade Santa Úrsula (USU); Centro Universitário Estadual da Zona Oeste (UEZO); Bio-Manguinhos (FIOCRUZ); Instituto de Pesquisa Clínica Evandro Chagas (IPEC, FIOCRUZ); Instituto Nacional de metrologia (INMETRO); Instituto Militar de Engenharia (IME) Instituto Nacional de Cardiologia (INC). 7. Santa Catarina Universidade Federal de Santa Catarina (UFSC, SC). 6

7 The ALs relies on the participation of leading researchers in different fields who work at each institution: AL1. Associated Laboratory of Virus and Cancer Structural Biology Coordinator: Jerson Lima Silva, Instituto de Bioquímica Médica/UFRJ. AL2. Associated Laboratory of Structural Biology of Cardiac and Amyloidogenic Proteins Coordinator: Débora Foguel, Instituto de Bioquímica Médica/UFRJ. AL3. Associated Laboratory of Proteins Structure Determination by NMR Coordinator: Fábio Almeida, Instituto de Bioquímica Médica, UFRJ. AL4. Associated Laboratory of Pharmacologic Proteomic Coordinator: Russolina Zingali, Instituto de Bioquímica Médica, UFRJ. AL5. Associated laboratory of Nuclear Magnetic Resonance, Organic Synthesis and Molecular Modeling Coordinator: José Daniel Figueroa Villar, Instituto Militar de Engenharia (IME) AL6. Associated Laboratory of Proteins and Proteomic Heterologous Expression Coordinator: Hernán Terenzi, Universidade Federal de Sta Catarina (UFSC) AL7. Associated Laboratory of Proteins Biochemistry Coordinator: Carlos H. Inácio Ramos, Universidade Estadual de Campinas (UNICAMP) AL8. Associated Laboratory of Macromolecules Crystallization Coordinator: Marcelo Santos Castilho, Universidade Federal de Bahia (UFBA) AL9. Associated Laboratory of Cellular Ultrastructure Hertha Meyer Coordinator: Wanderley de Sousa, Instituto de Biofísica Carlos Chagas Filho (UFRJ) AL10. Associated Laboratory of Genomic, Proteomic, Modeling and Nanoscopy of Biological Systems Coordinator: Paulo Mascarello Bisch, Instituto de Biofísica Carlos Chagas Filho (UFRJ) AL11. Associated Laboratory of Microscopy Coordinator: Thaís Cristina Souto Padrón, Instituto de Microbiologia Prof Paulo de Goes (UFRJ) AL12. Associated Laboratory of Cellular Ultrastructure Coordinator: Marlene Benchimol, Universidade Santa Ursula (USU) AL13. Associated Laboratory of Structural Biothecnology Coordinator: Celso B. Sant'Anna Filho, Instituto Nacional de Metrologia (INMETRO) 7

8 AL14. Associated Laboratory of Structural Biology Coordinator: Edilene Oliveira da Silva, Universidade Federal do Pará (UFPA) AL15. Associated Laboratory of Microscopy CETENE Coordinator: Christina Alves Peixoto, Fundação Oswaldo Cruz and Centro de Tecnologias Estratégicas do Nordeste (FIOCRUZ, CETENE - Pernambuco) AL16. Associated Laboratory of Molecular and Cellular Cardiology Coordinator: Antonio Campos de Carvalho, Instituto de Biofísica Carlos Chagas Filho (UFRJ) AL17. Associated Laboratory of Ion transport physiology in health and disease Coordinator: Adalberto Vieyra, Instituto de Biofísica Carlos Chagas Filho (UFRJ) AL18. Associated Laboratory of Immunology Coordinator: Júlio Scharfstein, Instituto de Biofísica Carlos Chagas Filho (UFRJ) AL19. Associated Laboratory of Cellular and Molecular Neurology Coordinator: Rosalia Mendez Otero, Instituto de Biofísica Carlos Chagas Filho (UFRJ) AL20. Associated Laboratory of Inflammation and Metabolism Coordinator: Fernando Augusto Bozza, Instituto de Pesquisa Clínica Evandro Chagas (IPEC-FOC) 8

9 The headquarters of the INBEB are located on the main campus of the Universidade Federal do Rio de Janeiro (UFRJ) (Figure1). Figure 1: INBEB location. During the first 18 months of operation, the INBEB sought to maximize its infrastructure, which includes equipment for procedures such as nuclear magnetic resonance (NMR), electron microscopy, atomic force microscopy, NMR imaging, and bioluminescence imaging. The facility is organized into three units, in wich each has its own headquarter building: 1) CNRMN, or Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas (Jiri Jonas National Center for Nuclear Magnetic Resonance) (CENABIO I);. Figure 2: NMR spectrometers room (CENABIO I) 2) CENABIO II, which houses the equipment for small animal bioimaging; 3) CENABIO III, which will be constructed with funds provided by Pro-INFRA at UFRJ (FINEP/MCT) to centralize the equipment available for electronic, confocal, multiphoton, and atomic force microscopy. The equipments are not only available for use by the groups that belong to the INBEB, but also by the general scientific community both within Brazil and 9

10 abroad. They are frequently utilized by our Mercosur colleagues, who are developing projects that require the use of our existing infrastructure. The INBEB focuses on the use of multidisciplinary approaches for the main research areas of the ALs, integrating studies to allow a better understanding of the structure of macromolecules. These studies include how macromolecules associate to form complexes and complex biological structuresthat are then organized into the different cell types that constitute the different tissues and organs that, in turn, form a definitive, living organism. In general, the INBEB program has specifically focused on several central themes of contemporary biotechnology and biomedicine: (1) the study of macromolecules involved in infectious diseases, neurodegenerative illnesses, and cancer; (2) the study of important viruses, such as Dengue fever, yellow fever, and others; (3) the study of complex structures found in protozoan parasites that are the agents responsible for causing relevant illnesses such as Leishmaniasis, Chagas disease, malaria, and toxoplasmosis; (4) monitoring the evolution of viral and protozoan parasite infections in small experimental animals and their behavior in animals undergoing experimental chemotherapy; and (5) the study of the in vivo behavior of stem cells in order to analyze their biodistribution, localization, and function as cellular therapies for degenerative diseases. We have also extended our interaction with the business sector through a partnership with the Instituto D OR (IDOR) in order to expand our ability to do translational research. Through this partnership, IDOR researchers have access to the small animal bioimaging infrastructure at the INBEB and the AL researchers have access, when necessary, to an array of human imaging equipment in the Rede D OR. The division that focuses on the elucidation of macromolecular structure has consolidated its NMR equipment facility, which was originally part of the CNRMN (Figure 2). Utilizing the resources for the INBEB equipment, the Bruker DRX 600 MHz spectrometer was upgraded to the digital system AVANCE. This now allows the potential for full use of four channels, inverse triple resonance probes, and the inverse triple resonance cryoprobe. With this upgrade, this equipment is now state-of-the-art and its sensitivity and resolution are equivalent to that of a new spectrometer. The Bruker Avance III 800 MHz spectrometer, which has four channels and an inverse triple resonance probe, also had its utility amplified and diversified. Resources from the project have allowed for the 10

11 maintenance of the spectrometers to be performed and for support equipment to be acquired (e.g., backup power supplies equipments and air conditioners). These additions were crucial to allow the NMR Center to be kept open 24 hours per day for use by the members of the INCT and by a large number of researchers, who are not affiliated with the INBEB. The Bruker DRX 400-MHz wide-bore instrument, which is equipped with three channels, inverse triple resonance probes, a broadband inverse probe, and magic angle spinning (MAS) for investigation of solid samples has also been widely used. The microscopy division has a variety of types of equipment, currently spread among several UFRJ laboratories, that will be transferred to the Central Unit of the INBEB (CENABIO 3 - Figure 1), including: 1) two confocal microscopes; 2) a multiphoton microscope; 3) a multiphoton microscope with a fluorescence correlation spectroscopy system (FCS); 4) a total internal reflection fluorescence (TIRF) microscope; 5) an atomic force microscope; 6) a conventional scanning microscope; 7) a high resolution scanning microscope with a cryo-stage; 8) two conventional transmission electron microscopes; 9) two analytical transmission electron microscopes, which use an X-ray emission spectrum and energy loss of electrons; and 10) a transmission electron microscope capable of operating at 200 KV that is equipped with a cryo-stage and an automated cryo-electron micrograph system. After the receipt of this vast array of equipment, the CENABIO III will be the most complete microscopy facility in Latin America. An environmental scanning electron microscope was purchased with INBEB resources (Figure 3). Together with the electron microscope (ME 200 KV with cryotomography), which was purchased with UFRJ Proinfra resources, it increased Figure 3: Environmental Scanning Electron Microscope our ability to perform complex microscopy experiments. 11

12 The array of microscopy equipment that will be available will allow us to visualize proteomic structures, such as amyloid fibers, viral particles, bacteria, and protozoans. It will also allow us to track a single viral particle within a living cell, enabling us to determine the route it takes during the infectious process. For example, Figure 4 shows an electronic micrograph of pseudocysts of the parasite Trichomonas interacting with host cells. This image was provided by AL12, which is coordinated by Prof. Marlene Benchimol. Figure 4: EM imgae of Trichomonas pseudocists. The small animal imaging division required the most resources and investments during the consolidation process. The construction of CENABIO II was ultimately completed (Figure 5), and INBEB resources were used to install the electrical wiring and to buy equipments required to support the imaging equipment. Figure 5: CENABIO building. 12

13 The 7-Tesla magnet used for magnetic resonance imaging (MRI) of small animals has been installed (Figures 6 and 7) and has been available since May of This equipment allows researchers to perform morphological and functional analyses of organs and systems in live animals (especially mice and rats, the most often used experimental animals in biomedical research). Figure 6: Inside view of CENABIO II building. Nuclear magnetic resonance imaging is a non-invasive, nondestructive technique that allows investigators to monitor the morphology and in some cases the organ function of animals over time without sacrificing the experimental animal (Figures 7 and 8). acquired Other previously bioimaging equipments have been moved to the new CENABIO II building, which contains all of the appropriate infrastructure that is necessary to allow its use by INBEB researchers and external users. We installed a 110-KVA generator and backup power supplies to protect the equipments and to ensure that there are no interruptions in the operations. Figure 7: 7 Tesla MRI Equipment for small animals installed in CENABIO II. 13

14 Anatomic images of high spacial resolution (0.1 x 0.1 x 1.0 mm 3 ) RM 7.0T Figure 8: MR Image (7 T) of a Mouse Brain. Paiva et al., 2007 The following pieces of equipment have been completely installed: 1) high-resolution ultrasound equipment that was specifically manufactured to acquire high resolution images from small animals, enabling the visualization of embryonic development in mice by monitoring organs such as the heart, liver, and kidneys, among others; and 2) a bioluminescence and fluorescence detection system for use in live animals that allows us to visualize cells labeled with enzymes (such as luciferase) that activate luminescent molecules, such as luciferin, or fluorescent labels (Figure 9). Labeling cells or pathogens will allow us to track their dissemination when they are injected into live animals. Figure 9: Bioluminescence Imaging Equipment. 14

15 Scintigraphy equipment for small animals (PET/SPECT) is being purchased (with funds from MS-Decit/FAPERJ) to make the facility more complete. This equipment will allow us to detect radiolabeled molecules and cells at a resolution of 2 millimeters. The equipment will be coupled to a computerized tomography scanner, which will allow us to overlay 3D SPECT images (single photon emission computerized tomography) with the computerized tomography images in real time. This equipment is particularly useful because it will allow us to label molecules and study their biodistribution, a technique that is especially important when one is evaluating drugs. We would like to emphasize the level of cooperation that has been established with the researchers, doctors, and physicists at the D'Or Institute for Research and Teaching (IDOR) and the Rede Labs-D'Or. This has been crucial in initiating research activities related to small animal magnetic resonance imaging at the INBEB. This partnership was established in June 2009, with Dr. Fernanda Tovar Moll, who is responsible for coordinating all of the installation activities and use of the 7-Tesla magnet for MRI studies, an especially important role. Dr. Moll is the director of research at IDOR and is responsible for implementing IDOR projects in collaboration with the INBEB, especially projects that involve MRI studies in human beings (located at IDOR) and small animals (located at the INBEB). A great level of interactionwas established among the different ALs during the consolidation process of CENABIO II. This process included the participation of groups outside the UFRJ, such as LA20, which are coordinated by Dr. Fernando Bozza, who was heavily involved in the design and installation of the animal facility as well as the assembly of the Life Support and Monitoring System for Small Animals. The CENABIO animal facility has been fully implemented and inaugurated at the 2 nd Annual Meeting of INBEB in November All of the required equipment, such as an exhaust hood, autoclave system, and air-conditioning system, were fully installed. We also coordinated efforts to acquire the Life Support and Monitoring System for Small Animals. The combined resources of the INBEB and the FAPERJ made it possible to refit the Inflammation and Metabolism Laboratory with an experimental apparatus that is capable of providing life support to small animals. We were able to 15

16 acquire a mechanical ventilator microprocessor that is compatible with our magnetic resonance imaging equipment and a complete monitoring system that uses modules to record arterial pressure, electrocardiographic tracings, and body temperature as well as to monitor the anesthetic and the inhalational anesthesia systems. With this instrument, we now have the complete infrastructure that is necessary to provide small animal life support similar to that currently used at the respiratory physiology laboratory. Their experimental protocols that involve the ventilation of rats for about two hours use equipment that is similar to that acquired by the INBEB. 16

17 2. SCIENCE HIGHLIGHTS 17

18 As is evident from the results of the second year of the project, there is an ever-increasing use of diverse methodologies in the fields of structural biology, microscopy, and animal imaging. The members of the INBEB ALs have published 375 papers, and several of these resulted from extensive collaborations between researchers within a single AL or collaboration between different ALs. The INBEB members are integrated into graduate programs, several of which were given a grade of 6 or 7 by the CAPES evaluation. To date, a total of 78 master s dissertations and 43 doctoral theses have been completed by INBEB members. The Associate Laboratories are integrated into the three large performance areas of the INBEB: 1) analysis of the structure of macromolecules; 2) microscopy; and 3) small animal imaging. During the first year of the project, there was a great deal of collaboration between the different study areas and between the ALs. The following is a brief summary of the results: 18

19 AL1 ASSOCIATED LABORATORY OF STRUCTURAL BIOLOGY OF VIRUSES, PRION AND CANCER. Coordinator: Jerson Lima Silva, Instituto de Bioquímica Médica, UFRJ. Members: Andréa Cheble Oliveira André MO Gomes Yraima Moura L Cordeiro Davis Fernandes Ferreira Claudia Vitoria M. Gallo Luciane Pinto Gaspar The central subject of our research is to understand the mechanisms of protein folding, protein misfolding, protein-protein interactions and supramolecular assembly. Our aim is to decode how these processes are related to the normal physiological function of the proteins and to the development of diseases, such as virus infections, prion and other neurodegenerative diseases and cancer. Exploiting spectroscopic tools such as fluorescence and NMR, our work with high pressure in biochemistry and structural biology has yielded a wealth of new data and testable models concerning new concepts for the folding and association of proteins, virus assembly, protein misfolding and aggregation. We have demonstrated that the entropic nature of protein interactions and the changes in hydration are crucial in the assembly of virus particles and amyloid aggregates. The studies of the stability of virus particles using high pressure have resulted in a new method for obtaining antiviral vaccines and other applications. Combining 19

20 biophysical (high pressure) and structural biology tools, we have found that changes in hydration and cavity distribution in the interior of the proteins play a key role in different biological processes such as viral membrane fusion, prion conversion and loss of function of the tumor suppressor protein. In the case of prion protein, involved in transmissible spongiform encephalopathies, the prion protein becomes less hydrated when converts to the aggregated, scrapie-like isoform and we have demonstrated Using structural and cellular biology approaches, we have investigated the interaction of the prion protein with biological ligands, such as nucleic acids, glycosaminoglycans, the co-chaperone hop/sti1 and copper, and their participation in the conversion of PrP to the scrapie isoform (Figure 1). Moreover, although several compounds have been evaluated for their ability to inhibit this conversion, to date there is no effective therapy for the prevention or reduction of the disease that this process can be catalyzed by nucleic acids. Among our most important findings is the description that prions have other accomplices, such as nucleic acids and glycosaminoglycans, which chaperone their activity in converting the PrP C into the disease-causing isoform. We highlight below the most important findings of our Associated Laboratory: 1. Prion Diseases. Transmissible Spongiform Encephalopathies (TSE) embody a group of neurodegenerative diseases that affect humans and other mammals. They occur when the native prion protein (PrP C ), an alpha-helical rich protein, is converted into an infectious misfolded isoform. This isoform, the scrapie PrP (PrP Sc ), forms aggregates, leading to neurodegeneration. It has been proposed that the spontaneous conversion from PrP C to PrP Sc is prevented by a high energetic barrier and changes in the activation energy, like the presence of a catalyst, would lead to prion conversion. Among the proposed catalysts, our group has characterized nucleic acid molecules as effective inducers of such process. Figure 1- Energy and volume diagram of PrP misfolding. PrP C (left) can misfold into an isoform rich in beta sheet structure capable of forming toxic and infectious aggregates (PrP Sc ) (right). The transition between the species is separated by a large energetic barrier. I and U represent intermediate and unfolded states. An adjuvant factor would lower the free-energy barrier, triggering formation of PrP Sc. PrP C has a larger solvent-accessible surface area than the misfolded/aggregated species, and the folding pathway also exhibits a kinetic barrier in the activation volume (inset, modified from ref. 15). The pressure-denatured states of -rprp (PrP C ) and - rprp (PrP Sc -like) are denoted as U and U, respectively. progression. Therefore, the selection and evaluation of new compounds that might inhibit PrP aggregation is also an important goal when studying such devastating diseases. Previous studies have shown that antimalarial compounds, such as quinoline and acridine derivatives, have an important anti-scrapie activity. We have synthesized new aminoquinoline compounds and evaluated their anti-prion activity in aggregation- 20

21 inhibition assays. Our results show that two of the selected compounds can significantly inhibit the aggregation of this prion protein hydrophobic domain, through light scattering, thioflavin-t binding assays and transmission electron microscopy. Moreover, we verified that these aminoquinolines are not toxic to neuroblastoma cells in culture. Therefore, such aminoquinolines might be considered as candidates for the further development of therapeutics to prevent the development of prion diseases. We also found that murine recombinant PrP interacts with low-molecular-weight heparin (LMWHep) at ph 7.4 and 5.5. The soluble complex of LMWHep-rPrP is resistant to aggregation induced by RNA, which raises the possibility to use glycosaminoglycans in anti-scrapie therapy. 2. Structure and function of the tumor suppressor protein p53 and its association with malignant tumors. TP53 mutation is the most frequent genetic alteration (20-50%) in breast cancer. Most mutations of TP53 are located in the DNA-binding domain and may result in loss of DNA contact or structural change, leading to three possible protein activity alterations: dominant negative, loss of function and gain of function. It was suggested by our group and others that the altered p53 conformation results in protein aggregation. Evidence that the three amyloid formation might participate in the malignant process. Aggregation of p53 would act as a sink to sequester native protein into the inactive conformation, replicating the structural information, very much like a prion (Figure 2). The search for molecules that preclude the formation of the misfolded conformation, which may ultimately lead to the prevention of tumor development, is a major goal in cancer research. We have used aptameric nucleic acids as an alternative to prevent aggregation and to rescue activity (Figure 2). A more stable variant of p53 would shift the equilibrium toward the soluble and active form of the protein. Because nucleic acids are susceptible to various modifications (such as phosphorothioate), which alter in vivo processing without affecting target discrimination, our study paves the way for the application of aptameric nucleic acids in cancer. To search for correlation Figure 2. Stabilization of p53c upon sequence-specific DNA binding and recovery of misfolded aggregated species of p53c. (A) Structure of p53c bound to DNA (PDB entry domains of p53 form amyloid-like 2ABY). (B) Full-length p53 is stabilized against pressure denaturation upon DNA binding as measured by fluorescence: p53 (blue circles), consensus-bound p53 (red aggregates is quite striking, making squares), and poly(gc)-bound p53 (green triangles). Open symbols are values after it tempting to speculate that p53 return to atmospheric pressure. (C) Cognate DNA rescues the native conformation of p53c after misfolding and aggregation. Fluorescence of wt p53c at atmospheric pressure (solid black line); after the first cycle of pressurization in the absence of DNA (red line); after DNA addition at atmospheric pressure (blue line); and after the second pressure cycle in the presence of DNA (green line). (D) Proposed 21 model for p53c aggregation. Conversion of native, active p53 (blue circles) into aggregates (red squares) in the cytoplasm (upper panels). Nuclear DNA is represented in purple.

22 between p53 aggregation and malignancy, we analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5 to 10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p < ). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and antip53. Our results show that mutant p53 colocalizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo (Figure 3). 3. Targeting Viruses of Medical Importance. In the last two years, our group has used as study models several animal and human viruses of great medical importance, such as Hepatitis C (HCV), Yellow Fever (YFV), Dengue (DENV), Mayaro (MAYV), Ebola and Influenza viruses, since there is an urgent need to develop new approaches to prevent and treat the diseases caused by these viruses. The overall proposal of our group is to study the problem of macromolecular recognition, using different variables as high hydrostatic pressure, temperature and chemical denaturants, and employing various structural and spectroscopic methods such as fluorescence spectroscopy, nuclear magnetic resonance, circular dichroism, calorimetry, molecular dynamics simulations, and new approaches and technologies of microscopy and fluorescence spectroscopy, such as multiphoton microscopy and fluorescence Figure 3- Immunofluorescence for p53 aggregates. (A) N 55 - normal tissue showing the absence of p53 aggregates in a mammary duct, which is expected. (B) T74 tumoral tissue with wild type p53, showing the absence of p53 aggregates. (C) T21 - tumoral tissue with R273H mutation presenting p53 aggregates, mainly in the cytoplasm. Green fluorescence p53; red fluorescence aggregates; yellow flurescence p53 and aggregates co-localization. correlation spectroscopy, field a pioneered by us in the state of Rio de Janeiro. The use of pressure inactivate nonenveloped to and enveloped viruses is being evaluated to be used in the preparation of vaccines, such as against Fever, Yellow Dengue, Foot-and-Mouth disease virus and 22

23 Influenza (Figure 4). Most of the studies have the explore the structural biology of HCV particles Figure 4- Pressure inactivation of picornaviruses. (A) Structure of human rhinovirus 14 (HRV14). (B) Scheme of pressure inactivation of HRV14. participation of Biomanguinhos, a State Company in FIOCRUZ. As a result of interaction with Biomanguinhos, we filed a patent entitled Method for Stabilized Vaccine Production. The present invention proposes to solve the problem of lack of stability of polio vaccine strains (serotypes 1, 2 and 3) present in the attenuated vaccine (Sabin). The use of hydrostatic pressure makes it possible to obtain the stability of the three strains, indicating that the addition of any chemical agent would become totally unnecessary. The results were also published in the journal "Vaccine". The Hepatitis C Virus, the leading cause of liver disease, chronically affects 3% of word population. HCV entry and assembly are complex processes with many steps still unknown. So, the main aim of our work is to enabling a better understanding of the infection cycle of this virus and thereby helping in the development of new antiviral drugs. We have analyzed the structure of a HCV fusion peptide candidate (HCV ) and its interaction with micelles and lipid membranes, besides the viral capsid assembly in vitro. Our results show that the peptide induces lipid vesicle aggregation and interacts with micelles in a process enthalpically driven involving Trp residues. The peptide also becomes structured, suggesting its participation in the entry process of HCV. We also show that the assembly process is highly cooperative and driven by the neutralization of basic residues. MAYV, YFV and DENV are viruses transmissible by mosquitoes, responsible for causing diseases of major global impact. The principal objective of this work is to evaluate 23

24 virus-cell interaction, investigating the importance of cholesterol (Chol) and the fusion peptide for infection, the participation of heparan sulfate (HS) for virus entry and the cellular mechanisms involved in the apoptosis process. Since there is no treatment for the infection with these viruses, the discovery of effective drugs to control these infections becomes an important strategy and has an extreme scientific interest. We have evaluated the importance of Chol and its dependence to the membrane fusion process of DENV-2. We found that depletion of viral Chol promoted a significant reduction of approximately 3 logs in viral titer. It is known that membrane fusion occurs by exposure of a hydrophobic segment (FLA ), the fusion peptide (FP), present in the glycoprotein of flaviviruses. Using two conserved FPs, we found that the interaction involved Trp residues independently of the ph and partially dependent on negative charge. We have also developed studies on the interaction of the Ebola fusion domain with target membranes by NMR, atomic force microscope and calorimetry. Apoptosis is a response involved in cytopathogenicity during infection by DENV and YFV. Our results show that DENV and YFV induce high rate of cell death only after 120 h and 72 h of infection, respectively. The process of programmed cell death was demonstrated by different methods. We evaluated the loss of integrity of m and the importance of opening the channel VDAC, suggesting the involvement of mitochondrial pathway in the apoptotic process. Our studies also demonstrate that the induction of apoptosis by YFV occurs in a p53- dependent manner. Influenza viruses are important threats to human healthy. Among different types, we work with H3N8, an avian Influenza virus, which was originally isolated from birds, later found in horses. We study the stability, inactivation and immunogenic capacity of H3N8 by using high hydrostatic pressure (HHP) as main tool. Our present goal is to assess the immunogenic capacity of these pressurized particles in murine model. The initial results are very promissing, showing good humoral and mucosal answer. All our data reinforce the potential of HHP in the development of vaccines against avian influenza with low cost, safety and promissing good immune response. The challenge of elucidating the mechanisms of entry and assembly/disassembly of the virus and cellular response involved in infection may represent an advance in the rational development of vaccines and new inhibitors based on structure, mode of interaction and function of viral proteins. The group of Dr. Davis Ferreira has worked on the antiviral properties of new molecules against Flavi and Alphaviruses. More than twenty new molecules were tested, some with promising antiviral activity. The group is also evaluating the profile of vertebrate and invertebrate cells infected with Dengue virus by Proteomics and Glycoproteomics, in collaboration. In collaboration with North Carolina State University, novel studies have been performed on the structure of Dengue virus using cryoelectron microscopy. 24

25 Group publications ( ): 1. Silva JL, Vieira TC, Gomes MP, Bom AP, Lima LM, Freitas MS, Ishimaru D, Cordeiro Y, Foguel D. (2010) Ligand Binding and Hydration in Protein Misfolding:Insights from Studies of Prion and p53 Tumor Suppressor Proteins. Acc Chem Res. 43: Silva, JL, Gomes, MPB, Vieira, TCRG and Cordeiro, Y (2010) Functional and Pathological Roles of Prion-Nucleic Acid Interactions. Frontiers in Bioscience, 15: Macedo B, Kaschula CH, Hunter R, Chaves JAP, van der Merwe JD, Silva JL, Egan TJ and Cordeiro Y. (2010) Synthesis and anti-prion activity evaluation of aminoquinoline analogues. Eur J Med Chem 45: Ano Bom AP, Freitas MS, Moreira FS, Ferraz D, Sanches D, Gomes AM, Valente AP, Cordeiro Y, Silva JL. (2010) The p53 core domain is a molten globule at low ph: Functional implications of a partially unfolded structure. J Biol Chem. 285: Cano-Estrada A, Vázquez-Acevedo M, Villavicencio-Queijeiro A, Figueroa-Martínez F, Miranda- Astudillo H, Cordeiro Y, Mignaco JA, Foguel D, Cardol P, Lapaille M, Remacle C, Wilkens S, González-Halphen D. Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp. Biochim Biophys Acta (8): Oliveira, G. A. P. ; Costa, E. S.; Freitas, M. S.; Dutra, F. F. ; Maia, S. F. ; Guerra, M. C. ; Tabernero, M. D. ; Borojevic, R. ; Otazu, I. B. ; Silva, Jerson L. (2010). Positive response to imatinib mesylate therapy for childhood chronic myeloid leukemia. Braz J Med Biol Res 43: Souza TLF, Sanches D, Gonçalves RB, Pita SSR, Pascutti PG, Bianconi ML, Almeida FCL, Silva JL and Oliveira AC. (2010). Conformational selection, dynamic restriction and the hydrophobic effect coupled to stabilization of the BIR3 domain of the human X-linked inhibitor of apoptosis protein by the tetrapeptide AVPI. Biophys Chem. 152: Braga AC, Follmer C, Palhano F, Khattar E, Freitas MS, Romão L, Di Giovanni S, Lashuel HA, Silva JL, and Foguel D (2010). The Anti-Parkinsonian Drug Selegiline Delays the Nucleation Phase of Alpha-Synuclein Aggregation Leading to the Formation of Non-Toxic Species. J Mol Biol, in press (Nov 1 [Epub ahead of print] PubMed PMID: ). 9. Levy CB, Stumbo AC, Ano Bom APD, Portari E, Cordeiro Y, Silva JL, and De Moura-Gallo CV (2010). Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors. Int J Biochem Cell Biol, in press (Nov 4 [Epub ahead of print] PubMed PMID: ). 10. Real-Hohn A, Zancan P, Da Silva D, Martins ER, Salgado LT, Mermelstein CS, Gomes AM, Sola-Penna M (2010). Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1- kinase association within the membrane of human erythrocytes. Biochimie. 92(5): Epub 2010 Feb Oliveira MF, Galvao Araujo JM, Ferreira OC Jr, Ferreira DF, Lima DB, Santos FB, Schatzmayr HG, Tanuri A, Ribeiro Nogueira RM (2010). Two lineages of dengue vírus type 2, Brazil. Emerg Infect Dis. 16(3): PubMed PMID: Vieira, T. C. R. G., Reynaldo, D. P., Gomes, M. P. B., Almeida, M. S., Cordeiro, Y. and Silva, J. L. (2010). Heparin Binding by Murine Recombinant Prion Protein Leads to Transient Aggregation and Formation of RNA-Resistant Species. J Amer Chem Soc, accepted. 13. Freitas, M. S., Follmer, C., Costa, L. T., Vilani, C., Bianconi, M. L., Achete, C. A., and Silva, J. L. (2010). Measuring the Strength of Interaction between the Ebola Fusion Peptide and Lipid Rafts: Implications for Membrane Fusion and Virus Infection. PLoS ONE, accepted. 14. Sousa Jr., I. P., Carvalho, C. A. M., Ferreira, D. F., Weissmüller, G., Rocha, G. M., Silva, J. L., and Gomes, A. M. O. (2010). Envelope lipid-packing as a critical factor for the biological activity and stability of alphavirus particles isolated from mammalian and mosquito cells. J Biol Chem, accepted. 15. Romano SA, Cordeiro Y, Lima LM, Lopes MH, Silva JL, Foguel D, Linden R. (2009). Reciprocal remodeling upon binding of the prion protein to its signaling partner hop/sti1. FASEB J. 23(12): Epub 2009 Aug Silva JL, Oliveira AC. (2009). Science and technology to combat dengue virus. An Acad Bras Cienc. 81(4): Silva, JL and Foguel, D (2009) Hydration, cavities and volume in protein folding, aggregation and amyloid assembly. Phys. Biol. 6: (1-12). 18. Ishimaru D, Ano Bom AP, Lima LM, Quesado PA, Oyama MF, Gallo CV, Cordeiro Y, Silva JL (2009) Cognate DNA stabilizes the tumor suppressor p53 and prevents misfolding and aggregation. Biochemistry 48: Palhano FL, Rocha CB, Bernardino A, Weissmuller G, Masuda CA, Montero-Lomelí M, Gomes AM, Chien P, Fernandes PM, Foguel D (2009). A fluorescent mutant of the NM domain of the yeast prion Sup35 provides insight into fibril formation and stability. Biochemistry 48: Silva, JL, Lima, LMTR, Foguel, D and Cordeiro, Y (2009) Response to Radulescu and Brenig: Infectious nucleic acids in prion disease: halfway through. Trends Biochem. Sci., 34: Marques, AF, Cordeiro, Y, Silva, JL, and Lima, LMTR (2009). Enhanced prion protein stability coupled to DNA recognition and milieu acidification. Biophys. Chem. 141(2-3): Fricks, A., Oestreicher, E., Cardozo, L., Feihrmann, A., CORDEIRO, Y., Dariva, C., Antunes, O. A. C. (2009). Effects of compressed fluids on the activity and structure of horseradish peroxidase. The Journal of Supercritical Fluids 50: Guimarães, D. P., Oliveira, I. M., de Moraes, E., Paiva, G. R., Souza, D. M., Barnas, C., Olmedo, D. B., Pinto, C. E., Faria, P. A., DE MOURA GALLO, C. V., Small, I. A., Ferreira, C. G., HAINAUT, P. (2009). Interferon-inducible guanylate binding protein (GBP)-2: A novel p53-regulated tumor marker in esophageal squamous cell carcinomas. International Journal of Cancer 124: Falagan-Lotsch, P., Rodrigues, M. S., Esteves, V., Vieira, R., Amendola, L. C., Pagnoncelli, D., Paixão, J. C., DE MOURA GALLO, C. V. (2009). XRCC1 gene polymorphisms in a population sample and in women with a family history of breast cancer from Rio de Janeiro (Brazil). Genetics and Molecular Biology 32: Ferreira E, Mendes YS, Silva JL, Galler R, Oliveira AC, Freire MS, Gaspar LP (2009). Effects of hydrostatic pressure on the stability and thermostability of poliovirus: A new method for vaccine preservation. Vaccine 27(39):

26 Book Chapters: 1. Omar Lupi ; Ivan Semenovitch ; Fabricio Lamy ; FERREIRA, D. F.. Propriedades Gerais dos Herpesvírus. In: Omar Lupi; Ivan Semenovitch; Fabricio Lamy. (Org.). Infecções por Herpesvírus. 1 ed. Rio de Janeiro: Editora Guanabara Koogan, 2010, v., p Omar Lupi ; Ivan Semenovitch ; Fabricio Lamy ; FERREIRA, D. F.. Diagnóstico Virológico. In: Omar Lupi; Ivan Semenovitch; Fabricio Lamy. (Org.). Infecções por Herpesvírus. 1 ed. Rio de Janeiro: Editora Guanabara Koogan, 2010, v., p Deborah Schechtman & Claudia de Moura Gallo. Desenho racional de drogas em Oncologia Molecular, editores Carlos Gil Ferreira e José Claudio Casali, editora Ateneu, 2010 Patents: 1. Use of hydrostatic pressure to inhibit and reverse protein aggregation and facilitate protein refolding. Inventors: Anne Skaja Robinson, Clifford R. Robinson, Debora Foguel, Jerson Lima Silva. USPTO 7,615,617 Patent number: ; Issue date: Nov 10, METHOD FOR STABILIZED VACCINE PRODUCTION. GASPAR, Luciane Pinto, FREIRE, Marcos da Silva, DE OLIVEIRA, Andréa Chelbe, DA SILVA, Jerson Lima. Patent record available from the World Intellectual Property Organization (WIPO). WO (A1)

27 AL2 ASSOCIATE LABORATORY OF STRUCTURAL BIOLOGY OF CARDIAC AND AMYLOIDOGENIC PROTEINS. Coordinator: Débora Foguel, Instituto de Bioquímica Médica/UFRJ. Members: Martha Sorenson Luis Mauricio Lima Fig 1: TTR quaternary structure. Each monomer is shown in a different color. Protein Misfolding Diseases caused by Tranthyretin: Protein misfolding diseases include a broad range of pathologies in which proteins fail to fold properly or to remain in their folded state. Many protein misfolding diseases, generically termed amyloidoses, are characterized clinically by the presence of proteinaceous insoluble amyloid material, the amyloid fibril. Amyloid fibrils share a common conformation, rich in cross-β structure formed by intertwined layers of β- sheets extending parallel to the fibril axis. Our group has been studying transthyretin (TTR), a 55-kDa homotetrameric protein composed of identical 127-residue subunits with a predominantly β-sheet structure and it is found in human plasma and cerebrospinal fluid (CSF) (Fig.1). Wild-type TTR (wt-ttr) is responsible for senile systemic amyloidosis, a disease that affects 10% of people over 80- years old and is characterized by heavy amyloid deposits in the heart. More than 80 point mutations of TTR are involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomiopathy (FAC) and central nervous system amyloidosis 27

28 (CNSA). Among the variants of TTR, V30M and L55P are the most important because of their high frequency of occurrence and the aggressiveness of the symptoms they evoke. A25T, on the other hand, is one of the few TTR mutations associated with a rare type of amyloidosis that is restricted to the CNS and is characterized by amyloid fibril deposition in leptomeningeal and subarachnoid vessels. T119M is a non amyloidogenic variant alleviating the symptoms of the V30M mutation. Several hypotheses have been proposed to explain the amyloidogenic properties of TTR. The most accepted one presupposes the dissociation of the tetramers into a monomeric, partially folded state, which is aggregation prone. Our group has characterized an altered tetramer of TTR (T4*, pink circles) which is aggregation prone (Fig. 2). Five decades of evidence support the interaction of TTR with Zn 2+ in the biological milieu but nothing is known of the accompanying structural correlates. The observation that concentrations of Zn 2+ in the plasma of healthy individuals are approximately μm, while the Zn 2+ :TTR apparent dissociation constant (Kd app ) is 1 μm, suggests that TTR may circulate as a complex with Zn 2+ in plasma. High concentrations of Zn 2+ and Cu 2+ can trigger TTR amyloid formation in vitro, and chelating agents disrupt these amyloid structures. Recently, through the use of X-ray crystallography and NMR we have shown that TTR presents three canonical Zn 2+ -binding sites. Zn 2+ binding induces structural perturbations that lead to TTR aggregation and decreases its affinity for retinol binding protein one of its partner in the plasma. Our data showed that profound structural perturbation take place on the a-helix upon Zn2+ binding that lead to exposition of a hydrophobic segment that could be the site for protein protein interaction and fibril formation. Fig. 2: Proposed mechanism to explain TTR aggregation. Below are summarized the main findings of our group regarding TTR. Most of the projects reported below were conducted by the groups of Dr. Foguel and Trambaioli: Searching for T4* in a close to physiological condition: Trapping the monomer of T119M for its use as a therapeutic strategy against TTR related amyloidoses: Nowadays, FAP patients are treated through liver transplant whereby a patient s liver expressing a TTR mutation (such as L55P) is replaced by one that expresses the wt protein. Besides, there is no treatment for patients bearing CNSA-related TTR mutations. Many studies have focused on developing effective and selective TTR ligands that can prevent TTR dissociation and aggregation through tetramer stabilization in vitro. 28

29 Recently, our group has shown that a combination of high hydrostatic pressure (HHP) with subdenaturing concentrations of urea facilitates the dissociation of T119M. At 1 o C (ph 5.0), in the presence of 4 M urea, T119M irreversibly dissociates and denatures in less than 30 min at 3,000 bar in a concentration-dependent fashion. These unfolded monomers (here called M T119M,U ) remain in solution as long as urea (4 M) is kept in the buffer, but upon dilution of the urea (0.4 M) at ph 7.0, they refold initially into folded monomers (M T119M, F ) and then slowly (hours) into native tetramers. T119M monomers presented a similar thermodynamic stability to that of the wt monomer, suggesting that most of the differences in stability between the tetramers of T119M and wt TTR can be attributed to the inter-subunit contacts inside the tetramers and not to the intra subunit contacts of the monomers. Our data also show that T119M monomers can be successfully incorporated into amyloidogenic tetramers, even when the exchange is performed in a more physiological environment, such as the plasma; these monomers render the resultant heterotetramers less amyloidogenic. The data presented here are relevant for understanding the T119M folding and association reactions and provide a protocol for producing T119M monomers that function as inhibitors of TTR aggregation when incorporated in tetramers; this protocol may be a new strategy for treating TTR diseases. Structural determination of A25T, the most amyloidogenic variant of TTR: The variant A25T of TTR is involved in leptomeningeal amyloidosis, which is characterized by amyloid fibril deposition in leptomeningeal and subarachnoid vessels. Very little data are available in the literature regarding the folding and aggregation pathways of this specific variant of TTR. Our group has determined its x-ray structure in the apo form and when bound to thyroxine - a TTR natural ligand-, and to flufenamic acid (FA), a NSAID. Different from what has been observed with other TTR variants, the structure of A25T showed profound alterations in relation to the wt-ttr structure, not only in the vicinity of position 25, but spread throughout the whole tetrameric structure. The two ligands were able to correct part of the structural alterations caused by the mutation. Combining an array of techniques, we analyzed A25T aggregation kinetics in buffer at ph 5.0, 6.0 and 7.0, as well as at physiological environments such as human plasma and CSF. AFM revealed that, at these ph values, A25T formed aggregates with different morphologies and susceptibility to proteinase K digestion. A25T was also able to aggregate massively at CSF and in plasma forming in the former case amyloid fibrils with a peculiar morphology. FA and T4 blocked completely the aggregation of A25T in CSF. Through the use of high hydrostatic pressure, we showed that A25T is 3.1 kcal/mol less stable than L55P. We also showed that amyloid formation in plasma and CSF arrested several protein components present in these complex milieu. 29

30 Cardiomiopathies caused by Defective Muscle Proteins (Sorenson s laboratory): The main focus of the muscle research group of AL2 is on defective proteins that lead to cardiomyopathies, as well as associated mechanisms of action of normal contractile and regulatory proteins. A project involving human cardiac troponin C (TnC) mutants that are associated with hypertrophic cardiomyopathies has led to a characterization of Ca 2+ off-rates from the regulatory Ca-binding sites I and II of TnC. Increasing complexity of the reconstituted system (from isolated TnC to the whole troponin complex to filaments reconstituted with actin and troponin to filaments containing actin, troponin and myosin cross-bridges ([subfragment 1]) produced progressively closer approximations of the increase in Ca 2+ sensitivity observed with intact skinned fibers containing these mutants. For these experiments our fast-kinetics capabilities were combined with the fluorescently labeled human cardiac mutants and other myofibrillar proteins produced by our collaborators at Univ. of Miami and our doctoral student DP Reynaldo performed the stopped-flow experiments (Pinto et al, 2010). The data show that cross-bridges contribute significantly to the cardiomyopathic phenotype, even though the mutation occurs in TnC and not in myosin. These same mutants (A8V, C84Y, D145E and E134D) are under analysis in Rio for changes in the C-domain of TnC, a region normally associated only with anchoring the protein on the thin filament. A second project aimed at characterizing a dilated cardiomyopathy double mutation of human cardiac TnC (E59D/D75Y) successfully demonstrated that there are ~25% fewer sites available for cross-bridges to bind to the thin filament when the double mutant is used to reconstitute the filaments in vitro. These results, which involved titrating fluorescently labeled thin filaments with myosin heads, explain why there is a reduction in isometric force when skinned myocytes contain the double mutant (Dweck et al, 2010). An additional project led to characterization of the importance of redox state for a cysteine residue located in the central helix of skeletal muscle TnC. When this cysteine is fully reduced, TnC s affinity for the thin filament is greater than when it is oxidized. This means that redox state may modify the ability of TnC to transmit messages along its axis from the thin filament (bearing cross-bridges) up through the C-domain to the N-domain, where Ca regulation occurs (Pinto et al, 2010). Biological and Medicinal Chemistry of Infectious and Degenerative Diseases (Mauricio s Laboratory): Laboratory for Pharmaceutical Biotechnology at the School of Pharmacy at UFRJ: Diabetes and amyloidosis amylin is a peptide secreted by islet beta cells in conjunction with insulin, involved in the regulation of several metabolic properties. Therapeutic use of amylin is limited by its low water solubility and propensity to form amyloid deposits. We have developed a nanostructured confined polymeric system (NPhIAPP) from which amylin is sustained 30

31 and controlled released. Pharmacological evaluation shows that subcutaneous administration of NPhIAPP is able to reduce glycemia in mices for long periods. We believe that amylin nanoconfinement in varying matrices is a promising approach for the therapeutic replacement of basal levels of amylin. Coagulopathies blood haemosthasis control affects several diseases. On of the most important enzymes in the blood cascade is thrombin. One of our projects focuses in the characterization of thrombin ligands as lead compounds in the treatment of Coagulopathies. We have recently characterized the mechanism of interaction between thrombin and suramin, a naphtyl sulfonated urea. Suramin interaction with thrombin is mediated through anion exosite II, in a dual mode: activation and inhibition. Crystallographic analysis revealed thrombin dimerization mediated by suramin, which was confirmed by small angle X-ray analysis. A larger investigation with a series of suramin analogues of varying sizes and substituents in the methyl group of suramin indicated that the inhibitory component correlates with a dimerization event, whilst the activation component is linked to direct binding of suramin to monomeric thrombin. We believe that other sulfonated compounds of natural occurrence may be involved in physiological modulation of thrombin, acting as a molecular switch between a procoagulant and inhibitory pathways. Nucleic acid interference nucleic acid recognition is a key regulatory during all stages of life and physiopathological processes. This fenomena is usually performed by large molecular protein assemblies, comprising a nucleic acid binding and recognition domain and one or more protein regions responsible for interaction with other molecular partners. Besides the dimension of the nucleic acid binding proteins, only a small segment is usually responsible for binding and recognition. Moreover, specific recognition is in part driven by a recognition code between nucleic acid base and aminoacid side chain. We have designed small peptides that target cognate DNA sequences according to the recognition code, which proved to be highly selective for the specific DNA sequences. Further optimization in peptide stability and derivatization are currently in development in order to optimize binding affinity and selectivity. The simplicity and easy of peptide design brings us on step toward the specific nucleic acid targeting with biologically compatible biopharmaceuticals for the use in varying biotechnological needs. Group publications ( ): 1.Pinto JR, Reynaldo DP, Parvatiyar MS, Dweck D, Liang JS, Jones MA, Sorenson MM, Potter JD (2010) Strong crossbridges potentiate the Ca 2+ affinity changes produced by HCM-cardiac troponin C mutants in myofilaments. A fast kinetic approach. J Biol Chem, under review. 2.Pinto JR, Sousa VP, Sorenson MM (2010) Redox state of troponin C cysteine in the D/E helix alters the C-domain affinity for the thin filament of vertebrate striated muscle. Biochim Biophys Acta Gen Subj, accepted 3.Dweck D, Reynaldo DP, Pinto JR, Potter JD (2010) A dilated cardiomyopathy troponin C mutation lowers contractile force by reducing strong myosin-actin binding. J Biol Chem. 285(23): Bomfim TR, Machado LE, Lima LM, Sorenson MM, Salerno VP.(2010) 2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1. Arch Biochem Biophys Sep 29. doi: /j.abb The binding of synthetic triiodo l-thyronine analogs to human transthyretin: Molecular basis of cooperative and non-cooperative ligand recognition. 31

32 Trivella DB, Sairre MI, Foguel D, Lima LM, Polikarpov I. J Struct Biol ahead of print]pmid: Novel Zn2+-binding sites in human transthyretin: implications for amyloidogenesis and retinolbinding protein recognition. Palmieri L de C, Lima LM, Freire JB, Bleicher L, Polikarpov I, Almeida FC, Foguel D. J Biol Chem. 2010, 8;285(41): Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1). Varejão N, Almeida Mda S, De Cicco NN, Atella GC, Coelho LC, Correia MA, Foguel D. Biochim Biophys Acta. 2010, 1804(9): Identification of archaeal proteins that affect the exosome function in vitro. Luz JS, Ramos CR, Santos MC, Coltri PP, Palhano FL, Foguel D, Zanchin NI, Oliveira CC. BMC Biochem. 2010, 27;11: Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: implications to tetramer stability and ligandbinding. Trivella DB, Bleicher L, Palmieri Lde C, Wiggers HJ, Montanari CA, Kelly JW, Lima LM, Foguel D, Polikarpov I. J Struct Biol (3): Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp. Cano-Estrada A, Vázquez-Acevedo M, Villavicencio-Queijeiro A, Figueroa-Martínez F, Miranda- Astudillo H, Cordeiro Y, Mignaco JA, Foguel D, Cardol P, Lapaille M, Remacle C, Wilkens S, González-Halphen D. Biochim Biophys Acta. 2010, 1797(8): Immunome and venome of Bothrops jararacussu: a proteomic approach to study the molecular immunology of snake toxins. Correa-Netto C, Teixeira- Araujo R, Aguiar AS, Melgarejo AR, De-Simone SG, Soares MR, Foguel D, Zingali RB. Toxicon. 2010, 15;55(7): Identification of a novel ligand binding motif in the transthyretin channel. Lima LM, Silva Vde A, Palmieri Lde C, Oliveira MC, Foguel D, Polikarpov I. Bioorg Med Chem. 2010, 1;18(1): Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins. Silva JL, Vieira TC, Gomes MP, Bom AP, Lima LM, Freitas MS, Ishimaru D, Cordeiro Y, Foguel D. Acc Chem Res. 2010, 16;43(2): Reciprocal remodeling upon binding of the prion protein to its signaling partner hop/sti1. Romano SA, Cordeiro Y, Lima LM, Lopes MH, Silva JL, Foguel D, Linden R. FASEB J. 2009,23(12): A fluorescent mutant of the NM domain of the yeast prion Sup35 provides insight into fibril formation and stability. Palhano FL, Rocha CB, Bernardino A, Weissmuller G, Masuda CA, Montero-Lomelí M, Gomes AM, Chien P, Fernandes PM, Foguel D. Biochemistry. 2009, 28;48(29): The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric. Villavicencio-Queijeiro A, Vázquez-Acevedo M, Cano-Estrada A, Zarco-Zavala M, Tuena de Gómez M, Mignaco JA, Freire MM, Scofano HM, Foguel D, Cardol P, Remacle C, González-Halphen D. J Bioenerg Biomembr. 2009, 41(1): Hydration, cavities and volume in protein folding, aggregation and amyloid assembly. Silva JL, Foguel D. Phys Biol. 2009, 10;6(1) 18. Predictions suggesting a participation of betasheet configuration in the M2 domain of the P2X(7) receptor: a novel conformation? Teixeira PC, de Souza CA, de Freitas MS, Foguel D, Caffarena ER, Alves LA. Biophys J. 2009, 96(3): Trapping the monomer of a nonamyloidogenic variant of transthyretin: exploring its possible use as a therapeutic strategy against transthyretin amyloidogenic diseases. Palhano FL, Leme LP, Busnardo RG, Foguel D. J Biol Chem. 2009, 16;284(3): Martinez, Leandro ; Souza, Paulo C. T. ; Garcia, Wanius ; Batista, Fernanda A. H. ; Portugal, Rodrigo V. ; Nascimento, Alessandro S. ; Nakahira, Marcel ; Lima, Luis M. T. R. ; Polikarpov, Igor ; Skaf, Munir S. On The Denaturation Mechanisms Of The Ligand Binding Domain Of Thyroid Hormone Receptors. Journal Of Physical Chemistry. B, V. 114, P , Lima, Luis Mauricio T R ; Figueira, Ana Carolina Migliorini ; Lima, Leonardo H.F. ; Ranzani, Americo T. ; Mule, Guilherme Dos Santos ; Polikarpov, Igor. Recognition By Thyroid Hormone Receptor Of Canonical Dna Response Elements. Biochemistry (Easton), V. 49, P , Souza, Fernando Gomes ; Marins, Jéssica Alves ; Pinto, José Carlos ; Oliveira, Geiza Esperandio ; Rodrigues, Cezar Manzini ; Lima, Luis Mauricio T. R.. Magnetic Field Sensor Based On A Maghemite/Polyaniline Hybrid Material. Journal Of Materials Science, P. 3000, Marques, A ; Cordeiro, Y ; Silva, J ; Lima, L ; Lima, Luis Mauricio T. R.. Enhanced Prion Protein Stability Coupled To Dna Recognition And Milieu Acidification. Biophysical Chemistry, P. 1-5, Lima, Luis Maurício T.R. ; Becker, Camila Franco ; Giesel, Guilherme Menegon ; Marques, Adriana Fonseca ; Cargneluti, Maria Thereza ; De Oliveira Neto, Mario ; Queiroz Monteiro, Robson ; Verli, Hugo ; Polikarpov, Igor. Structural And Thermodynamic Analysis Of Thrombin:Suramin Interaction In Solution And Crystal Phases. Bba. Proteins And Proteomics, V. 1794, P. 1-9, Ishimaru, D. ; Anobom, Ap ; Lima, Luis Mauricio T. R. ; Quesado, P. A. ; Galo, C ; Cordeiro, Y. M. L. ; Silva, Jerson L.. Cognate Dna Stabilizes The Tumor Suppressor P53 And Prevents Misfolding And Aggregation. Biochemistry (Easton), V. 48, P , Casanova, Fabiana ; Nakamura, Angelica ; Masuda, Hatsaburo ; Lima, Luis Mauricio T. R. ; Fialho, Eliane. Functionality Of Phosphorylated Vicilin Exposed To Chemical And Physical Agents.. Food Chemistry, V. 107, P ,

33 AL3 ASSOCIATE LABORATORY OF PROTEINS STRUCTURE DETERMINATION BY NMR. Coordinator: Fábio Almeida, Instituto de Bioquímica Médica, UFRJ. Members: Ana Paula Valente Ronaldo Mohana Borges José Ricardo Pires Marcius da Silva Almeida AL3 is directely related to CNRMN and the use of nuclear magnetic resonance as a tool to solve structure and dynamics of biomolecules, probe interactions and in the development of biologically active compund. As a group we have published 18 papers from January, 2009 to October, As a remarkable result by the group was the development of one anti-angiogenic compound (dlpr) in collaboration with M.D. Anderson Cancer Center. NMR and phage display were the techniques that enabled these achievements. The results were published in PNAS (Giordano et al., 2010) and were patented. The new generation of biologically active compounds developed during the 20th century relied on knowledge of enzymology and protein structure, and were based initially, on the understanding that protein-protein and small molecule-protein interactions occurred through a lock-and-key mechanism. Later, evidence suggested that this mechanism was usually followed by a conformational change, known as induced fit. Recent studies on protein dynamics, mainly by nuclear magnetic resonance (NMR) relaxation measurements, have shown that 33

34 proteins are not structured in a unique conformation. Rather, they frequently have regions of conformational diversity. Our group used a novel view of binding, put forward in by several research groups in the last 5 to 10 years. In the free state, protein regions displaying conformational diversity exhibit equilibria among pre-existing conformations. In the presence of a ligand, one of these conformations is stabilized, so that the ligand does not need to induce a new conformation. Upon ligand binding there is a population shift toward the bound conformational state. Conformational diversity of binding sites of several proteins has been measured and has important practical as well as thermodynamical consequences: binding sites can be mapped without prior knowledge of the ligand and also evolution of binding sites depends mostly on the free state, occurring at least partially independently of the ligand (Valente et al., 2006). Discrete bound conformations of the anticcidial peptide PW2 in dpc micelles (Fábio C. L. Almeida) Protein and peptide structure determination is strongly influenced by the internal dynamics. Proteins and peptides display flexible regions that undergo motions that are either thermal or slower, related to conformational diversity. The regions that display conformational diversity are frequently part of binding epitopes related to biological important events. Nevertheless, these regions are often challenging for the current methods for structural determination, which leads to timeaveraged structures. We have previously solved the structure of the anti-coccidial peptide PW2 in the presence of SDS, DPC and other mimics of membrane environment. Here we applied a diverse set of strategies of restrained molecular dynamics simulations (MD) of PW2 in explicit DPC micelles. The MD runs enabled the calculation of discrete conformational states, dissociating the effects of conformational averaging. Spectroscopic characterization of a The restrained MD of PW2 interacting with a 65 DPC molecules micelle. In the beginning of MD there is a fast increment of detected clusters (0 to 1.5ns). Between 1.5 to 7 ns there is a period of instability of the binding. After this there is a complete interaction of PW2 with the micelle surface with events of partial detachment showed by new cluster index increments (Gomes-Neto et al., 2010 in preparation). Truncated hemoglobin from the nitrogenfixing bacterium Herbaspirillum seropedicae (Fábio C. L. Almeida) The Herbaspirillum seropedicae genome sequence encodes a truncated hemoglobin typical of group II (Hs-trHb1) members of this family. We show that Histagged recombinant Hs-trHb1 is monomeric in solution, and its optical spectrum resembles those of previously reported globins. NMR analysis allowed us to assign 34

35 heme substituents. All data suggest that HstrHb1 undergoes a transition from an aquomet form in the ferric state to a hexacoordinate lowspin form in the ferrous state. The close positions of Ser-E7, Lys-E10, Tyr-B10, and His-CD1 in the distal pocket place them as candidates for heme coordination and ligand regulation. Peroxide degradation kinetics suggests an easy access to the heme pocket, as the protein offered no protection against peroxide degradation when compared with free heme. The high solvent exposure of the heme may be due to the presence of a flexible loop in the access pocket, as suggested by a structural model obtained by using homologous globins as templates. The truncated hemoglobin described here has unique features among truncated hemoglobins and may function in the facilitation of O2 transfer and scavenging, playing an important role in the nitrogen-fixation mechanism. (Razzera et al., 2009) From combinatorial peptide selection to drug prototype: targeting the vascular endothelial growth factor receptor pathway (Fábio C. L. Almeida) Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesisdependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated D(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models. In addition to its systemic activity, D(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retroinversion for rapid drug discovery and development. (Giordano et al., 2010, patent) Backbone dynamics of the antifungal psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy (Fábio C. L. Almeida/Ana Paula Valente) Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the socalled cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow 35

36 motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (peploop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH. Portrayal of complex dynamic properties of Sugarcane defensin 5 by NMR: Multiple motions associated with membrane interaction (Ana Paula Valente) Defensins are essentially ancient natural antibiotics with potent activity extending from lower organisms to humans. Sd5 is a recently described antifungal defensin that appears to be the result of a recent gain of function. We reported the solution NMR structure of Sd5 and characterized the backbone dynamics in the free state and in the presence of membrane models. 15 N relaxation dispersion measurements, indicate intrinsic conformational exchange processes, showing two clear distinct kex, 490 and 1800 s -1. These Specific Interaction of PSD1 with CMH. PSD1 first searches the two-dimensional conformational space of the membrane via coulombic atraction via a basic patch. Once the specific target in the membrane (CMH, in cyan) is reached, the protein flips and initiate its internalization. The resisues that participate in the specific binding to CMH are colored in red. multiple motions may be related to transient twisting or breathing of the -helix and -sheet. The stages of membrane recognition and disruption by Sd5 over a large time scale range were mapped and demonstrated that Sd5 in solution sampled an ensemble of different conformations, of which a subset is selected upon membrane binding. Defensins share similar structures, but we demonstrated here that their dynamics can be extremely diverse. Structure of SD5 by solution NMR 36

37 Mapping the interactions between a major pollen allergen and human IgE antibodies. (Ana Paula Valente) The interaction of specific IgE antibodies with allergens is a key event in the induction of allergic symptoms, thus representing an important target for therapeutic interventions in Type I allergies. We report here the solution NMR structure of Art v 1, the major mugwort pollen allergen. Art v 1 is the first protein structure with an allergenic defensin fold linked to a polyproline domain, which has not been identified in any reported allergen structure in the PDB. Moreover, the direct interaction of polyclonal IgE antibodies from an allergic patient has been mapped on the surface of an allergen for the first time. Our data provide the basis for the design of tools for safe and effective vaccination against mugwort pollen allergy. Specific recognition of Art v 1 to IgE antibodies. Design of hypoallergenic peptides. NMR Structures of Protein from Parasites and Vector Related to Neglected Infectious Diseases (José Ricardo M. Pires) The structures of several important proteins from infectious diseases were characterized as describe below. Structure and mode of action of microplusin, a copper II-chelating antimicrobial peptide from the cattle tick Rhipicephalus (Boophilus) microplus (José Ricardo M. Pires) Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. We described the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied the action mechanism of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also 37

38 demonstrated that microplusin affects M. luteus respiration, a copperdependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II. KSC Domain (José Ricardo M. Pires). Calpains are a big family of calcium dependent cisteine-proteases. They are modular proteins and contain, just before the catalytic domain, and extra domain that is exclusive for kinoplatids(ksc- Kinetoplastid-specific relatated do calpains). KSC does not show similarity to any protein with known function. We determined the solution structure of the T. cruzi (Access code Unitprot: Q4CYU3) protein. This protein contain only the domain KSC. The structure is a beta-sandwich, containing two beta-sheet. The folding is similar to known hydrolases but o far we could not demonstrate this funtion. The hydrolase activity is not probable since the amino acid signature is missing. We also studied the dynamics of the Structure of domain KSC: 15 lowest energy structure. Microplusine structure in solution proteins, which are well ordered in its majority, showing flexibility in the N- and C-terminal. The beta strands β5 e β6 show motion in the micro to millisecond timescale. Structure and function of Cancer-related proteins (Prof. Marcius S. Almeida) Representing an initiative in human cancer structural proteomics, the aim of this work is the structural and functional characterization of cancer-related proteins. The specific objective is the structural characterization of some of these targets, which are described bellow: BEX3 (Brain Expressed X-linked). BEX3 mediates apoptosis in response to Neurotrophin Growth Factor by interacting with the death domain of p75ntr and some other downstream mediators. Analysis by NMR, circular dichroism, and fluorescence spectroscopy showed that this protein has a high content of flexibly disordered regions in addition to significant secondary and tertiary structures. Small Angle X-ray Scattering indicates that BEX3 is a defined oligomer of approximately 200 kda, which would 38

39 embrace 14 subunits. Surface plasmon resonance binding experiments using Biacore X system revealed binding and dissociation of BEX3:1-106/p75-NTR with Kd of 1.4 um (per BEX3 subunit), which fits to an one-site binding curve (R2 = ). The binding of BEX3 to p75- NTR has been mapped by NMR. Programmed cell death 10 (PDCD10). PDCD10 was purified by hydrophobic interaction chromatography and gel filtration chromatography. The protein was obtained as a dimer. NMR experiments showed that this protein was structured, and circular dichroism showed a high content of alpha helix. Crystallization trials are being performed, and crystals with 3.6 Å resolution have been obtained. Two hybrid associated protein 1 (TWA- 1). TWA-1 has been purified by ion interaction chromatography followed by hydrophobic interaction chromatography and gel filtration chromatography. The protein has propensity to form instable aggregates. A condition has been found where the protein forms a stable dimer, and crystallization trials are being performed. Structure and function of Neurotrophic factors (Prof. Marcius S. Almeida) Conserved Dopamine Neurotrophic Factor (CDNF). This protein showed a neurotrophic activity on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat experimental model of Parkinson s Disease. CDNF has two domains; a N-terminal saposinlike domain, which may bind to membranes; and an presumably intrinsically unstructured C- terminal which contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus reduce the endoplasmic reticulum stress caused by incorrectly folded proteins. We show for the first time the production of a soluble monomer of CDNF in Escherichia coli. This recombinant CDNF, contains four disulfide bounds, and was able to protect the dopaminergic neurons isolated from the embryonic day 14 mouse mesencephalon from the damage caused by 6-OHDA. We are also characterizing the proteolytic activity that separates the two domains of CDNF. Glial cell-line Derived Neurotrophic Factor (GDNF). Our goal is the study of GDNF mimetic peptides interaction with their GFRalpha1 co-receptor (GDNF-Family Receptor alpha 1) to identify GDNF regions that activate this co-receptor and trigger biological activity, for development of small molecules to treatment of neurodegenerative disorders. DNA sequences encoding a mature GDNF, and its heel region (P9, a sequence of 15 amino acids which shows biological activity and binds to its co-receptor) and GFRalpha1 domains were subcloned into vectors for Escherichia coli. The best expression conditions have been tested to give the highest level of soluble recombinant protein production for subsequent purification of these various constructions. Recombinante and synthetic P9 were produced for structural analysis by Nuclear Magnetic Resonance and Circular Dichroism. Such as GDNF, P9 can be most biologically active as covalently linked homodimers. Thereby, we are also engaged on synthesis of tetrameric and dimeric dendrimers of P9. Activity assays for P9 using dopaminergic neurons of 14 days-old embryos or SH-SY5Y neuroblastoma cells have been used for 39

40 biological trials using 6-hydroxidopamine (6- OHDA) as a model for Parkinson s disease. Structure and Thermodynamics of DNA binding proteins and Virus Fusion Proteins (Prof. Ronaldo M. Borges) In the first goal, we correlated the oligomeric state of the proteins UmuD e UmuD, which function is mutagenesis and DNA repair in E. coli. The findings were published in PNAS (Simon et al., PNAS, 2008) in collaboraton with Dr. Graham C Walker from MIT (EUA). The second goal we could characterize the process of membrane fusion of the virus Dengue II. Dengue virus (DV) infection depends on a step of membrane fusion, which occurs in the acidic environment of the endosome. This process is mediated by virus surface envelope glycoprotein, in which the loop between residues D98-G112 is considered to be crucial, acting as a fusion peptide. We have characterized functionally and structurally the interaction between the DV fusion peptide and different model membranes by fluorescence and NMR. Its interaction was strongest in dodecylphosphocholine (DPC) micelles and anionic phosphatidylcholinephosphatidylglycerol vesicles, the only vesicle that was fused by DV fusion peptide. The threedimensional structure of DV fusion peptide bound to DPC micelles was solved by solution homonuclear NMR with an r.m.s.d. of 0.98 A. The most striking result obtained from the solution structure was the hydrophobic triad formed by residues W101, L107, and F108, pointing toward the same direction, keeping the segment between G102 and G106 in a loop conformation. The interaction of DV fusion peptide with phosphatidylcholinephosphatidylglycerol vesicles was also mapped by transfer-nuclear Overhauser enhancement (NOE) experiments, in which the majority of the NOE cross-peaks were from the hydrophobic triad, corroborating the DPC-bound structure. Substitution of the residue W101 by an alanine residue completely abolished membrane binding and, thus, fusion by the peptide and its NOE cross-peaks. In conclusion, the 15-residue DV fusion peptide has intrinsic ability to promote membrane fusion, most likely due to the hydrophobic interaction among the residues W101, L107, and F108, which maintains its loop in the correct spatial conformation. 3D conformation of the DV fusion peptide segment W101 F108 of the full-length E protein at different fusogenic conditions. In (a) and (c) are represented the DV fusion peptide in pre- and post-fusogenic states, respectively, solved by X-ray crystallography,7,8 and in (b) when it is bound to DPC micelles in the fusogenic state solved by NMR (our group). 40

41 Group publications ( ): 1. de Souza TL, Sanches D, Gonçalves RB, da Rochapita SS, Pascutti PG, Bianconi ML, de Almeida FC, Silva JL, de Oliveira AC. Conformational selection, dynamic restriction and the hydrophobic effect coupled to stabilization of the BIR3 domain of the human X-linked inhibitor of apoptosis protein by the tetrapeptide AVPI. Biophys Chem Aug 17. [Epub ahead of print] 2. Angeli, R., MEDEIROS, L. N., Sarzedas, C. G., E, Barreto Bergter, VALENTE, Ana Paula, E, Kurtenbach, ALMEIDA, F. C. L Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy. In Biochimica et Biophysica Acta. Biomembranes., v.1798, GIORDANO, Ricardo José, CARDOVILA, Marina, Salameh, A., ANOBOM, Cristiane Diniz, ZEITLIN, B. D., Hawke, D.H., VALENTE, Ana Paula, ALMEIDA, F. C. L., Nör, J.E., Sidman, R.L., PASQUALINI, Renata, ARAP, Wadih From combinatorial peptide selection to drug prototype (I): Targeting the vascular endothelial growth factor receptor pathway In Proceedings of the National Academy of Sciences of the United States of America., v.107, Razzera g, GADERMAIER, G., De Paula, V., ALMEIDA, Marcius da Silva, Egger, M., Jahn-Schmid, B., ALMEIDA, F. C. L., Ferreira, F., VALENTE, Ana Paula Mapping the interactions between a major pollen allergen and human IgE antibodies. In Structure (London)., v.18, PALMIERE, L. C., Lima, L.M.T.R, Freire, J.B., Bleicher, L., Polikarpov, I., ALMEIDA, F. C. L., FOGUEL, Debora Novel Zn2+-binding sites in human transthyretin: implications for amyloidogenesis and retinol binding protein recognition. In The Journal of Biological Chemistry (Print)., v.2010, Pereira da Silva, A.P., El-Bacha, T., Dos Santos, R.S., Da Silva, W.S., ALMEIDA, F. C. L., Dapoian, A.T., Galina, A Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate In Biochemical Journal (London)., v.417, Melo, M.N., Sousa F.J., Carneiro F.A., Castanho, M.A., VALENTE, Ana Paula, ALMEIDA, F. C. L., Dapoian, A.T., Mohana-Borges R Interaction of the Dengue Virus Fusion Peptide with Membranes Assessed by NMR: The Essential Role of the Envelope Protein Trp101 for Membrane Fusion. In Journal of Molecular Biology., v.392', Razzera g, GADERMAIER, G., ALMEIDA, Marcius da Silva, Ferreira, F., ALMEIDA, F. C. L., VALENTE, Ana Paula, ABREU, Hector Nicolas Seuanez Sequence-specific 1H, 15N and 13C resonance assignments of Art v 1: a proline-rich allergen of Artemisia vulgaris pollen. In Biomolecular NMR Assignments., v.3, Verly, R.M., De Moraes, C.M., Resende, JM, Aisenbrey C., Bemquerer, MP, Piló-Veloso, D., VALENTE, Ana Paula, ALMEIDA, F. C. L., Bechinger, B Structure and membrane interactions of the antibiotic peptide dermadistinctin K by multidimensional solution and oriented 15N and 31P solid-state NMR spectroscopy. In Biophysical Journal., v.96, Luciano Medeiros, Angeli R, Barreto-Bergter E., Valente, A.P., KURTENBACH, E., ALMEIDA, F. C. L Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction. In Biochimica et Biophysica Acta. Biomembranes., v.1798, Cruz AKM, Andrade GPV, Chavante SF, de Vasconcelos CL, Garcia RB, Leite EL, Valente, A.P., Sales MP, Oliveira FW Structural elucidation of n galactan from the eggs of mollusc Pomacea lineata In Carbohydrate Polymers., v.79, L.P. Cinelli, Andrade LR, Valente, A.P., MOURÃO, P. A. S Sulfated alpha-l-galactans from the sea urchin ovary: selective 6-desulfation as eggs are spawned. In Glycobiology (Oxford)., v.20, Ano Bom, A. P., Freitas M, Moreira FS, Ferraz D, Sanches D, GOMES, A. M., Valente, A.P., Y, C., SILVA, J. L The p53 core domain is a molten globule at low ph: Functional implications of a partially unfolded structure. In The Journal of Biological Chemistry (Print)., v.01, Angeli R, DaPaz NVM, Maciel JC, Araujo FFB, Paiva PGM, Calazans GMT, Valente, A.P., ALMEIDA, F. C. L., Coelho LCBB, Silva MPC, Correia MTS Ferromagnetic Levan Composite: an Affinity Matrix to Purify Lectin In Journal of Biomedicine and Biotechnology., v.01, Rezende CA, Silva FD, Daffre S, Pires JR. (2009) (1)H, (15)N and (13)C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin. Biomol NMR Assign. 3: Silva FD, Rezende CA, Rossi DC, Esteves E, Dyszy FH, Schreier S, Gueiros-Filho F, Barbosa C, Pires JR, Daffre S. (2009) Structure and mode of action of microplusin, a copper II chelating antimicrobial peptide from the cattle tick Rhipicephalus (Boophilus) Microplus. J Biol Chem. 284: Varejão N, Almeida MS, De Cicco NN, Atella GC, Coelho LC, Correia MA, Foguel D. Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1). Biochim Biophys Acta Sep;1804(9): Epub 2010 Jun DOMITROVIC, T. ; Kozlov G ; Freire, J.C.G. ; Masuda, C.A. ; ALMEIDA, M. S. Montero-Lomeli, M. ; Atella, G.C. ; Matta-Camacho, E. ; Gehring K ; KURTENBACH, E.. Structural and Functional Study of Yer067w, a New Protein Involved in Yeast Metabolism Control and Drug Resistance. Plos One, v. 5, p. e11163, DIAZ, A., FONTANA, E. C., TODESCHINI, A. R., SOULE, S., GONZALEZ, H., CASARAVILLA, C., PORTELA, M., MOHANA-BORGES, R., MENDONCA- PREVIATO, L., PREVIATO, J. O., FERREIRA, F. The Major Surface Carbohydrates of the Cyst: Mucin-Type- Glycans Decorated by Novel Galactose-Based Structures. Biochemistry (Easton), v. 48, p , 2009.

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43 AL4 Members: ASSOCIATE LABORATORY OF PHARMACOLOGIC PROTEOMIC. Robson Monteiro Márcia Soares Clarissa Damaso Bianca Cruz Neves Coordinator: Russolina Zingali, Instituto de Bioquímica Médica, UFRJ. The Proteomic Unity has been serving many laboratories from INBEB for the determination of mass from recombinant proteins to peptides in order to confirm correct expression and/or synthesis (Laboratories from Dr Foguel, Dr Almeida, Dr da Silva, Dr Bisch, etc). Furthermore, the unit has collaborated in projects that envisage the proteomic characterization of biological processes such as sugar cane wall analysis, interaction of mesenchimals and renal cells, Gluconacetobacter interaction with sugar cane, protein modification, etc... (Dr. Souza, Dr. Vieyra, Dr. Bisch, Dr. Soares, Dr Souto-Padron). Main research projects where the group participates: 1. Proteomics, Genomics and Bioinformatics in the study of the interaction between pathogenic and non-pathogenic microorganisms with their hosts. 2. Prospective proteomics. Results obtained for these projects: 43

44 Study of Gluconacetobacter diazotrophicus interaction with plant Recently as a consequence of previous results obtained with the analyses of Gluconacetobacter diazotrophicus we started the study of the interaction between the bacteria and Arabidopis thaliana. Arabidopsis thaliana was chosen as study model, because its genome was fully sequenced. Arabidopsis plants were inoculated with Gluconacetobacter in hydroponic medium, and after 3 days of inoculation the plants had been separated in leaves and roots. The extracts of both segments were submitted to 1D SDS- PAGE. The extracts of inoculated leaves showed to be enhanced by three major proteins bands between KDa. The trypsinized proteins have been analyzed using proteomics technologies. Some of them were identified being involved in metabolism process as aldolase, GAPDH and photosynthesis as PSBO2, LHCP AB 180 by ESI-Q-TOF. Mass spectra analyses resulted in the identification of 149 proteins most of them induced by the bacteria. We have demonstrated that the inoculation with G. diazotrophicus changes the pattern of protein expression of Arabidopsis leaf and root. Dengue virus infection In order to identify proteins and peptides associated with Dengue virus, the secreted proteins of HepG2 cells (infected or not) were analyzed by mass spectrometry. The identification of proteins >10 kda resulted in the publication by Higa et al., We also analyzed the pool of peptides (<10kDa) by ESI- Q-TOF. Up to now, 7 molecules were identified. The method for the analysis of peptide is in develepment to optimize the results and to identify more peptides. Furthermore we have also looked at the phosphorilation profile of infected cells. The phosphorilation profile of cellular proteins was establisehed for 30 min, 2 and 8 hours after the infection. Proteins were submitted to the Pro-Q Diamond staining that resulted in different profile of phosphorilation probably induced by the infection. The identification of this proteins by LC-MS/MS and also the purified phosphorilated peptides are under analysis. Snake venom proteomics The immunological recognition of B.jararacussu venom by the antisera was determined. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis (Figure 1). Western blots revealed that anti-jararacussu showed higher reactivity to L-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards snake venom serine proteases (SVSPs), PLA2, C-type lectin and cysteine-rich proteins. While anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs and Anticrotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA2. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA2. These results were published in Toxicon. Another venom that was explored by our group was from the snake genus Micrurus. To gain clues for outlining an alternative antiserum generation strategy based on immunization we 44

45 have characterized the proteome and the venom gland transcriptome of M. altirostris (Figure 2). The venom proteome contained around 50 proteins belonging to eight toxin families. Neurotoxins (73%) and PLA 2 s (14%) comprise the major toxin families. Other toxin groups included 3FTxs (7%), Kunitz-type inhibitors (2%), PIII-SVMPs (1%), CTLs (<0.1%), CRISP (<0.1%), and unknown molecules (4%). The transcriptome (from the pooled glands of an adult and a neonate specimens) contained 946 ESTs grouped into 384 clusters. Venomic and transcriptomic data provided full-length amino acid sequences for the major M. altirostris toxins. This information allowed homology 3Dmodel-based design of neurotoxin- and PLA 2 - derived peptide stretches as targets for antibody generation in rabbits. The capability of the corresponding antisera to neutralize the toxic activities of M. altirostris venom is being investigated in our laboratories. This results were obtained in collaboration with Dr Inacio Junqueira de Azevedo (Instituto Butantan) and Dr Juan Calvete (Instituto de Biomedicina de Valencia,Spain) The manuscript is in preparation. Analysis of protein-protein interaction Bothrojaracin is a protein from Bothrops jararaca venom that forms complex with thrombin, blocking biological activities. Here we analyze the extent of bothrojaracin-thrombin interaction using footprinting and ESI Q-TOF spectrometer. After the induction of oxidation in controled medium samples were digested using trypsin. MS/MS fragmentation reveal seven different oxidized peptides, with KDa, KDa, KDa, KDa, KDa, KDa and KDa for bothrojaracin molecules with at least one site of oxidation (table 2). Mass spectrum of thrombin showed eighth different oxidized peptides as KDa, KDa, KDa and KDa, with multiple sites of oxidation. Oxidation of the complex was performed with the same protocol and the mass spectra clearly demonstrate protected sites in the presence of bothrojaracin. We also performed deuterium exchange experiments for thrombin in the absence and presence of bothrojaracin; a new interacion site and conformational changes in thrombin molecule could be detected. We are also using molecular modeling to investifate the binding of disintegrin to integrins. Jararacin (JARC) and jarastatin (JAST) are disintegrins present in venom of Bothrops jararaca. They bind to and antagonize the adhesive functions of β3 integrin receptors, which complexes possess two members: αiibβ3, present in platelets, and αvβ3, widely distributed in various cells. In our study comparative analyses of the binding of these disintegrins to αiibβ3 and αvβ3 integrins were performed in silico, to observe the degree of affinity between these complexes. To perform this theoretical analysis, we constructed homology models of docking complexes using models of JARC, JAST and crystal structures of αiibβ3, αvβ3, and theoretically evaluated their interaction using a molecular modeling approach. Our results show that JARC has more interaction and more affinity with αiibβ3. On the other hand, JAST/αvβ3 presents more affinity than JARC/αvβ3. The results we obtained suggest that αiibβ3 has a higher affinity to structures 45

46 similar to JARC. On the other hand αvβ3 have affinity to structures such as the JAST. The analysis of complex dynamics is under evaluation in order to confirm our docking analysis. Exogenous factors as Anti-thrombotic and anti-cancer drugs Nitrophorin 2 (NP2) isolated from the blood sucking insect, Rhodnius prolixus is a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. The in vivo antithrombotic properties of NP2 were investigated. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (KD = 43 and 47 nm, respectively), and prolongs the aptt without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo, two distinct models of thrombosis in rats were carried out and demonstrate the effectiveness of this molecule. The antithrombotic effect lasted for up to 48 hours after a single i.v. dose. Notably, effective doses of NP2 potent and long-lasting inhibitor of arterial thrombosis with minor effects on haemostasis. Another protein studied by our group was the tick anticoagulant Ixolaris that had been previously proven to be an antithrombotic drug. We now have tested if it interferes with glioblastoma progression. We demonstrate that Ixolaris effectively blocked the in vitro TFdependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. did not increase the blood loss as evaluated by tailtransection model. NP2 showed to be a Fig. 1. 2D-PAGE of B. jararacussu venom and organization of proteins for identification by MS/MS. In the first dimension, 250 mg of B. jararacussu venom were applied to 3 10 IPG strips (7 cm) followed by an equilibrium step in non-reducing (A) or reducing conditions (B) (6 mm DTT and 2.5% w/v Iodoacetamide). The second dimension was carried out with 15% SDS- PAGE. The gels were stained with Coomassie blue G. The arrows indicate the spots that are different between the two conditions. The proteins spots submitted for identification were organized in groups and numbered with Arabic and Roman numbers in (C) 2D-PAGE under non-reducing and (D) reducing conditions. 46

47 B A C D Fig. 2- Reverse-phase HPLC separations of the Micrurus altirostris venom (A). Coomassie blue-stained SDS-AGE (B) showing the protein composition of the reverse-phase HPLC separated venom protein fractions displayed in the respective panel and run under nonreduced (upper panels) and reduced (lower panels) conditions. The composition of the Micrurus altirostris venom determined by venomics (C) and by venom gland transcriptomics (D). Group publications ( ): 1. Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: assessment of geographic variation and its implication on snakebite management. Boldrini-França J, Corrêa-Netto C, Silva MM, Rodrigues RS, De La Torre P, Pérez A, Soares AM, Zingali RB, Nogueira RA, Rodrigues VM, Sanz L, Calvete JJ. J Proteomics Aug 5;73(9): Comparative proteomic analysis of whole saliva from chronic periodontitis patients. Gonçalves Lda R, Soares MR, Nogueira FC, Garcia C, Camisasca DR, Domont G, Feitosa AC, Pereira Dde A, Zingali RB, Alves G. J Proteomics May 7;73(7): Immunome and venome of Bothrops jararacussu: a proteomic approach to study the molecular immunology of snake toxins. Correa-Netto C, Teixeira-Araujo R, Aguiar AS, Melgarejo AR, De- Simone SG, Soares MR, Foguel D, Zingali RB. Toxicon Jun 15;55(7): Keratinolytic activity of Bacillus subtilis AMR using human hair. Mazotto AM, Cedrola SM, Lins U, Rosado AS, Silva KT, Chaves JQ, Rabinovitch L, Zingali RB, Vermelho AB. Lett Appl Microbiol Jan;50(1): Effect of Moringa oleifera lectin on development and mortality of Aedes aegypti larvae. Coelho JS, Santos ND, Napoleão TH, Gomes FS, Ferreira RS, Zingali RB, Coelho LC, Leite SP, Navarro DM, Paiva PM. Chemosphere Nov;77(7): Engineering ecotin for identifying proteins with a trypsin fold. Sathler PC, Craik CS, Takeuchi T, Zingali RB, Castro HC. Appl Biochem Biotechnol Apr;160(8): Evaluation of sample preparation methods for the analysis of papaya leaf proteins through twodimensional gel electrophoresis. Rodrigues SP, Ventura JA, Zingali RB, Fernandes PM. Phytochem Anal Nov;20(6): Pharmacological action of tick saliva upon haemostasis and the neutralization ability of sera from repeatedly infested hosts. Reck J Jr, Berger M, Marks FS, Zingali RB, Canal CW, Ferreira CA, Guimarães JA, Termignoni C. Parasitology Sep;136(11): Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data. Valente RH, Guimarães PR, Junqueira M, Neves-Ferreira AG, Soares MR, Chapeaurouge A, Trugilho MR, León IR, Rocha SL, Oliveira-Carvalho AL, Wermelinger LS, Dutra DL, Leão LI, Junqueira-de- Azevedo IL, Ho PL, Zingali RB, Perales J, Domont GB. J Proteomics Mar 6;72(2): A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule. Pauer H, Ferreira Ede O, dos Santos-Filho J, Portela MB, Zingali RB, Soares RM, Domingues RM.

48 FEMS Immunol Med Microbiol Apr;55(3): Integrin inhibitors from snake venom: exploring the relationship between the structure and activity of RGD-peptides. Wermelinger LS, Geraldo RB, Frattani FS, Rodrigues CR, Juliano MA, Castro HC, Zingali RB. Arch Biochem Biophys Feb;482(1-2): Tissue factor expression on monocytes from patients with severe dengue fever. de Azeredo EL, Kubelka CF, Alburquerque LM, Barbosa LS, Damasco PV, Avila CA, Motta-Castro AR, da Cunha RV, Monteiro RQ.Blood Cells Mol Dis Sep 14. [Epub ahead of print] No abstract available. 13. Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis. Mizurini DM, Francischetti IM, Andersen JF, Monteiro RQ. Thromb Haemost Sep 13;104(6). [Epub ahead of print]pmid: Increased expression of tissue factor and protease-activated receptor-1 does not correlate with thrombosis in human lung adenocarcinoma. de Meis E, Azambuja D, Ayres-Silva JP, Zamboni M, Pinheiro VR, Levy RA, Monteiro RQ. Braz J Med Biol Res Apr;43(4): Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo. Calvo E, Tokumasu F, Mizurini DM, McPhie P, Narum DL, Ribeiro JM, Monteiro RQ, Francischetti IM. FEBS J Jan;277(2): Anticoagulant activity of a sulfated galactan: serpin-independent effect and specific interaction with factor Xa. Glauser BF, Rezende RM, Melo FR, Pereira MS, Francischetti IM, Monteiro RQ, Rezaie AR, Mourão PA. Thromb Haemost Dec;102(6): Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model. Carneiro-Lobo TC, Konig S, Machado DE, Nasciutti LE, Forni MF, Francischetti IM, Sogayar MC, Monteiro RQ. J Thromb Haemost Nov;7(11): Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines. Kirszberg C, Lima LG, Da Silva de Oliveira A, Pickering W, Gray E, Barrowcliffe TW, Rumjanek VM, Monteiro RQ. Melanoma Res Oct;19(5): Evidence for increased expression of tissue factor and protease-activated receptor-1 in human esophageal cancer. Ribeiro FS, Simão TA, Amoêdo ND, Andreollo NA, Lopes LR, Acatauassu R, Rumjanek FD, Albano RM, Pinto LF, Monteiro RQ. Oncol Rep Jun;21(6): Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner. Lima LG, Chammas R, Monteiro RQ, Moreira ME, Barcinski MA.Cancer Lett Oct 8;283(2): Structural and thermodynamic analysis of thrombin:suramin interaction in solution and crystal phases. Lima LM, Becker CF, Giesel GM, Marques AF, Cargnelutti MT, de Oliveira Neto M, Monteiro RQ, Verli H, Polikarpov I. Biochim Biophys Acta Jun;1794(6): Lung adenocarcinoma and antiphospholipid antibodies. de Meis E, Monteiro RQ, Levy RA. Autoimmun Rev May;8(6): Epub 2009 Jan 29.PMID: [PubMed indexed). 48

49 AL5 ASSOCIATE LABORATORY OF NUCLEAR MAGNETIC RESONANCE, ORGANIC SYNTHESIS AND MOLECULAR MODELING. Coordinator: José Daniel Figueroa Villar, Instituto Militar de Engenharia (IME) Members: Pedro Geraldo Pascutti Luzineide Wanderley Tinoco The work have been focused on the development of compounds with biological activity, with the main emphasis on potential drugs for the chemotherapy of neglected diseases, like malaria, leishmaniasis and leprosy, but also on other general disease like AIDS and cancer. Part of the work has also been directed to the development of agents for defense against chemical and biological weapons, mainly on infections caused by Yersinia pestis and antidotes for intoxication with organophosphorus compounds, like the so called war gases and pesticides. General Research Procedures and Strategies: In general, the potential drugs and defense agents are planned by molecular modeling methods, where the biological target structures, which are usually obtained by comparative molecular modeling, are used for docking and molecular dynamics process. The designed compounds are then synthesized using effective synthetic procedures. Sometimes becomes necessary to invent new reactions or new synthetic procedures. The evaluations of the prepared compounds are conducted in several different ways, being the 49

50 most effective ones the intermolecular interactions with the targets and their effect in their function. For example, the most common targets are enzymes, and the best evaluation of their potential inhibitors are carried out by enzyme kinetics and inhibition studies, which are conducted using spectroscopy methods, with emphasis on nuclear magnetic resonance and ultraviolet. Some tests are also conducted directly with the cell targets, principally with microorganisms, like Plasmodium and Leishmania cells. Also, in vivo tests are conducted in some cases. The enzymes that are the targets for the chemotherapy are obtained commercially or by heterologous expression. Antidotes for Intoxication with Pesticides and Warfare Agents 100 We have developed new reactivators of the enzyme acetylcholinesterase (AChE) inhibited with the very toxic organophosphorus compound paraoxon. These compounds are neutral oximes, which display a much better capacity of permeation of the hematoencephalic barrier and a better effect than the commercial drug pralidoxime. Simulation of the reactivation process using quantum mechanics procedures indicated that other nucleophiles, like hydrazones, have also potential to function as antidotes. Kinetic studies by NMR were able to show that the neutral oximes and hydrazones are effective inhibitors of AChE, with important potential as chemical defense agents and also as drugs for Alzheimer disease. Further reactivation tests by NMR also showed that these compounds are good reactivators of the inhibited AChE. Figure 1 shows the inhibition results obtained by NMR for one new hydrazone The mechanism of reactivation of AChE inhibited with war gases was effectively simulated using QM/MM, using RM1 to simulate 10 amino acids of the active site (146 atoms) including the phosphilated Ser203 and the oxime, as shown in Figure 2. The studies with Yersinia pestis were based on the peptide of interaction of human plasminogen (PLG) with the bacteria protein for invasion of human cells (PLA). This peptide was discovered by molecular modeling and its structure determined by NMR. The future studies are based on the experimental interaction of the peptide with Y. pestis PLA by NMR, in order to determine the interaction process and to discover forms to avoid the invasion of mammal cells by this bacterium. The structure of the peptide is shown in Figure 3. Ach(5) AcO(5) Ach(10) AcO(10) Ach(20) AcO(20) Ach(40) Ach(40) Figure 1: NMR Data of Inhibition of AChE with a Hydrazone: Signal intensity of the methyl groups of ACh and Ac vs. time (minutes). The kinetic tests were conducted with a 5 M concentration of the hydrazone and increasing concentration of the Acetylcholine (ACh). 50

51 Figure 2: QM/MM dynamics of the reactivation of AChE inhibited with tabun using pralidoxime (2-PAM), with expansion of the QM simulated part of the system. Ne Figure 3: Interaction of human PLG with Y.pestis PLA, with the interaction peptide displayed with a molecular surface. 51

52 glected Diseases The main targets for the development of new drugs for the treatment of neglected diseases are the enzymes dihydrofolate reductase (DHFR) of Plasmodium falciparum and Mycobacterium leprae, the nucleoside hydrolase of Leishmania donovani and chagasi. For DHFR in malaria, the structure of the complete enzyme DHFR-TS and their chemotherapy resistant mutants were used to design new type 2 antifolates with IC 50 from 80 to 120 pm. New compounds are actually being synthesized using a novel reaction prepared in our research group, as shown in Figure 4. The leishmania nucleoside hydrolase (NH) had its structure determined by molecular modeling and used to plan new effective inhibitors. The enzyme was expressed linked with maltose binding protein (MBP), a process that kept the enzyme active and with great stability and solubility. Enzyme kinetic studies with different compounds using ultraviolet spectroscopy lead to the discovery of two new inhibitors with IC 50 from 15 to 25 M. It was also found that the AIDS drug AZT is an effective inhibitor of NH, having potential to be used for the treatment of leishmaniosis. Figure 5 shows the NMR basic spectra used for the monitoring of the enzyme kinetics of NH. Figure 4: New synthetic procedure for the preparation of furopyrimidines as precursors of new antifolates for malaria and leprosy. Figure 5: Monitoring of NH kinetics of hydrolysis of inosine by NMR. 52

53 Group publications ( ): José Daniel Figueroa Villar 1. Gonçalves, Arlan da Silva; França, Tanos Celmar Costa; Figueroa-Villar JD; Pascutti, Pedro G. Conformational Analysis of Toxogonine, TMB-4 and HI-6 using PM6 and. Journal of the Brazilian Chemical Society (Impresso), v. 21, p , Silva, Manuela Leal da; Gonçalves, Arlan da Silva ; Batista, P. R. ; França, Tanos Celmar Costa; Pascutti, P; Figueroa-Villar JD ;. Design, docking studies and molecular dynamics of new potential selective inhibitors of Plasmodium falciparum serine hydroximethyltransferase. Molecular Simulation, v. 36, p. 5-14, Guimarães, Evelyn de Freitas; Rego ECP; Cunha HCM; Rodrigues JMR; Cunha VS; Figueroa-Villar JD. Validação de Metodologia Analítica para a Determinação de Hidrocarbonetos Policíclicos Aromáticos em Solução. Produto & Produção, v. 11, p , Gonçalves, Arlan da Silva; Fraga, C. A. M.; Figueroa-Villar JD; Pascutti, Pedro G. Molecular Dynamics Simulations and QM/MM Studies of the Reactivation by 2-PAM of Tabun Inhibited Human Acetilcholinesterase. Journal of the Brazilian Chemical Society (Impresso), v. 21, p. 1-11, Vieira, A. A.; Gomes, N. M.; Matheus, M. E.; Fernandes, P. D.; Figueroa-Villar JD. Synthesis and In Vivo Evaluation of 5-Chloro- 5-benzobarbiturates as Central Nervous System Depressants. Journal of the Brazilian Chemical Society (Impresso), v. 21, p. 1-8, Delfino, R. T.; Ribeiro, Tatiana Santana; Figueroa-Villar JD. Organophosphorus Compounds as Warfare Agents: A Review. Journal of the Brazilian Chemical Society, v. 20, p , Delfino, R. T.; Figueroa-Villar JD. Nucleophilic Reactivation of Sarin-inhibited Acetylcholinesterase: a Molecular Modeling Study. Journal of Physical Chemistry. B, v. 113, p , Figueroa-Villar JD; Tinoco, Luzineide Wanderley. Spin-lattice relaxation time in drug discovery. Current Topics in Medicinal Chemistry (Print), v. 9, p , Produção de Pedro Geraldo Pascutti 1. Gomes, Diego E. B.; Lins, Roberto D.; Pascutti, Pedro G.; Lei, Chenghong; Soares, Thereza A. The Role of Nonbonded Interactions in the Conformational Dynamics of Organophosphorous Hydrolase Adsorbed onto Functionalized Mesoporous Silica Surfaces. Journal of Physical Chemistry. B, v. 114, p , Batista, Paulo Ricardo; Robert, Charles Herbert; Maréchal, Jean-Didier; Hamida- Rebaï, Meriam Ben; Pascutti, Pedro Geraldo; Bisch, Paulo Mascarello; Perahia, David. Consensus modes, a robust description of protein collective motions from multiple-minima normal mode analysis application to the HIV-1 protease. PCCP. Physical Chemistry Chemical Physics, v. 12, p , Valiente, Pedro A.; Gil L., Alejandro; Batista, Paulo R.; Caffarena, Ernesto R.; Pons, Tirso; Pascutti, Pedro G. New parameterization approaches of the LIE method to improve free energy calculations of PlmII-Inhibitors complexes. Journal of Computational Chemistry, v. xx, p. n/a-n/a, Figueirêdo, P.H.; Moret, M.A. ; Pascutti, P. G. ; Nogueira Jr., E. ; Coutinho, S.. Self-affine analysis of protein energy. Physica. A (Print), p , Soares, Rosemberg O.; Batista, Paulo R. ; Costa, Mauricio G.S. ; Dardenne, Laurent E. ; Pascutti, Pedro G. ; Soares, Marcelo A.. Understanding the HIV-1 protease nelfinavir resistance mutation D30N in subtypes B and C through molecular dynamics simulations. Journal of Molecular Graphics & Modelling, v. xxx, p. x, Guizado, Teobaldo R. Cuya; Louro, Sonia R. W.; Pascutti, Pedro G. ; Anteneodo, Celia. Solvation of anionic water-soluble porphyrins: A computational study. International Journal of Quantum Chemistry, p. n/a-n/a, Gonçalves, A. S.; França, T. C. C.; Figueroa-Villar, J. D.; Pascutti, Pedro G. Molecular Dynamics Simulations and QM/MM Studies of the Reactivation by 2-PAM of Tabun Inhibited Human Acethylcolinesterase. Journal of the Brazilian Chemical Society (Impresso), v. 00, p. 1-11, de Souza, Theo Luiz Ferraz ; Sanches, Daniel ; Gonçalves, Rafael Braga ; da RochaPita, Samuel Silva ; Pascutti, Pedro Geraldo ; Bianconi, M. Lucia ; de Almeida, Fabio Ceneviva Lacerda ; Silva, Jerson L. ; de Oliveira, Andréa Cheble. Conformational selection, dynamic restriction and the hydrophobic effect coupled to stabilization of the BIR3 domain of the human X-linked inhibitor of apoptosis protein by the tetrapeptide AVPI. Biophysical Chemistry (Print), p. 1-10, Bernanrdi, R. C.; Gomes, D. E. B.; Taft, C. A.; Ota, A. T.; Pascutti, P. G. Molecular Dynamics Study of Biomembrane-Local Anesthetics Interactions. Molecular Physics, v. 107, p , Moret, M. A.; Santana, M. C.; Zebende, G. F.; Pascutti, P. G. Self-similarity and protein compactness. Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics (Print), v. 80, p , Produção de Luzineide Wanderley Tinoco 1. Merlino, Alicia; Benitez, Diego; Chavez, Santiago; Da Cunha, Jonathan; Hernández, Paola; Tinoco, Luzineide W. ; Campillo, Nuria E. ; Páez, Juan A.; Cerecetto, Hugo; González, Mercedes; Tinoco, Luzineide Wanderley. Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma cruzi-inhibitors bearing benzofuroxan 53

54 and benzimidazole 1,3-dioxide core scaffolds. MedChemComm, v. 1, p , Valente, Ligia M.M.; da Paixão, Djavan; do Nascimento, Adriana C.; dos Santos, Priscila F.P.; Scheinvar, Leia A.; Moura, Mirian R.L. ; Tinoco, Luzineide W. ; Gomes, Luiz Nelson F; da Silva, Joaquim F.M.; Tinoco, Luzineide Wanderley. Antiradical activity, nutritional potential and flavonoids of the cladodes of Opuntia monacantha (Cactaceae). Food Chemistry, v. 123, p , Malta, L. F. B.; Senra, J. D.; Tinoco L. W.; Medeiros, M. E.; Antunes, O. A. C.. Chiral Recognition of 2-Hydroxypropyl-alphacyclodextrin Towards DLTryptophan. Letters in Organic Chemistry, v. 6, p , Borges, R. M.; Tinoco, L. W. ; Dias Filho, J.; Barbi, N. S.; SILVA, A. J. R. Two New Oleanane Saponins from Chiococca alba (L.) Hitch. Journal of the Brazilian Chemical Society (Impresso), v. 20, p , Figueroa-Villar, Jose D.; Tinoco, L. W. Spin-Lattice Relaxation Time in Drug Discovery and Design. Current Topics in Medicinal Chemistry (Print), v. 9, p ,

55 AL6 ASSOCIATE LABORATORY OF PROTEINS AND PROTEOMIC HETEROLOGOUS EXPRESSION. Coordinator: Hernán Terenzi, Universidade Federal de Sta Catarina (UFSC) Major lines of investigation Lipolytic enzymes applications Biofuels Food technology Lipases Chemistry Detergent Lipases have emerged as key enzymes in swiftly growing biotechnology, owing to their multi-faceted properties, which find usage in a wide array of industrial applications, such as food technology, detergent, chemical industry and biomedical sciences (1).These enzymes usually exhibit good chemioselectivity, regioselectivity, enantioselectivity and possess a broad substrate specificity exhibiting optimum activities over a wide range of temperatures (1-55

56 2). Lipases also catalyze esterification, transesterification, acydolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides (2-4). In the last months, our group published two papers (5-6) on purification and biochemical characterization of lipases and one paper leading with esterase activity (7). We also characterized structural features of a Staphylococcus xylosus lipase and its need of a metal cofactor to thermal stability and enzymatic activity (unpublished data), which becomes extremely important for design of the biocatalysis process. Therefore, the knowledge of characteristics such stability at high temperatures and metal tolerance of lipases enables the development and application of these proteins in industrial processes. Proteomic Analysis of neuroprotection and pre-condiotioning induce by N-Methyl-D- Aspartate (NMDA) in mice hippocampi Exposure of neuronal cells to exogenous or endogenous toxicants can affect many biological processes such as the cell signaling. Cells respond to these insults by modulating protein expression and posttranslational modifications and the deregulation of protein expression is associated with many human diseases such as cancer, neurodegenerative disorders and acute events such as ischemia, traumatic injury epilepsy. cerebral brain and On the other hand, a brief toxic stimulus allows the cells to acquire tolerance to a more severe insult, known as preconditioning,it has been reported in a wide variety of models. In this context, the administration of a NMDA sublethal dose has been used as a model of chemical preconditioning against several lethal later injuries. Studies have shown that 24 hours after administration of NMDA in mice, a neuroprotective effect is observed in approximately 50% of animals when exposed to quinolinic acid (QA), after this period, the protective effect is reversed, again causing convulsions and damage to neuronal tissue. Despite the enormous progress in recent decades, the cellular and molecular mechanisms that involve the preconditioning have yet to be elucidated. One of the more recent approaches to extend this knowledge is neuroproteomics (Study of brain proteins in a specific condition or treatment) In this work we aim to identify the proteins (and therefore which signaling pathways) involved in NMDA pre-conditioning and neuroprotection, in mice treated with NMDA for different period of time using 2D Figure 1: 2DE gel of hippocampal total protein of mice treated with NMDA and control sample. Arrows indicate absence/presence spots, circles indicate density differences comparing the two treatments 56

57 gels analysis and mass spectrometry. The 2DE gels of total proteins from mice hippocampi obtained showed some differences in the proteome profile for each treatment (1, 24 and 72 hours). Further analyses to identify proteins by mass spectrometry are underway. Chemical Hydrolases Interactions between biomacromolecules, like DNA and proteins, with synthetic small molecules remains a field of great interest in inorganic biochemistry, for the development of new therapeutic agents for cancer or molecular tools for biotechnology. Our research group, in collaboration with many institutions in the south and southeastern Brazil, carries out research into DNA interaction with artificial metal complexes. The techniques used for these purposes range from agarose and polyacrylamide gel electrophoresis to spectroscopic methods, recently involving the use of circular dichroism spectroscopy. In the last two years, we reported the complex-dna interaction of several transition and lanthanides metal complexes in high impact scientific journals. Some of these complexes were developed to mimic the active site of some enzymes (bioinspired complexes). We highlight two papers published this year reporting the DNA cleavage by mononuclear complexes of copper 7 and iron 8 exclusively in UV-light, with no evidence of cleavage in dark conditions. These works contributed to the development of new drugs for photodynamic therapy of cancer, since the discover of new compounds able to provoke citotoxic effects only in presence of light with no toxic effects in its absence is urgently needed. At least other three copper(ii) complexes with same properties are being studied at this time. 57

58 In addition, we report a new example of metal complex capable of hydrolyze phosphodiester bonds in the DNA and peptide bonds in proteins, i.e., a complex whose activity shows catalytic promiscuity. To our knowledge, this is the third example of metal complex with this property. Our efforts also concentrate on the development of DNA cleavage agents in heterogeneous catalyst systems, i.e., immobilizing the catalyst in specific surfaces like in silica. This approach favors the re-use of the catalyst as already performed by many biotechnological procedures. Group publications ( ): 1. Brod, Fábio Cristiano Angonesi ; Pelisser, Márcia Regina ; Bertoldo, Jean Borges ; VERNAL, Javier ; Bloch, Carlos ; Terenzi, Hernán ; Arisi, Ana Carolina Maisonnave. Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase. Molecular Biotechnology, v. 44, p , de Souza, Bernardo ; BORTOLUZZI, Adailton J. ; Bortolotto, Tiago ; Fischer, Franciele Luane ; Terenzi, Hernán ; Ferreira, Dalva E. C. ; Rocha, William R. ; NEVES, Ademir. DNA photonuclease activity of four new copper(ii) complexes under UV and red light: theoretical/experimental correlations with active species generation. Dalton Transactions (2003. Print), v. 39, p. 2027, Brod, Fábio Cristiano Angonesi ; VERNAL, Javier ; Bertoldo, Jean Borges ; Terenzi, Hernán ; Arisi, Ana Carolina Maisonnave. Cloning, Expression, Purification, and Characterization of a Novel Esterase from Lactobacillus plantarum. Molecular Biotechnology, v. 44, p , Silva, Priscila P. ; Paula, Flávia C.S. ; Guerra, W. ; Silveira, Josianne N. ; Botelho, Françoise V. ; Vieira, Leda Q. ; Bortolotto, T. ; Fischer, Franciele L. ; Bussi, G. ; Terenzi, H. ; MAIA, Elene Cristina Pereira ; Pereira-Maia, Elene C.. Platinum(II) compounds of tetracyclines as potential anticancer agents: cytotoxicity, uptake and interactions with DNA. Journal of the Brazilian Chemical Society (Impresso), v. 111, p , Piovezan, Clovis ; Jovito, Rafael ; BORTOLUZZI, Adailton J. ; Terenzi, Herna?n ; Fischer, Franciele L. ; Severino, Patricia C. ; Pich, Claus T. ; Azzolini, Gisele G. ; Peralta, Rosely A. ; Rossi, Liane M. ; NEVES, Ademir. Heterodinuclear Fe Zn -Bioinspired Complex Supported on 3-Aminopropyl Silica. Efficient Hydrolysis of Phosphate Diester Bonds. Inorganic Chemistry, v. 49, p , Camargo, Maryene A. ; NEVES, Ademir ; SZPOGANICZ, Bruno ; BORTOLUZZI, Adailton J. ; Fischer, Franciele L. ; Terenzi, Herna?n ; Castellano, Eduardo E.. Synthesis, Structure, and Phosphatase-Like Activity of a New Trinuclear Gd Complex with the Unsymmetrical Ligand H L As a Model for Nucleases. Inorganic Chemistry, v. 49, p , Souza, Bernardo de ; Xavier, Fernando R. ; Peralta, Rosely A. ; BORTOLUZZI, Adailton J. ; Conte, Gilmar ; Gallardo, Hugo ; Fischer, Franciele L. ; Bussi, Giselle ; Terenzi, Hernán ; NEVES, Ademir. Oxygen-independent photonuclease activity of a new iron(ii) complex. Chemical Communications (London Print), p. 11, Menegatti, Angela C.O. ; Tavares, Carolina P. ; VERNAL, Javier ; Klein, Catia S. ; Huergo, Luciano ; Terenzi, Hernán. First partial proteome of the poultry pathogen Mycoplasma synoviae. Veterinary Microbiology (Amsterdam. Print), p , Kolling, Deise Juliana ; Bertoldo, Jean Borges ; Brod, Fábio Cristiano Angonesi ; VERNAL, Javier ; Terenzi, Hernán ; Arisi, Ana Carolina Maisonnave. Biochemical and Structural Characterization of Two Site- Directed Mutants of Staphylococcus xylosus Lipase. Molecular Biotechnology, p. 3-8, Mascarello, Alessandra ; Chiaradia, Louise Domeneghini ; VERNAL, Javier ; Villarino, Andrea ; Guido, Rafael V.C. ; Perizzolo, Paulo ; Poirier, Valerie ; Wong, Dennis ; Martins, Priscila Graziela Alves ; NUNES, Ricardo José ; Terenzi, H.. Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth. Bioorganic & Medicinal Chemistry, p. 1-7, SOLETTI, R C ; VERNAL, J ; Terenzi, H. ; Anderluh, G. ; Borges, H.L. ; Gabilan, N. H. ; Moura Neto, V.. Inhibition of MAPK/ERK, PKC and CaMKII Signaling Blocks Cytolysin-induced Human Glioma Cell Death. Anticancer Research, v. prelo, p. 1-5, Camargo, Maryene A. ; NEVES, Ademir ; BORTOLUZZI, Adailton J ; SZPOGANICZ, Bruno ; FISCHER, Franciele Luanne ; TERENZI, H ; Serra O ; Eberlin M. Efficient Phosphodiester Hydrolysis by Luminescent Terbium(III) and Europium (III) complex. Inorganic Chemistry, v. 49, p. 425, PERALTA, Rosely A ; BORTOLUZZI, Adailton J ; SZPOGANICZ, Bruno ; Brandão, T. A. S. ; CASTELLANO, Eduardo ; OLIVEIRA, Mauricio César Bof de ; SEVERINO, Patricia Cardoso ; TERENZI, H ; NEVES, Ademir. Catecholase and DNase Activities of Copper(II) Complexes Containing 58

59 Phenolate-type Ligands. Journal of Physical Organic Chemistry, v. 23, p , Ecco G ; VERNAL, Javier ; RAZZERA, G. ; Martins, Priscila Graziela Alves ; TERENZI, H. Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) activity is modulated by S- nitrosylation. Chemical Communications (London Print), v. prelo, p. 1, Puhl, Ana&nbsp ; Giacomini, Cecilia ; Irazoqui, Gabriela ; Batista?Viera, Francisco ; Villarino, Andrea ; Terenzi, Hernán. Covalent immobilization of tobacco-etch-virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins. Biotechnology and Applied Biochemistry, v. 53, p , Cangahuala-Inocente, Gabriela Claudia ; Villarino, Andrea ; Seixas, Daniela ; Dumas- Gaudot, Eliane ; Terenzi, Hernán ; GUERRA, Miguel Pedro. Differential proteomic analysis of developmental stages of Acca sellowiana somatic embryos. Acta Physiologiae Plantarum, v. 31, p , Oliveira, Mauricio C. Bof ; Mazera, Deise ; Scarpellini, Marciela ; SEVERINO, Patricia Cardoso ; NEVES, Ademir ; TERENZI, Hernan. Mononuclear Cu?Phenolate Bioinspired Complex is Catalytically Promiscuous: Phosphodiester and Peptide Amide Bond Cleavage. Inorganic Chemistry, v. 48, p , Rey, Nicolás A. ; NEVES, Ademir ; Silva, Priscila P. ; Paula, Flávia C.S. ; Silveira, Josianne N. ; Botelho, Françoise V. ; Vieira, Leda Q. ; Pich, Claus T. ; Terenzi, Hernán ; Pereira-Maia, Elene C.. A synthetic dinuclear copper(ii) hydrolase and its potential as antitumoral: Cytotoxicity, cellular uptake, and DNA cleavage. Journal of Inorganic Biochemistry, v. 103, p , Xavier, Fernando R. ; NEVES, Ademir ; Casellato, Annelise ; Peralta, Rosely A. ; BORTOLUZZI, Adailton J. ; SZPOGANICZ, Bruno ; Severino, Patricia C. ; Tomkowicz, Zbigniew ; Ostrovsky, Sergei ; HAASE, Wolfgang ; Ozarowski, Andrew ; Krzystek, Jerzy ; Telser, Joshua ; SCHENK, Gerhard ; GAHAN, Lawrence R. ; Terenzi, Hernán. Unsymmetrical Fe Co and Ga Co Complexes as Chemical Hydrolases: Biomimetic Models for Purple Acid Phosphatases (PAPs). Inorganic Chemistry, v. 48, p , Matiollo, Camila ; VERNAL, Javier ; Ecco, Gabriela ; Bertoldo, Jean Borges ; Razzera, Guilherme ; de Souza, Emanuel M. ; Pedrosa, Fábio O. ; Terenzi, Hernán. A transthyretin-related protein is functionally expressed in Herbaspirillum seropedicae. Biochemical and Biophysical Research Communications, p , Ecco, Gabriela ; VERNAL, Javier ; Razzera, Guilherme ; TAVARES, Carolina ; Serpa, Viviane Isabel ; Arias, Santiago ; Marchini, Fabricio Klerynton ; Krieger, Marco Aurélio ; Goldenberg, Samuel ; Terenzi, Hernán. Initial characterization of a recombinant kynureninase from Trypanosoma cruzi identified from an EST database. Gene (Amsterdam), v. 448, p. 1-6, NEVES, Ademir ; BORTOLUZZI, Adailton J ; SOUZA, Rafael Jovito de ; PERALTA, Rosely A ; SOUZA, B. ; SZPOGANICZ, Bruno ; JOUSSEF, Antonio Carlos ; TERENZI, H ; SEVERINO, Patricia Cardoso ; FISCHER, Franciele Luanne ; SCHENK, Gerhard ; RILEY, Mark J. ; GAHAN, Lawrence R.. Catalytic Promiscuity: Catecholase-like Activity and Hydrolytic DNA Cleavage Promoted by a Mixed-Valence FeIIIFeII complex. Journal of the Brazilian Chemical Society (Impresso), v. press, p. 1-10,

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61 AL7 Members: ASSOCIATE LABORATORY OF PROTEINS BIOCHEMISTRY. Ljubica Tasic Coordinator: Carlos H. Inácio Ramos, Instituto de Química (UNICAMP) Ana Olívia Tiroli This associated Laboratory studies molecular chaperones, a family of proteins involved with the folding of other proteins and with the cellular homeostasis. Thus, these proteins have increasing importance for the study of conformational diseases. These diseases include neurodegenerative diseases, several types of cancer, diseases involved with ageing and others. The proposal is focused on human chaperonas but we are also studying chaperons of biotechnological interest, as sugar cane, orange and Xanthomonas (involved with the citrus canker). Thus, this proposal may generate inputs that not only benefit the basic science but may also lead to new therapies. Another important line of study is the forces involved in the stability of proteins and with the formation of amyloides (present in conformational diseases). Metabonomic analysis based on 1H NMR and chemometrics in bipolar disorder Bipolar disorder, formerly known as manic-depressive psychosis, is one of the most debilitating and common psychiatric disorders worldwide. It is characterized by recurrent mood disturbances with periods of depression, mania, hypomania and mixed states. In this work, a metabonomics study employing 1 H NMR and 61

62 chemometrics was performed to detect molecular changes in human blood serum samples by comparing the metabolic profiles of healthy subjects (control group/ n = 25), patients being treated for bipolar disorder with lithium (n = 15), and patients being treated for bipolar disorder with other medications not including lithium (n = 10). 1 H NMR data were transported to a data matrix, and chemometrics analyses, based on interval principal component analysis (ipca) and partial least-squares discriminant analysis (PLS-DA), were performed using MATLAB 6.5 software. After further processing the data, the investigated groups (control and patients with bipolar disorder under different treatments) could be distinguished according to their metabolic profiles, and the main differential metabolites found were lipids, lipid-metabolismrelated molecules (acetate, choline, and myoinositol), and some key amino acids (glutamate, glutamine). This strategy showed significant potential for exploring pathophysiological and toxicological features involved in bipolar disorder and our results suggest that some of the 24 identified metabolites may be linked to lithium and other medication provoked metabolic changes or may even be directly related to the disorder. This work can contribute to find the way for future studies aiming at identifying potential biomarkers for bipolar disorder. Supramolecular Interactions in Captopril and Cyclodextrins complexes: Physico-chemical and Angiotensin-I-converting enzyme Inhibition Evaluations Captopril (CAP) was the first angiotensin-i-converting enzyme (ACE) inhibitor developed and it is largely used in hypertension treatment. On the other hand, cyclodextrins (CD) are cyclic oligosaccharides whose cone-shaped cavity allows formation of non-covalent inclusion complexes with appropriately sized host molecules thus modifying quests physical, chemical, and biological properties. We have reported the physicochemical characterization and in vivo ACE inhibition evaluation of seven CAP/CD complexes. The inclusion complexes (IC) were prepared by spray drying, freeze drying, kneedling, or lyophilization methods, and characterized applying the nuclear magnetic resonance (NMR), Fourier-transformed infrared (FTIR) spectroscopy, X-ray diffraction (DRX), differential thermal analysis (DSC), and scanning electron microscopy (SEM) techniques. In vivo assays compared the captopril and CAP/CD complexes (0.5 mg/kg, or 0.09 mg/kg, n=4-7) administration as to evaluate the ACE inhibition by continuously infusion of angiotensin I (Ang I, 30ng/50µL/min) in conscious Wistar rats. The physicochemical analysis demonstrated complete amorphization and complexation of captopril and cyclodextrins, indicating the substitution of water molecules inside the CDs cavity by CAP. During the infusion of Ang I, the administration of all CAP/CD complexes induced a reduction in mean arterial pressure (MAP) similar to that observed upon captopril administration. The nanoparticles obtained by kneading method (CAP / -CD:KM) showed a potent and longlasting inhibitory activity (~22h) upon the Ang I pressor effect. Therefore, obtained results suggest that the inclusion complex of captopril and -CD can function as a novel 62

63 antihypertensive formulation that may improve captopril therapeutic use by reducing its oral dose administration in one per day thus providing better life conditions for almost 15% of word population that suffers from hypertension. (A) (B) Figure 1. Proposed structure for captopril and -CD in CAP/ -CD:KM inclusion complex (IC). The CAP is completely included in -CD cavity; -CD s upper and broader cone side accommodates CAP that is emerged by thiol moiety seen from two different perspectives (A) and (B). The hydrogen atoms in green (H-9), purple (H- 4) and red (H-8) on CAP molecule are the most altered by complexation as measured in ROESY NMR and seen by CICS. Main publications: Borges, J.C., Ramos, C.H.I. (2009). Characterization of nucleotide-induced changes on the structure of human 70 kda heat shock protein Hsp70.1 by analytical ultracentrifugation. BMB Reports., 42, Hsp70s assist in the process of protein folding through nucleotide-controlled cycles of substrate binding and release by alternating from an ATP-bound state in which the affinity for substrate is low to an ADP-bound state in which the affinity for substrate is high. It has been long recognized that the two-domain structure of Hsp70 is critical for these regulated interactions. Therefore, it is important to obtain information about conformational changes in the relative positions of Hsp70 domains caused by nucleotide binding. In this study, analytical ultracentrifugation and dynamic light scattering were used to evaluate the effect of ADP and ATP binding on the conformation of the human stress-induced Hsp70.1 protein. The results of these experiments showed that ATP had a larger effect on the conformation of Hsp70 than ADP. In agreement with previous biochemical experiments, our results suggest that conformational changes caused by nucleotide binding are a consequence of the movement in position of both nucleotide- and substratebinding domains. Summers, D.W., Douglas, P.M., Ramos, C.H.I., Cyr, D.M. (2009). Polypeptide transfer from Hsp40 to Hsp70 molecular chaperones. Trends in Biochem. Sc. 34, Heat shock protein 40 (Hsp40) cochaperones assist in cellular protein folding and degradation through the binding and delivery of non-native proteins to heat shock protein 70 (Hsp70). The mechanism for substrate transfer from Hsp40s to Hsp70 is unknown. Two recent studies provide new details that shed light on novel mechanisms for substrate recognition by 63

64 Heat shock protein 40 (Hsp40) co-chaperones assist in cellular protein folding and degradation through the binding and delivery of non-native proteins to heat shock protein 70 (Hsp70). The mechanism for substrate transfer from Hsp40s to Hsp70 is unknown. Two recent studies provide new details that shed light on novel mechanisms for substrate recognition by Hsp40s and a common mechanism for polypeptide transfer to Hsp70. Hsp40s and a common mechanism for polypeptide transfer to Hsp70. Tiroli-Cepeda, A.O., Ramos, C.H.I. (2010). Heat causes oligomeric disassembly and increases the chaperone activity of small heat shock proteins from sugarcane. Plant Physiol. Biochem. 48, Small heat shock proteins (shsp) constitute an important chaperone family linked to conformational diseases. In plants, shsps prevent protein aggregation by acting as thermosensors and to enhance cell stress tolerance. SsHsp17.2 and SsHsp17.9 are the most highly expressed class I shsps in sugarcane. They exist as dodecamers at 20 _C and have distinct substrate specificities. Therefore, they are useful models to study how class I SHsps work. Here we present data on the effects of heat on the oligomerization and chaperone activity of SsHsp17.2 and SsHsp17.9. Using several biophysical and biochemical probes, we show that the effects of heat are completely reversible, an important property for proteins that act at heat shock temperatures. SsHsp17.2 and SsHsp17.9 dodecamers dissociated to dimers at temperatures ranging from 40 to 45 _C and this dissociation was followed by enhanced chaperone activity. We conclude that high temperature affects the oligomeric state of these chaperones, resulting in enhanced chaperone activity. 64

65 Group publications ( ): 1.Correa, D.H.A., Ramos, C.H.I. (2009) The use of circular dichroism spectroscopy to study protein folding, form and function. African J. Biochem. Res. 3, da Silveira, Pires, D.E.V., Melo, R.C., Ribeiro, C., Veloso, C.J.M., Lopes, J.C.D., Meira Jr, W., Neshich, G., Ramos, C.H.I., Habesh, R., Santoro, M.M. (2009). Protein cutoff scanning: a comparative analysis of cutoff dependent and cutoff free methods for prospecting contacts in proteins. Proteins: Struct. Funct. Bioinfo. 74, Gava, L., Ramos, C.H.I. (2009) Human 90 kda heat shock protein Hsp90 as a target for cancer therapeutics. Curr. Chem. Biol. 3, Borges, J.C., Ramos, C.H.I. (2009). Characterization of nucleotide-induced changes on the structure of human 70 kda heat shock protein Hsp70.1 by analytical ultracentrifugation. BMB Reports., 42, Lira, CBB; Gui, KE; Perez, AM; da Silveira, RCV; Gava, L.M., Ramos, CHI; Cano, MIN (2009). DNA and heparin chaperone the refolding of purified recombinant Replication Protein A subunit 1 from Leishmania amazonensis. Biochimica et Biophysica Acta - General Subjects., 1790, Summers, D.W., Douglas, P.M., Ramos, C.H.I., Cyr, D.M. (2009). Polypeptide transfer from Hsp40 to Hsp70 molecular chaperones. Trends in Biochem. Sc. 34, Quaresma, A.J.C., Bressan, G.C., Gava, L.M., Lanza, D.C.F., Ramos, C.H.I, and Kobarg, J (2009). Hnrnpq interacts with hsp70/bip and co-localizes with it upon pma, tapsigargin and heat treatment in the endoplasmic reticulum. Experimental Cell Research, 315, Matavel, A., Fleury, C., Oliveira, L., Molina, F., De Lima, M.E., Cruz, J. Cordeiro, M. Richardson, M. Ramos, C.H.I. Beirão, P. (2009) Structure and activity analysis of two spider toxins that alter sodium channel inactivation kinetics. Biochemistry, 48, Correa, D.H.A., Ramos C.H.I. (2009). Insigths on the structure of amyloid fibrils from site-directed mutagenesis. Protein Pept. Lett.. 16, Durán, N.; Durán, M.; Tasic, L.; Marcato, P. D. Nanocrystal technology in pharmaceuticals. Química Nova 2010, 33(1), Sussulini, A.; Prando, A.; Maretto, D. A.; Tasic, L.; Poppi, R. J.; Arruda, M. A. Z.; Banzato, C. Metabolic Profiling of Human Blood Serum from Treated Patients with Bipolar Disorder Employing H-1 NMR Spectroscopy and Chemometrics. Analitical Chemistry 2009, 81(23), Gonçalves, D.C., Gava, L.M., Ramos, C.H.I. (2010). Human Hsp70/Hsp90 organizing protein (Hop) D456G is a mixture of monomeric and dimeric species. Protein Pept. Lett.. 17, Tiroli-Cepeda, A.O., Ramos, C.H.I. (2010). Heat causes oligomeric disassembly and increases the chaperone activity of small heat shock proteins from sugarcane. Plant Physiol. Biochem. 48, Fan, A.C.Y., Gava, L.M., Ramos, C.H.I., Young, J.C. (2010). Human mitochondrial import receptor Tom70 functions as a monomer. Biochem. J. 429, Fattori, J.; Prando, A.; Martini, A.M.; Rodrigues, F.H.S.; Tasic, L. (2010) Bacterial Secretion Chaperones. Protein & Peptide Letters, (Accepted) 16.Oliveira, C.Z.; Filho, N.A.S.; Menaldo, D.L.; França, J.B.; Giglio, J.R.; Calderon, L.A.; Stabeli, R.G.; Rodrigues, F.H.S.; Tasic, L.; Silva, S.L.; Soares, A.M. - Type Phospholipase A 2 Inhibitor from Bothrops jararacussu Snake Plasma. Current Topics Topics in Medicinal in Medicinal Chemistry (CTMC),. In press. 65

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67 AL8 ASSOCIATE LABORATORY OF MACROMOLECULES CRYSTALLIZATION. Coordinator: Marcelo Santos Castilho, Universidade Federal de Bahia (UFBA) Members: Tânia Fraga Barros Luciana Veiga Barbosa Associate laboratory number 8, from Universidade Federal da Bahia, has focused on two main research projects that aim to develop novel drugs against cryptococcosis and Alzheimer s Disease (AD), by means of modern in silico tools, such as docking, homology modeling, pharmacophore searches and QSAR model development. We have described some recent results on how mutations in lanosterol 14- alfa demethylase from C. neoformans are related to azole drugs resistance, as well as the development of QSAR models that hint at chemical and structural features that might be useful to develop drugs that are effective against fluconazole resistant C. neoformans strains. Furthermore, we describe the use of pharmacophore searches that lead to the selection of potential inhibitors of human betasecretase (BACE-1), a well known target for AD drug development and the use of hologram QSAR to further investigate the structureactivity relationship for a series of known BACE-1 inhibitors. Cryptococcus neoformans, the causative agent of cryptococcosis, has received much attention due to its increased incidence among HIV positive patients and the limited number of drugs available. Thus there is an urgent need for novel antifungal drugs, which has been amplified by the emergence of resistance to fluconazol, the treatment of choice for this disease. One of the main causes of resistance do 67

68 fluconazol is decreased affinity of lanosterol 14- alfa demethylase (14 -DM) towards the drugs, as a consequence of mutated residues. Knowledge about the interaction profile of drugs into the binding site provides valuable information to circumvent this kind of problem, however there is no crystallographic structure of C. neoformans 14 -DM available. In such cases, it is possible to gain some structural insight from homology based models that are built from the aminoacid sequence of the the target structure and the 3D coordinates from a related protein, whose sequence identity is greater than 30%. Using the coordinates from 14 -DM from Figure 1 - Homology models of mutant 14 -DMs that are resistant to fluconazole. A) 95 % of all 17 mutant strains studied show mutations in a-helices highlighted in yellow and red, which are considered as hot spots for mutation. The majority of mutations alter HEME positioning (B steric clash between Ser (Gly in the wild type) and propionic side chain of heme) or binding profile in the 14 -DM binding site (C the mutation from His Wild-type - to Tyr - resistant straindecreases heme anchoring ) thus preventing azole drugs from binding. Homo sapiens (PDB id: 3LD6) and Trypanosoma cruzi (PDB id: 2CIB), models with acceptable stereochemical features were built. The final models highlight the hot spots for mutation and explain why such modifications might alter the drug affinity towards C. neoformans 14 -DM (Figure 1). In order to further investigate this matter, crystallographic studies with wild-type and mutant 14 -DM are being undertaken. Meanwhile, we decided to explore ligand-based in silico tools that might be helpful to design more effective antifungals. Aiming to contribute to this goal, 2D QSAR studies were carried out for a series of 33 azoles derivates that had been tested against a fluconazole-resistant C. neoformans strain. The best PLS model shows good fit and internal consistence (r 2 = 0.89 and q 2 (LOO)=0.82, with 3 PCs) and average predictive power (r 2 pred= 0.73) (Figure 2). Analysis of regression vector suggests that halogens linked to aromatic carbons and the mean electrotopological state of the molecules are important to the biological activity. Aiming to further investigate the structureactivity relationship for this series of molecules, hologram QSAR modeling was also carried out. The best model (A/B/C/H/Ch) shows excellent fit and internal consistence (r 2 = 0.97 and q 2 (LOO)= 0.79, with 6 PCs), but very poor predictive power (r 2 pred=0.28). This result suggests that fragment based QSAR models (i.e molecular holograms) have limited predictive power, whereas descriptor-based QSAR models can be useful not only to shed some light on the structure-activity relationship 68

69 of azole derivates that were evaluated against fluconazole-resistant C. neoformans strains, but also to guide the design of novel and more potent antifungals. R 2 = 0.89, Q 2 (LOO)=0.82 AND R 2 PRED= 0.73 (3 PCS) Figure 2-2D Descriptor-based QSAR model for a series of azole derivatives that were assayed against fluconazoleresistant C. neoformans. The best model show good fit and predictive ability. Alzheimer's disease (AD) is the leading cause of cognitive decline in elders, affecting today more than 37 million people around the world. AD symptoms are related to the death of neuronal cells due to formation of neurofibrilar tangles and amyloid plaques (amyloid hypothesis). However, currently available drugs for AD treatment do not target this issue. Instead, it focus on palliative measures that prevent acetylcholine from being metabolized in the neuronal junction. Therefore, development of novel anti-ad drugs is of utmost significance. Recent approaches to develop anti-ad drugs have targeted BACE-1, a key enzyme in the formation of insoluble amiloyd precursor peptide, which is the main constituent of neurofibrilar tangles. Taking this information into consideration, we carried out QSAR model development to further investigate the structural and chemical features that are crucial for BACE- 1 inhibition. In order to do so, we selected a diverse dataset of 122 hydroxy-ethylamine (HEA) derivatives, whose inhibitory potency (IC 50 ) against BACE-1 ranges from 4208 to 8.70 nm. Exploratory analysis using principal component analysis (PCA ) shows that 3 PCs hold 66.4% from the original information and that the first PC accounts for HEA potency against BACE-1. The best PLS model shows good internal consistency (r 2 = 0.80 e q 2 = 0.80) and predictive power, r 2 pred= 0.75) (Figure 3-left panel). With the intention to facilitate QSAR model analysis, fragment based QSAR models (HQSAR) were also developed using molecular holograms. The best HQSAR (A/B/C ) model presented q² = 0,77 (Leave-One-Out), r² = 0,89 and r 2 pred =0, 88 (Figure 3-right panel). The synergic interpretation of contribution maps, from HQSAR, and regression vector plot (from classical 2D-QSAR), suggests that electron deficiency around sulfone moiety contributes negatively to potency. This result will be useful in the design of novel BACE-1 inhibitors. As part of our ongoing project to discover novel BACE-1 inhibitor we developed a pharmacophore model (Figure 4) that was employed, along with ROCS to screen a dataset of 1 Million lead-like compounds. In order to explore the chemical diversity of this database, hits that resemble HEA scaffold were discarded. This strategy afforded 15 compounds that will be assayed against BACE-1. 69

70 Figure 3 2D descriptor-based (left panel) and fragment-based (right panel) QSAR models for hydroxy-ethylamine (HEA) derivatives that inhibit BACE-1. Both models show good statistical parameters and predictive power Figure 4 - Structure-based pharmacophore model for BACE-1 inhibitor superposed on the reference molecule used by ROCS and the 3 top scoring hits selected in the virtual screening protocol carried out in lead-like molecules available in zinc database (www.zinc.docking.org ). Group publications (2010): Pereira, H. M.; Rezende, M. M.; CASTILHO, M. S.; OLIVA, G.; Garratt, R. C. Adenosine binding to lowmolecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme from. Acta Crystallographica. Section D, Biological Crystallography, v. 66, p , Castilho, M. S.; Postigo, M. P.; Pereira, H. M.; OLIVA, G.; Andricopulo, A. D. Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies. Bioorganic & Medicinal Chemistry, v. 18, p , MOTA, S. G. R.; BARROS, T. F.; CASTILHO, M. S. In vitro screening and chemometrics analysis on a series of azole derivatives with fungicide activity against Moniliophthora perniciosa. Journal of the Brazilian Chemical Society (Impresso), v. 21, p , Postigo, M. P.; Guido, R. V. C.; OLIVA, G.; Castilho, M. S.; Pitta, I.; Albuquerque, J. F. C.; Andricopulo, A. D. Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening. Journal of Chemical Information and Modeling, v. 50, p , SANTOS, S. C.; FERREIRA F. S.; DAMIÃO, A. O.; BARROS, T. F.; ROSSI-ALVAA, J.C.; FERNANDEZ, L. G. AVALIAÇÃO DA ATIVIDADE ANTIBACTERIANA DOS EXTRATOS DE Avicennia schaueriana (Stapf & Leechman). Revista Brasileira de Farmacognosia (Impresso), v. 20, p , JUIZ, P. J. L.; ALVES, R. J. C.; BARROS, T. F. Uso de produtos naturais como coadjuvante no tratamento da doença periodontal. Revista Brasileira de Farmacognosia (Impresso), v. 20, p , REIS, A. L. V; VELOZO, E. S.; FERRER, S. R.; GURREIRO, H. M. N.; BARROS, T. F.. Avaliação da atividade antimicrobiana de duas espécies de Rutaceae do Nordeste Brasileiro. Revista Brasileira de Farmacognosia (Impresso), v. 20, p , Note: The results described herein will be submitted to publication within 3 months 70

71 AL9 ASSOCIATE LABORATORY OF CELLULAR ULTRASTRUCTURE HERTHA MEYER. Coordinator: Wanderley de Souza, IBCCF/UFRJ Members: Tecia de Carvalho Rossiane Vommaro Maria Cristina Motta Márcia Attias Narcisa Cunha e Silva Kildare Miranda Acidocalcisomes and Contractile vacuole Electron tomography of the contractile vacuole of wild type and mutant epimastigote forms of T. cruzi, incubated or not in hypo and hyperosmotic conditions and submitted to cryofixation and freeze substitution, is an ongoing project. Preliminary results showed that the structure of the contractile vacuole complex of T. cruzi, formed by a central vacuole (the bladder) surrounded by a network of tubules and vesicles (the spongiome), may suffer dramatic changes upon hyposmotic treatment, potentially involving fusion mechanisms between the spongiome tubules and the central vacuole. These fusion events reduce the size and number of the tubules and lead to the enlargement of the bladder. These data are in agreement with our previous results that show large bladders in TcVps34 osmoregulation efficient mutants (see below). Freeze-fracture analysis of the contractile vacuole region was carried out in cells submitted to hypospomotic treatment. The structural remodeling of the complex by deepetching technique is currently under investigation. The functional role of a PI3 kinase (TcVps34) in osmoregulation and receptor mediated endocytosis in T. cruzi was studied in wild type cells and cells overexpressing Vps34 products, submitted or not to hyposmotic treatment. Results showed that OE cells were 71

72 more efficient in regulatory volume decrease and less efficient in receptor mediated endocytosis. Cells overexpressing TcVps34 showed special structural characteristics, such as the presence of a large and functional contractile vacuole (Schoijet et al., JBC. 283, ). The potential interaction of acidocalcisomes with the contractile vacuole in Trypanosoma cruzi and other organelles in protozoan parasites has led to the identification of a novel organelle in Toxoplasma gondii with similar characteristics to the plant vacuole that has been named Toxoplasma PLV (Miranda et al., Mol. Microbiol. 76, ). Functional analysis showed that this organelle contains an aquaporin channel, proton pumps and a proteolytic activity (cathepsin L) and is formed only in extracellular parasites. The localization of PDEC2 in T. cruzi, using antibodies against TcPDEC2 showed that this phosphodiesterase is mainly localized in the contractile vacuole complex (CVC) region, preferentially in the spongiome. Functional analysis and expression of truncated sequences showed that a FYVE domain is required for the correct docking of the TcPDEC2 in the CVC and that it down regulates the regulatory volume decrease (Schoijet et al., Mol. Microbiol. 2010, in press). Endosymbiosis in Trypanosomatids. Biochemical, molecular, ultrastructural and computational methods were used to to identify and characterize the C. deanei endosymbiont porin. A search of the annotated symbiont genome database identified a sequence that presents identity to porins of the Bordetella genus. The protein s primary sequence presents Electron tomography of a T. gondii tachyzoite form showing the PLV (green), selected as the cover of Molecular Microbiology porin features and the secondary structure prediction indicates a β-barrel pattern with 18 transmembrane domains. Antiserum produced against the recombinant porin revealed that this protein is mainly located in the symbiont envelope, forming porin channels with slight cation-selectivity when reconstituted in a lipid bilayer. Taken together, our data show that the C. deanei endosymbiont has a porin channel that is phylogenetically and structurally similar to classical porins of Gram-negative bacteria. Endocytosis in T. Cruzi The endocytic pathway of of T. cruzi epimastigotes is comprised by two entry sites, the cytostome and the flagellar pocket, an early endosomal network and reservosomes, the final compartment, which stores exogenous proteins 72

73 and lipids besides enzymes produced by the parasite. We have published the proteome of isolated reservosomes in LC-MS/MS analysis identified a total 442 T. cruzi-specific proteins; of these, 286 had predicted function and 156 were classified as hypothetical proteins. We could confirm the presence of several proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins and others. We have localized the protein immunologically similar to BILBO-1, that have been recently described as involved in endoexocytosis and essential for flagellar pocket biogenesis in T. brucei. In T. cruzi epimastigotes, BILBO-1 was found at the flagellar pocket and flagellum. Electron tomography and dual beam scanning electron microscopy are been used to study the distribution of the endosomal compartments within the parasite and have already revealed that several reservosomes can fuse with the early endosomal network simultaneously. Reservosomes also appear to fuse directly with the endoplasmic reticulum, defining a new degradative pathway for internal proteins. We have isolated the crystalloid lipid inclusions of reservosomes and demonstrated, using GC-MS, that they are formed by high amounts of cholesterol. Different from mammal cells, T. cruzi epimastigotes are able to mobilize cholesterol from crystals. Paraflagellar Rod (PFR) Structure Understanding the structural aspects of the flagellum may be important for the identification of novel targets for therapeutic intervention. Our group used atomic force microscopy (AFM) to examine the ultrastructure of Trypanosoma cruzi, obtaining valuable information on the organisation of the flagellar sub-structure. AFM images revealed novel flagellar components such as the presence of periodically-spaced protrusions organised along a flagellar furrow and oriented through the major flagellar axis between the axoneme and the paraflagellar rod. The nature and functional role of this structure are still unknown, although the hypothesis that the furrow might physically separate the two distinct domains of the flagellar membrane that comprise the surface of the axoneme and the paraflagellar rod (PFR) has been raised. To test whether the furrow was present or not only in PFR-bearing flagella, different protists containing or lacking the PFR, were analysed by AFM. Analysis of T. cruzi, Trypanosoma brucei and Herpetomonas megaseliae, which present distinct PFRs, showed similar and equivalent furrows along the main axis of their flagella, whereas Crithidia deanei, Giardia lamblia and Tritrichomonas foetus (in which the PFR is reduced or absent) lacked a furrow. Our results strongly suggest that the flagellar furrow is a characteristic feature of PFR-containing flagella and opens new perspectives for its functional role in the definition of sub-domains on the flagellar membrane.(rocha GM et al.,micron 41:939-44) Interaction of protozoa with host cells Trypanosoma cruzi- Dynasore acts as a potent inhibitor of endocytic pathways. We observed that, in macrophages and LLC-MK2 the parasite internalization was drastically diminished when we used 100 mm dynasore. T. 73

74 cruzi adhesion index was unaffected. By scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages using methyl-betacyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. (Barrias ES et al. PloS One 5:7764, 2010) Toxoplasma gondii- Among the diverse signaling events which take place during egress, kinases seem to play a crucial role. Although parasite egress induction was only slightly affected by wortmanin and staurosporin, the specific inhibitor of tyrosine kinase, genistein, blocked the exit of parasites to more than 50%. The actin polymerization inhibitor cytochalasin D also blocked the induced egress of T. gondii. Dynasore that blocks dynamin had little or no effect on egress of T. gondii. Participation of host cell microdomains in adhesion and internalization of T. gondii. The participation of cholesterol enriched microdomains in invasion of T. gondii into LLC-MK2 and murine macrophages was evaluated through transient depletion of host cells cholesterol with either methyl-betacyclodextrin (MβCD); Filipin. Cholera Toxin B subunit (GM1 marker) and Lidocaine were also A murine macrophage (green) engulfes a tachyzoite of Toxoplasma gondii (pink). Field emission scanning electron microscopy (Marcia Attias and Karla Dias). Active invasion by the parasite is inhibited by Methyl-Bcyclo dextrin, a drug that sequesters plasma membrane cholesterol. However, the macrophage ability to phagocytose is mantained. Active invasion starts by the conoid. In this situation it is pointed to the oposite end, indicating the passive role of the parasite. tested. Adhesion to macrophages was not affected by any of the drugs, but was significantly diminished in LLC-MK2 cells. This effect was not reversible. On the other hand, invasion was diminished in all instances for both cell types. Ultrastructural organization of parasitic protozoa- The conoid, in Toxoplasma gondii is a small cone-shaped structure composed of a spiral of microtubules, two intraconoidal 74

75 microtubules, and two polar rings. It shows a mechanical activity in invasion of host cells. We combined Transmission (TEM) and Field Emission Scanning (FESEM) Electron Microscopy techniques. For TEM fixation was in 2.5% glutaraldehyde and 1% tannic acid in 0.1M cacodylate buffer, Post fixation was carried out in 1% O s O 4 in the same buffer. For FESEM the membrane was extracted with nonionic detergents. We can already show that the conoid is more cylindrical than conoidal and in its retracted position remains under the posterior polar ring and surrounded by the inner pellicle. Tiny bridges connecting the conoid to the lateral portion of the posterior polar ring were also observed. 3-D reconstruction of tachyzoites of T. gondii and Morphometric analysis of secretory organelles- At present, all information on the inner organization of T. gondii has been obtained from random ultrathin sections, freeze fracture replicas, and partial 3-D reconstruction from serial sections or electron-tomography. Using the slice and view tool of FEI's Helios DualBeam (FIB/SEM) a 3D observation of whole tachyzoites, it was possible to conjugate a high level of resolution with the whole view of the cell. 14 rhoptries were counted, but only 4 reached the inside of the conoid. Several 70 nm vesicles were seen aligned with the central pair of microtubules of the conoid. These vesicles are present in free and in vacuole contained tachyzoites. Micronemes were seen in a radial distribution around the conoid. Dense granules were scattered in the cytoplasm. Rhoptries were grouped in one side of the cell and the necks made a sinuous route up to the conoid and micronemes were also concentrated around the base of the conoid. Stereological measurements showed that rhptries occupied 2-3% of the total cell volume, while micronemes filled less than 0.5% of it. Dense granules filled the higher volume: 4-5%. In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like 3D- reconstruction of Crithidia deanei. was obtained FIB tomography. The close association between the endosymbiont (green) and the nucleus (light blue) can be seen in figures A and C. The plasma membrane is in dark blue and the flagellum in lilac. The endosymbiont changes its shape during the cell cycle from a rod-shape (Fig. B) to a more constricted or dividing form, which is associated to the host cell nucleus (Fig. C arrowhead). After division both bacteria are simetrically distributed in relation to the nucleus (Fig. D). E- Electron dense area, in points where the symbiont is closely associated to the nuclear surface (Fig. E-arrowhead). Bar = 0.5 mm. 75

76 Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with threedimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. (Motta et al. PLos One, 5(8): e12415, 2010) Experimental Chemotherapy in Trypanosomatids Trypanosoma cruzi - Epimastigotes treated with 20, 40 and 50 µm of posaconazole and observed by FESEM showed extensive loss of integrity of the plasma membrane with parasite body deformation. Moreover, the flagellar pocket also demonstrated deformation. Observations by TEM of epimastigote treated with posaconazole in same concentrations showed swollen and vacuolization in mitochondria, intense changes of the Golgi complex, cytoplasm vacuolization and alterations in the flagellum. In intracellular amastigote form treated with posaconazole, cytoplasm vacuolization and plasma membrane shedding were observed. Amastigotes treated with posaconazole plus amiodarone showed similar vacuolization in the cytoplasm, changes in Golgi complex and intense plasma membrane shedding. T.cruzi expressing luciferase was used to evaluate the progression of Chagas Disease in the murine model. The luciferase gene (firefly, Promega) was amplified by PCR and cloned in a vector Topo II blunt. The luciferase gene was removed from TOPO vector and cloned in the integrative expression vector of T.cruzi, ptrex (Vásquez & Levin, 1999). Next, the linearized plasmid was inserted in the epimastigote forms by eletroporation. The metacyclogenesis of genetic modified epimastigotes (GMO) was carried out in the LLC-MK2 cells. The trypomastigote forms (GMO) were used to infect mice(10 5 /animal) and the infection could be monitorized by the the detection of bioluminescence produced by the infected tissue and organs, utilizing the IVIS Lumina image system (Xenogen). New tests are being performed to evaluate the sensibility of this methodology. The effects of different topoisomerase inhibitors and DNA binding drugs were tested on the cellular proliferation and ultrastructure of the Trypanosoma cruzi epimatigote form. Blastocrithidia culicis was used as a comparative model, with a more relaxed kdna organization. Our results showed that the eukaryotic topoisomerase I inhibitors, camptothecin and rebeccamycin, were the most effective compounds in T. cruzi proliferation arrest. The eukaryotic topoisomerase II inhibitor, mitoxantrone, but not merbarone, was effective against cell proliferation. The prokaryotic topoisomerase II inhibitors, norfloxacin and enoxacin, targeted the kinetoplast specifically, thus promoting ultrastructural kdna rearrangement in B. culicis. Among DNA binding drugs, berenil caused remarkable kdna 76

77 disorganization. With the exception of camptothecin, there have been no previous evaluations of the compounds tested here on trypanosomatid ultrastructure. We conclude that inhibitors of the same class have different effects on trypanosomatid proliferation and ultrastructure. The results obtained in this work may help us to reveal the mechanism of action of different topoisomerase inhibitors in trypanosomatids. Leishmania amazonensis- Our group has evaluated the effects of 13 phospholipids analogues and dinitroanilines (trifluralin and oryzalin) against promastigotes and intracellular amastigotes of Leishmania amazonensis. The miltefosine and trifluralin have also evaluated in vivo against murine models of cutaneous leishmaniasis by L. amazonensis infection. From assays in vitro with phospholipids analogues, only one compound, the TC 95, was chosen, because the IC 50 values were lower than the values obtained to the other compounds. The effect of dinitroaniline was also studied, because the TC 95 is a hybrid molecule between miltefosine and trifluralin. The results of antiproliferative effects for promastigotes and intracellular amastigotes showed that the TC 95 has a higher leishmanicidal activity than the dinitroanilines and miltefosine. Treatment with TC 95 caused changes in the structure of plasma membrane observed by scanning electron microscope and also in its integrity showed by fluorometry assays with Sytox Blue. Using light and electron microscopy, we could observe changes in the morphology, cell division at the stage of cytokinesis, accumulation of lipid bodies and important lesions in organelles such as mitochondrion, endoplasmic reticulum and Golgi complex at concentrations and times of treatment lower than those of dinitroanilines. The in vivo assay miltefosine was effective when the mice were treated orally with doses of 40 and 50 mg/kg/day for 21 days. Treated-mice presented a significant reduction in the size of the lesions, which disappeared and did not grow again after a hundred days of the end of treatment. However, the treatment performed with trifluralin was ineffective, since it was not possible to observe reduction in size of the lesions in the infected-mice. Thus, these studies suggest that TC 95, a phospholipid analogue, is a compound with an interesting leishmanicidal activity against L. amazonensis in vitro and that miltefosine is effective for the treatment of experimental cutaneous leishmaniasis caused by L. amazonensis. Experimental Chemotherapy in Toxoplasmosis 8 thiolactomycin (TLM) analogues were tested against tachyzoite-infected LLC-MK 2 cells. The TLM analogues demonstrated anti-t. gondii activity, arresting tachyzoite proliferation with IC 50 values in the micromolar level after 24 h and 48 h of treatment. Metabolic labelling of extracellular parasites treated with TLM analogues using [ 3 H]acetate demonstrated that these drugs affected acylglycerol synthesis. The rapid reduction of parasite load suggests that these compounds have selective cytotoxic effects against T. gondii. Transmission electron microscopy demonstrated that TLM analogues interfered with membrane-bounded organelles and parasite division and this in turn affected parasite development and survival (Martins- Duarte et al., Parasitol Int. 58:411-15, 2009). 77

78 The activity of the antifungals fluconazole (FLZ) and itraconazole (ITZ) was also tested against T. gondii in mice infected with the Me49 strain. As previously reported for ITZ, FLZ also demonstrated a selective effect against T. gondii in vitro; the IC(50) values obtained for FLZ were 8.9 microm and 3.1 microm after 24h and 48 h of treatment, respectively. A 10-day treatment of mice with orally or intraperitoneally administered 20mg/kg/day FLZ showed a significant survival difference compared to untreated mice. The administration of 20mg/kg/day ITZ significantly reduced the brain cyst burden compared to untreated mice but did not exert significant protection against death. The results obtained in this work are rather promising as ITZ and FLZ are safe and low-cost drugs available on the market (Martins-Duarte et al., Exp Parasitol 124:466-9, 2010) Group publications ( ): 1. Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi. Rocha, GM; Seabra, SH; Miranda, K; Cunha-e-Silva, N; Carvalho, TMU; De Souza, W. Parasitology International, p. 1-10, Calcium and polyphosphate-containing acidic granules of sea urchin eggs are similar to acidocalcisomes but are not the targets for NAADP. Ramos, I; Miranda, K; Pace, D; Verbist, K; Lin, F-Y; Zhang, Y; Oldfield, E Machado, E; De Souza, W; Docampo, R. Biochemical Journal, 2010, 429(3): Calcium- and polyphosphate-containing acidocalcisomes in chicken egg yolk. Ramos, I; Miranda, K; Ulrich, P; Ingram, P; LeFurgey, A; Machado, E; De Souza, W; Docampo, R. Biology of the Cell,102, , Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole. Miranda, K; Pace, DA; Cintron, R; Rodrigues, JCF; Fang, J; Smith, A; Rohloff, P; Coelho, E; de Haas, F; De Souza, W; Coppens, I; Sibley, D; Moreno, SNJ. Molecular Microbiology, 76, , Dynasore, a dynamin inhibitor, inhibits Trypanosoma cruzi entry into peritoneal macrophages. Barrias, E S; Reignault, LC ; De Souza, W; Carvalho, T MU. PLoS One ;5(1):e Encystation process of Giardia lamblia:morphological and regulatory aspects. Silvestre JB; Lemgruber L ; De Souza, W. Archives of Microbiology, 554, 1-9, Evaluation of three novel azasterols against Toxoplasma gondii. Martins-Duarte S, Lorente S, Magaraci, Ludovic, Gilbert I, de Souza W and Vommaro RC. Veterinary Parasitology in press 8. Heterogeneity in the sensitivity of microtubules of Giardia lamblia to the herbicide oryzalin. Terra, LL ; Campanati, L; De Souza, W. Parasitology Research, 436, , Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids. Lopez ML ; Vommaro, RC; Zallis M ; De Souza, W; Blair S; Segura C. Parasitology International 2010; 59(2): Interaction of the monoxenic trypanosomatid Blastocrithidia culicis with the Aedes aegypti salivary gland. Nascimento, MTC; Garcia, MCF; Pereira, K; Pinto Da Silva, LH; Atella, G; Motta, MCM; Saraiva, E. Acta Tropica,113, , Melanin in Fonsecaea pedrosoi: a trap for oxidative radicals. Cunha, MML; Franzen, AJ; Seabra, SH; Herbst, MH; Vugman, NV; Borba, LP; De Souza, W; Rozental, S. BMC Microbiology (Online), 10, 80-89, Microscopic analysis of calcium ionophore activated egress of Toxoplasma gondii from the host cell. Caldas, LA; De Souza, W; Attias, M. Veterinary Parasitology,167, 8-18, Neolignans from plants in northeastern Brazil (Lauraceae) with activity against Trypanosoma cruzi. Cabral, MMO; Maia, GLA; Chaves, MCO; Braga, MV; De Souza, W Soares, ROA. Experimental Parasitology, 124, , New details on the fine structure of the rhoptry of Toxoplasma gondii. Lemgruber L, Lupetti P, de Souza W, Vommaro RC. Microscopy Research and Technique in press. 15. Organização Estrutural da Forma Taquizoíta de Toxoplasma gondii. De Souza, W; Martins- Duarte, ES; Lemgruber L; Attias, M; Vommaro RC. Scientia Medica (PUCRS), 20: 40, Review on Trypanosoma cruzi: Host Cell Interaction. De Souza W, Carvalho TMU, Barrias ES. Int J Cell Biol. 2010; pii: Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi. Rocha, GM; Teixeira, DE; Miranda, K; Weissmüller, G; Bisch, PM; De Souza, W. Plos One, 5, e11407, The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus. Motta MC, Catta-Preta CM, Schenkman S, de Azevedo Martins AC, Miranda K, de Souza W, Elias MC. PLoS One. 2010; 5(8):e The fine structure of the Acanthamoeba polyphaga cyst wall. Lemgruber L ; Lupetti, P; De Souza, W; Vommaro, RC; Azevedo- Rocha, B. FEMS Microbiology Letters, 305, , Toxoplasma gondii: Fluconazole and itraconazole activity against toxoplasmosis in a murine model. Martins-Duarte, ES; Lemgruber, L; De Souza, W; 78

79 Vommaro, RC. Experimental Parasitology, 124, , Visualization of the flagellar surface of protists by atomic force microscopy. Rocha, GM; Miranda, K; Weissmüller, G; Bisch, PM; De Souza, W. Micron. 2010;41(8): A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia. Abodeely M, DuBois KN, Hehl A, Stefanic S, Sajid M, DeSouza W, Attias M, Engel JC, Hsieh I, Fetter RD, McKerrow JH. Eukaryotic Cell, 8, , Brazilian contribution for a better knowledge on the biology of Toxoplasma gondii. De Souza, W; Damatta, RA; Attias, M. Memórias do Instituto Oswaldo Cruz, 104, , Characterization in vivo and in vitro of a strain of Leishmania (Viannia) shawi from the Amazon Region. Ramos PK, Diniz JA, Silva EO, Quaresma JA, Saraiva EM, Seabra SH, Atella GC, de Souza W. Parasitol Int. 2009; 58(2): Cidofovir inhibits genome encapsidation and affects morphogenesis during the replication of vaccinia virus. Jesus DM, Costa LT, Gonçalves DL, Achete CA, Attias M, Moussatché N, Damaso CR. J Virol. 2009; 83(22): Cryptococcus neoformans cryoimmunoelectronmicroscopy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronxylomannan. Oliveira DL, Nimrichter L, Miranda K, Frases S, Faull KF, Casadevall A, Rodrigues ML. Fungal Genet Biol (12): Distinct acetylation of Trypanosoma cruzi histone H4 during cell cycle, parasite differentiation, and after DNA damage. Nardelli SC, Chagas da Cunha JP, Motta MCM, Schenkman S. Chromosoma, 118, , Dynamin inhibitor impairs Toxoplasma gondii invasion.caldas LA, Attias M, de Souza W. FEMS Microbiology Letters, 301, , Electron Microscopy and Cytochemistry Analysis of the Endocytic Pathway of Pathogenic Protozoa. De Souza W, Sant Anna C, Cunha-e-Silva NL. Progress in Histochemistry and Cytochemistry, 44, , Expression and subcellular localization of kinetoplast-associated proteins in the different developmental stages of Trypanosoma cruzi. Cavalcanti, DP; Shimada, MK; Probst, CM; Souto-Pádron, T; De Souza, W; Goldenberg, S; Fragoso, SP; Motta, MCM. BMC Microbiology (Online), 9, , Giardia lamblia: Characterization of ecto-phosphatase activities. A; Arayaa, JE ; González, J. Parasitology International, 58, , Particularities of mitochondrial structure in parasitic protists (Apicomplexa and Kinetoplastida). De Souza W, Attias M, Rodrigues JC. International Journal of Biochemistry & Cell Biology, 41, , Phylogenetic Analyses Based on Small Subunit rrna and Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Ultrastructural Characterization of Two Snake Trypanosomes: Trypanosoma serpentis n. sp. from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus. Viola LB, Attias M, Takata CS, Campaner M, De Souza W, Camargo EP, Teixeira MM. J Eukaryot Microbiol. 2009; 56(6): Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescens D- mannose sensitive fimbriae. Moraes CS, Seabra SH, Albuquerque-Cunha JM, Castro DP, Genta FA, de Souza W, Brazil RP, Garcia ES, Azambuja P. Exp Parasitol. 2009;122(2): Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs. De Souza W, Rodrigues JC. Interdiscip Perspect Infect Dis. 2009; Structural organization of Trypanosoma cruzi. De Souza W. Mem Inst Oswaldo Cruz. 2009; 104 Suppl 1: Subcellular proteomics of Trypanosoma cruzi reservosomes. Sant'anna C, Nakayasu ES, Pereira MG, Lourenço D, de Souza W, Almeida IC, Cunha-e-Silva NL. Proteomics, 9, , Thiolactomycin analogues as potential anti-toxoplasma gondii agents. Martins-Duarte ES, Jones SM, Gilbert IH, Atella GC, De Souza W, Vommaro RC. Parasitol Int ;58(4): Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation. Villanova GV, Nardelli SC, Cribb P, Magdaleno A, Silber AM, Motta MC, Schenkman S, Serra E. International Journal for Parasitology, 39, , Trypanosoma cruzi: parasite shed vesicles increase heart parasitism and generate an intense inflammatory response. Trocoli Torrecilhas AC, Tonelli RR, Pavanelli WR, da Silva JS, Schumacher RI, De Souza W, Cunha-e-Silva NL, de Almeida Abrahamsohn I, Colli W, Manso Alves MJ. Microbes and Infection, 11, 29-39, Amazonas JN, Cosentino-Gomes D, Werneck-Lacerda A, Pinheiro AAS, Lanfredi-R 33. Glucose uptake in the mammalian stages of Trypanosoma cruzi. Silber, AM; Tonelli, RR; Lopes, CG; Cunha-e-Silva, N; Torrecilhas, AT; Schumacher, RI; Colli, W; Alves, MJM. Molecular and Biochemical Parasitology 168, , HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis. Santos, LO; Marinho, FA; Altoé, EF; Vitorio, BS; Alves, CR; Britto, C; Motta, MCM; Branquinha, MH; Santos, ALS; Davila-Levy, C. Plos One, 4, , Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome. Gutierrez, B; Osorio, L; Motta, MCM; Huima- Byron, T; Erdjument- Bromage, H; Muñoz, C; Sagua, H; Mortara, R; Echeverria, 79

80 Book Chapters: 1. Cunha e Silva, N. L. Sant Anna, C., Pereira, M.G., De Souza, W. Reservosomes of Trypanosoma cruzi. In W. de Souza (ed.), Structures and Organelles in Pathogenic Protists, Microbiology Monographs 17, DOI / _5, Springer-Verlag, Berlin Heidelberg, 2010, p Carvalho, TMU; De Souza, W. Microscopia de Fluorescência. Marcadores de Organelas. In: Wanderley de Souza. (Org.). Microscopia óptica: Fundamentos e Aplicações às Ciências Biomédicas. 1 ed. Rio de Janeiro: Sociedade Brasileira de Microscopia e Microanálise, 2010, 3. Fontes, A., Thomaz, A.A., Cesar C.L., De Souza, W. Microscopia de Óptica não Linear ou Raman. In: Wanderley de Souza. (Org.). Microscopia óptica: Fundamentos e Aplicações às Ciências Biomédicas. 1 ed. Rio de Janeiro: Sociedade Brasileira de Microscopia e Microanálise, De Souza, W. Microscopia de Fluorescência de Alta Resolução. In: Wanderley de Souza. (Org.). Microscopia óptica: Fundamentos e Aplicações às Ciências Biomédicas. 1 ed. Rio de Janeiro: Sociedade Brasileira de Microscopia e Microanálise, Miranda, K.; Gomes, F. Deconvolução de imagens. In: Wanderley de Souza. (Org.). Microscopia óptica: Fundamentos e Aplicações às Ciências Biomédicas. 1 ed. Rio de Janeiro: Sociedade Brasileira de Microscopia e Microanálise, 2010, p De Souza, Wanderley; de Carvalho, Tecia Maria Ulisses; Barrias, Emile S. Ultrastructure of Trypanosoma cruzi and its interaction with host cells. In: Jenny Telleria; Michel Tibayrenc. (Org.). American Trypanosomiasis. Chagas Disease. One hundred years of Research, 2010, p De Souza, W; Miranda, K ; Cunha e Silva, N.L.; Souto-Padron, T. A Review on the Ultrastructure of Trypanosoma cruzi. In: Antonio Teixeira, Marina Vinaud, Ana Maria Castro. (Org.). Emerging Chagas Disease. 1 ed. Oak Park IL: Bentham Science Publishers, 2009, v. 1, p

81 AL10 Coordinator: Paulo Mascarello Bisch, IBCCF/UFRJ Members: Gilberto Weissmuller Geraldo Antônio Cidade ASSOCIATE LABORATORY OF GENOMIC, PROTEOMIC, MODELING AND NANOSCOPY OF BIOLOGICAL SYSTEMS General Remarks: Following the tradition of our group, along these two years we have introduced and developed new tools and extended our collaboration with others groups inside the INCT, but also with some outside groups including international partners. We point out also that younger scientists of our group (G. Weissmuller and A.B. Pacheco) have initiated and conducted collaborations in an independent way, showing their effectiveness in propose and develop their own scientific work. We list below the studies that resulted in publications in the last two years. Consensus modes, a robust description of protein collective motions from multipleminima normal mode analysis application to the HIV-1 protease. Paulo Ricardo Batista, Charles Herbert Robert, Jean-Didier Maréchal, Meriam Ben Hamida-Rebaï, Pedro Geraldo Pascutti, Paulo Mascarello Bisch and David Perahia., Phys. Chem. Chem. Phys., 2010, 12, A new method to analyse large and collective motions in protein was proposed by our group in collaboration with Prof. David Perahia from University Paris-Sud. Although molecular dynamics is now reliable in the hundred nanosecond scale, even for large systems, most of the important motions in the 81

82 millisecond scale, involved, for instance, in the catalytic activity of enzymes, are still difficult to be attained and analysed. The proposed method combines a short dynamics, typically 50 ns, with a normal modes analysis, given a robust picture of important motions involved in catalytic activity, as exemplified in the article for the HIV-1 protease. Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi. Gustavo Miranda Rocha, Dirceu Esdras Teixeira, Kildare Miranda, Gilberto Weissmüller, Paulo Mascarello Bisch, Wanderley de Souza., PLoS ONE 5(6): e (2010). The unusual combination of Electron Microscopy with Atomic Force Microscopy was used in a fruitfull collaboration with the group of Prof. Wanderley de Souza by showing within molecular details the structural changes during flagellar beating in T. cruzi. Our work is pioneer showing the powerful combination of these techniques. Unravelling the molecular basis of the selectivity of the HIV-1 fusion inhibitor sifuvirtide towards phosphatidylcholinerich rigid membranes. Henri G. Franquelim A. Salomé Veiga, G. Weissmüller, Nuno C. Santos and Miguel A.R.B. Castanho, Biochimica et Biophysica Acta (BBA) Biomembranes, Volume 1798, Issue 6, June 2010, Pages One of the crucial steps on the virus invasion of cells is the virus fusion with the cell lipid membrane. The combination of several spectroscopic techniques, including atomic force spectroscopy, a method now currently used by our group, was used to study the interaction of a HIV fusion inhibitor with lipid films, in a collaborative work with the group of Dr. Castanho from University of Lisbon. The paper reveals important structural details of this interaction and the mechanism of inhibition, which should be used as a guide to design new and more efficient inhibitors. Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae. Goulart, Carolina L. ; Lery, Letícia M.S. ; Diniz, Michelle M.P. ; Vianez-Junior, João L. ; Neves-Ferreira, Ana Gisele C. ; Perales, Jonas ; Bisch, Paulo M ; von Krüger, Wanda M.A. FEMS Microbiology Letters, 298 (2009) As a consequence of previous proteomic analysis, we have identified a new putative phosphoporin involved in the response of phosphate starvation. We have made a detailed molecular analysis of the expression and modeling the structure of this new porin, which should have an important role on the adaptation during human colonization by V. cholerae. The studies of the expression conditions, bioinformatics and computer modeling have helped us to confirm the function of the new protein and have shown the effectiveness of such approach. Effects of light intensity and light quality on growth and circadian rhythm of saxitoxins production in Cylindrospermopsis raciborskii (Cyanobacteria). Ronaldo Leal Carneiro, Maria Elisângela Venâncio dos Santos, Ana 82

83 Beatriz Furlanetto Pacheco and Sandra Maria Feliciano de Oliveira e Azevedo. Journal of Plankton Research, Volume 31, Number 5, Pages , Covering a new subject, we have started a new collaboration with the group of Dr. Sandra Azevedo about toxin production in Cyanobacteria, a very important contaminant of waters, especially in our region. The initial work concerns the conditions of toxin production as a function of light radiation of the cyanobacteria culture. Our groups will investigate proteome and protein expression regulation of thir bacteria. Group publications ( ): 1- Paulo Ricardo Batista, Charles Herbert Robert, Jean-Didier Maréchal, Meriam Ben Hamida-Rebaï, Pedro Geraldo Pascutti, Paulo Mascarello Bisch and David Perahia. Consensus modes, a robust description of protein collective motions from multipleminima normal mode analysis application to the HIV-1 protease, Phys. Chem. Chem. Phys., 2010, 12, Gustavo Miranda Rocha, Dirceu Esdras Teixeira, Kildare Miranda, Gilberto Weissmüller, Paulo Mascarello Bisch, Wanderley de Souza. Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi, PLoS ONE 5(6): e (2010). 3- Henri G. Franquelim A. Salomé Veiga, G. Weissmüller, Nuno C. Santos and Miguel A.R.B. Castanho, Unravelling the molecular basis of the selectivity of the HIV-1 fusion inhibitor sifuvirtide towards phosphatidylcholine-rich rigid membranes. Biochimica et Biophysica Acta (BBA) Biomembranes, Volume 1798, Issue 6, June 2010, Pages Goulart, Carolina L. ; Lery, Letícia M.S. ; Diniz, Michelle M.P. ; Vianez-Junior, João L. ; Neves-Ferreira, Ana Gisele C. ; Perales, Jonas ; Bisch, Paulo M ; von Krüger, Wanda M.A. Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae. FEMS Microbiology Letters, 298 (2009) Ronaldo Leal Carneiro, Maria Elisângela Venâncio dos Santos, Ana Beatriz Furlanetto Pacheco and Sandra Maria Feliciano de Oliveira e Azevedo. Effects of light intensity and light quality on growth and circadian rhythm of saxitoxins production in Cylindrospermopsis raciborskii (Cyanobacteria). Journal of Plankton Research, Volume 31, Number 5, Pages ,

84 84

85 AL11 ASSOCIATE LABORATORY OF MICROSCOPY. Coordinator: Thaís Cristina Souto Padrón, IMPPG/UFRJ Member: Ulisses Lins This group consists of 2 laboratories involved in structural and cellular biology: 1 - Laboratório de Biologia Celular e Ultraestrutura - coordinated by Dr. Thaïs Souto-Padrón focus its attention to the study of the structural organization of parasitic protozoa such as Trypanosoma cruzi and Leishmania and their interaction with hostcells. The main subjects of our interest are: Analysis of the effects of drugs that interfere in the endocytic /exocytic pathways in T. cruzi and Leishmania and their potential effects in the modulation of cell surface molecules- This subject is complemented by the studies of parasite-host cell interaction. In this topic we have analyzed the effect of Bromoenol lactone (BEL) an inhibitor of the PLA2 activity in the morphology and in the intracellular traffic of endocytic tracers and surface molecules of the promastigote forms of Leishmania amazonensis. This study is developed by Anne Cristine Silva Fernandes that completed her master theses in july 2010 and continue in the same subject in her PhD studies. The student is completing the structural analysis of the effect of BEL on the ultrastructure of L. amazonensis in order to present a tridimensional model based in serial sections analysis. Figure 1. 85

86 Experimental chemotherapy in trypanosomatids- We have analyzed the effects of natural compounds such as snake and bee venons on the proliferation and ultrastructure searching to define the kind of cell death process involved in parasite death. This topic has been developed by 2 students: Camila Marques Adade that has recently concluded her PhD theses (July 2010) and continues in the laboratory as a Pos- Doc student; The second student is Gabriela Santos Ferreira das Chagas, an undergraduated student co-oriented by Camila Adade that works with bee venons. Recently, Camila published a paper and a review about chemotherapy in T. cruzi. Figure 2 The analysis of shedding process in T. cruzi- The ultrastructure and immunocytochemical detection of the components of the shedding vesicles from differente strains of the parasite. In this topic we have analyzed the ultrastructure, composition (presence of proteases and antigens), and signaling pathways involved in the process of shedding of different vesicles by trypomastigote and amastigote forms of T. cruzi. This topic has been conducted by Roberta Ferreira Cura das Neves, now a PhD student that presented her master theses, also related to shedding process, in August Roberta co-orients an undergraduated student, Grazielle Lima Cruz in the same topic. During the last 12 months, Roberta has been analyzing the presence of metalloproteases in the shedding vesicles from trypomastigotes of different strains of T. cruzi and the effect of cell surface ligands, such as cationized ferritin and concanavalin A, in stimulating shedding process. Nowadays the students are accumulating shedding vesicles to analyze their ultrastructure in criofracture replicas. Ultrastructural analysis of trypanosomatids isolated from fishes and toads- This topic is developed by a PhD student, Moara Lemos that analyses at the scanning and transmission electron microscopes the ultrastructure of trypanosomes obtained from the blood of Brazilian fishes and toads. The development of a tridimensional model using tomography is in the planes for the next year. Figure 1: Cytochemical detection of acid phosphatase in promastigotes of L. amazonensis. A- control - the electrondense precipitate indicative of the acid phosphatase activity on the flagellar (red arrow), flagellar pocket (blue arrow) and on the cell body membrane (black arrow) and inside the pocket where it is released; B and C- cells previously treated with BEL. After treatment with 2.5 mm BEL for 1 h, there was a decrease in the intensity of staining on the membrane of the flagellum (red arrow), and on the flagellar pocket membrane (blue arrow ). The activity observed on the cell body membrane parasite also appears to vary in intensity (black arrow). In MVBs the electrondense precipitate can be observed in internal lumen of the organelle and on the membrane of the vesicles inside it. Treatment with BEL results in the redistribution of staining all the Golgi cisterns (G). The precipitate is also present in tubules (yellow arrow) and vesicles neat the flagellar pocket (green arrow). bf-flagellar pocket, G-Golgi, multivesicular bodies, MVB, multivesicular tubules-tmv. Bars =300nm. 86

87 Figure 2. Effect of Cvv venom on the ultrastructure of Trypanosoma cruzi trypomastigotes observed using transmission (A, F J) and scanning (B E) electron microscopy. Parasites were treated with 0 3 μg/ml Cvv venom for 1 day. (A, B) Control parasites presenting a typical morphology with normal characteristics of the nucleus (N) and kinetoplasts (k). (C E) Treated parasites presenting swollen and twisted cell bodies (white star in C and D), loss of membrane integrity and cell lysis (E and H). The main changes in the ultrastructure of the trypomastigotes observed by TEM were shrinkage of the nuclear membrane (arrowhead in the inset and in F), the presence of clear areas in the cytoplasm (star in F), blebs budding from the cell body (arrow in G) and from the flagellar membrane (arrows in F and I), and the presence of swollen organelles (G). (J) Note the swollen mitochondria (black star). Scale bars: A, F J=300 nm; B E=2 μm. Adade et al., Parasitology Laboratório de Ultraestrutura e Biologia Celular de Procariotos - coordinated by Dr. Ulysses Lins focus its attention to the study of the biology, diversity and biomineralization in magnetotactic bacteria. The main subjects of our interest are: Biology and diversity of magnetotactic bacteria- During the period, we advanced in the description of magnetotactic bacteria in extreme environments. To achieve that goal we analyzed samples collected from sediments with extreme conditions: high temperature, low temperature, high salinity and high sulfur content. At least four new types of magnetotactic bacteria were discovered and their morphological (ultrastructure, mineral analysis of the magnetosomes) and phylogenetic (16S rdna sequencing) characteristics were described (Figure 3). Biomineralization of magnetosomes- We have studied the biomineralzaition of magnetosomes in magnetotactic bacteria for several years. We have studied the purity of the magnetosome particles and have shown that they can incorporate small amounts of magnese in their crystalline structure (Figure 4). Cultivation of magnetotactic bacteria- Magnetotactic bacteria are fastidious microorganisms. But, to advance in the understanding of the cell biology it is mandatory to grow these cells in pure cultures. For that we have established a collaboration effort with Professor Dennis Bazylinski from University of Nevada, LV, EUA. Dr. Bazylinski is one of the world leading experts in magnetotactic microorganisms. So far, we have been able to cultivate at least three strains of marine and freshwater magnetotactic bacteria. Also, we are now trying the isolate and grow one strain from sediments collected in Brazil.

88 Figure 3: Ultrastructure of cells of strain LO-1. A) DIC light microscope image showing the numerous, large, highly refractile, intracellular inclusions within a cell of strain LO-1. B) Transmission electron microscope (TEM) image of an unstained LO-1 cell showing large globular inclusions and magnetosomes. C) Elemental spectra of an inclusion (beam focused at white star) and backgroundof the cell (beam focused at black star) using energy dispersive x-ray spectroscopy analysis. Note that the globular inclusion is sulfur-rich and appear to be similar to the type of sulfurcontaining inclusions typical of sulfide-oxidizing bacteria. D) TEM image of a stained thin-section of a cell of LO-1 showing the complex tripartite cell wall composed of the cytoplasmic membrane (at black arrow), the outer membrane (at grey arrow) and the external amorphous layer (at white arrow). The latter might represent some type of polysaccharide layer. The empty inclusions appear to be the same shown in F below. E and F) TEM images of a thin section of a stained LO-1 cells showing the two types of numerous inclusions present in LO-1 cells. Those in E show some degree of extraction during fixation (shown as holes (at star)) and could be the sulfur-rich inclusions described above. Note the smaller inclusions shown in F have an electron-dense periphery with a less dense center. Figure 4 - High-resolution elemental maps of iron, oxygen and manganeseof a magnetosome from a 24-h manganese-exposed sample, obtained using the threewindow method. Scale bar = 100 nm.

89 Group publications ( ): Thaïs Souto-Padrón: 1) Da Silva CV, Kawashita SY, Probst CM, Dallagiovanna B, Cruz MC, da Silva EA, Souto-Padrón TC, Krieger MA, Goldenberg S, Briones MR, Andrews NW, Mortara RA. Characterization of a 21kDa protein from Trypanosoma cruzi associated with mammalian cell invasion. Microbes Infect. 11(5): , ) Soares JA, Leite FG, Andrade LG, Torres AA, De Sousa LP, Barcelos LS, Teixeira MM, Ferreira PC, Kroon EG, Souto-Padrón T, Bonjardim CA. Activation of the PI3K/Akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication. J. Virolology 83(13): , ) Cavalcanti DP, Shimada MK, Probst CM, Souto-Padron TC, De Souza W, Goldenberg S, Fragoso SP, Motta MC. Expression and subcellular localization of kinetoplast-associated proteins in the different developmental stages of Trypanosoma cruzi. BMC Microbiol. 9(1): 120, ) Adade CM, Cons BL, Melo PA, Souto-Padrón T. Effect of Crotalus viridis viridis snake venom on ultrastructure, and intracellular survival of Trypanosoma cruzi. Parasitology, IN PRESS, available on line 5) Adade CM & Souto-Padrón T. Contributions of Ultrastructural Studies on Cell Biology of Trypanosomatids: Targets for Anti- Parasitic Drugs. The Open Parasitology Journal, 2010, 4, IN PRESS 6) Vermelho AB, Nogueira de Melo AC, Soares RA, Alviano DS, Souza EP, Souto-Padrón T, Lopes AH, Rodrigues GC, Aguiar AP, Pereira MC, Ferreira-Pereira A, Rosa MS, Meirelles MNL, Alviano CS. Trypanosoma cruzi peptidases: An overview. The Open Parasitology Journal, 2010, 4, IN PRESS 7) Lopes AH, Dias F, Gomes MT, Capcci G, Zimmermann LT, Alves e Silva TL, Souto-Padrón T, and Vermelho AB. Trypanosomatids: Odd organisms, devastating diseases. The Open Parasitology Journal, 2010, 4, IN PRESS 8) Lopes AH, Gomes MT, Dutra FL, Vermelho AB, Meyer-Fernandes JR, Silva-Neto MAC, Souto-Padrón T, Vieira DP. Intracellular Signaling Pathways Involved in Cell Differentiation in Trypanosomatids. The Open Parasitology Journal, 2010, 4, IN PRESS CHAPTER BOOK Wanderley de Souza, Kildare Miranda, Narcisa Leal Cunha e Silva and Thaïs Souto- Padrón. A Review on the Ultrastructure of Trypanosoma cruzi. in EMERGING CHAGAS DISEASE, CHAPTER FIVE Pp eisbn: , 2009 Editors: Antonio Teixeira Marina Vinaud Ana Maria Castro Ulysses Lins: 1) C.N. Keim, U. Lins and M. Farina. Manganese in biogenic magnetite crystals from magnetic bacteria. FEMS Microbiology Letters 292: , ) C.C.P. Hardoim, R. Costa, F. Araújo, E. Hajdu, R. Peixoto, E. Miranda, U. Lins; A.S. Rosado, and J.D. van Elsas. Diversity of bacteria in the marine sponge Aplysina fulva in Brazilian coastal waters. Applied and Environmental Microbiology 75: , ) V. Bernardo, S. Q.C. Lourenço, R. Cruz, L.H. Monteiro-Leal, L. E. Silva, D. R. Camisasca, M. Farina, and U. Lins. Reproducibility of immunostaining quantification and description of a new digital image processing procedure for quantitative evaluation of immunohistochemistry in pathology. Microscopy and Microanalysis 15: , ) J.L. Martins, T.S. Silveira, K.T. Silva, R.L. Sobrinho, M.C. Bernardes, A.S. Rosado, U. Lins. Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon. International Microbiology 12: , ) M. L. E. Gutarra,; M. G. Godoy, J. N. Silva, I. A. Guedes, U. Lins, L. R. Castilho, D. M. G. Freire. Lipase production and morphology in solid-state and submerged fermentations. Biotechnology Journal. 4: , ) K. P. Lam, A.P. Hitchcock,, M. Obst, J. R. Lawrence, G.D.W. Swerhon,, G. G. Leppard, T. Tyliszczak, C. Karunakaran, J. Wang, K. Kaznatcheev, D. Bazylinski, U. Lins. Characterizing magnetism of individual magnetosomes by X-ray magnetic circular. Chemical Geology, 270: , ) J. P.Albuquerque, C. N. Keim, U. Lins. Comparative analysis of Beggiatoa from hypersaline and marine environments. Micron 41: , ) A.M. Mazotto,, S.M. Lage Cedrola, U. Lins, A. S. Rosado, K.T. Silva, J.Q. Chaves, L. Rabinovitch, R.B. Zingali,, A.B. Vermelho. Keratinolytic activity of Bacillus subtilis AMR using human hair. Letters in Applied Microbiology 50: 89-96, ) C. T. Lefreve, F. Abreu, U. Lins, D. Bazylinski. Non-magnetotactic multicellular prokaryotes from low saline, nonmarine aquatic environments and their unusual negative phototactic behavior. Applied and Environmental Microbiology 76: , ) C.T. Lefevre, F. Abreu,; M.L. Schmidt, U Lins, R.P. Frankel, B.P. Hedlund, D. Bazylinski, Moderately thermophilic 89

90 magnetotactic bacteria from hot springs in Nevada USA. Applied and Environmental Microbiology 76: , ) L. G. Abraçado, F. Abreu, C. N. Keim, A. P. C. Campos, U. Lins, M. Farina. Magnetosome chain superstructure in uncultured magnetotactic bacteria. Physical Biology in press, ) R. Sobrinho, U. Lins, M. Bernardes. Geochemical characteristics related to the gregite producing multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis in a hypersaline lagoon. Geomicrobiology Journal in press, ) C.T. Lefevre, R.P Frankel, F. Abreu, U. Lins, D. Bazylinski. Cultureindependent characterization of a novel, uncultivated magnetotactic member of the Nitrospirae phylum. Environmental Microbiology in press, CHAPTER BOOK 1) U. Lins, D. Bazylinski, D. (2009). Magnetotaxis. In: Moselio Schaechter. (Org.). Encyclopedia of Microbiology. 3 ed.eua: Elsevier Inc., pp

91 AL12 ASSOCIATE LABORATORY OF CELLULAR ULTRASTRUCTURE. Coordinator: Marlene Benchimol, Universidade Santa Ursula (USU) The main purposes of the project were followed, as shown below: 1) Several drugs were used to compare the behavior of the trichomonas under interaction with host-cells. Several different host cells were used, such as: (a) oviduct cells obtained from cows, and formation of a primary cell culture, (b) MDCK cells, (c) Caco Cells. Interactions were performed with different strains of T. vaginalis and T. foetus. We have one article already published and another one under submission. 2) We have followed the behavior of pseudocysts, a form of trichomonas which internalize the flagella under stress conditions. For this, we have collaborated with Dr. Carlos Campero, from Argentina, who provided several fresh trichomonas isolates directly taken from bulls and cows in Argentina. We have obtained several results which were published in one article. The aim of the present study is to verify whether T. foetus pseudocysts are encountered in naturally infected bulls. In an attempt to clarify this question, fresh preputial samples obtained from seven mature bulls, naturally infected with T. foetus, were analyzed using complementary techniques, such as videomicroscopy, fluorescence microscopy, scanning and 91

92 transmission electron microscopy. The analyses revealed that approximately 55% of the parasites were in pseudocyst form at each preputial sample, whereas approximately 25% of T. foetus displayed pear-shaped body. 3) We have isolated organelles using cell fractionation form trichomonas. The main organelles were: hydrogenosomes, costa and Golgi. One article is in preparation, showing two specific proteins found in trichomonas Golgi. 4) The antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol and 24(R,S),25- epiminolanosterol, known inhibitors of 24(25) - sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) cell wrinkling and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines MDCK showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis. 5) We have published an important article concerning the effects of jasmonates in trichomonas. Jasmonates are a group of small lipids that are produced in plants and function as stress hormones. We tested this drug against Trichomonas vaginalis and we demonstrated using flow cytometry, JC-1 and scanning and transmission electron microscopy that MJ induced the cell death of T. vaginalis parasites. 6) Concerning Giardia lamblia, we have obtained gerbils to in vivo infection. Processes of encystation and excystation were performed in vitro, successfully. We have used immunocytochemistry to compare the behavior of the encystation specific vesicles (ESV) and try to find the granules responsible for the carbohydrates portion of the cyst wall. Using immunofluorescence we detected two granules populations, which is an important result. In addition, cytochemistry for acid phosphatase demonstrated a positive reaction in a distinct sub-population of vesicles localized in Giardia when they are under excystment. 7) In another report we showed the cytopathic effect of Trichomonas vaginalis, using oviduct cells. It occurs due to mechanical stress and subsequent phagocytosis of the necrotic cells. The investigation was done using a primary culture of bovine oviduct epithelial cells (BOECs), grown either in monolayers or as floating cells. Trophozoites displaying different virulence levels were co-incubated with BOECs for times varying between 1 min and 48 h. Analyses were performed using videomicroscopy, scanning and transmission electron microscopy, colourimetric assays and 92

93 cytochemistry. Injury was observed as early as 1 h after incubation, while after 12 h the host cells were severely damaged when a fresh trichomonad isolate was used. Trichomonads attack the host cells by clustering around them. Mechanical stress on the microvilli of the host cells was observed and appeared to induce plasma membrane damage and cell death. After membrane injury and lysis, fragments of the necrotic cells were ingested by trichomonads. Phagocytosis occurred by trichomonads avidly eating large portions of epithelial cells containing the nucleus and other organelles, but living or intact cells were not ingested. Necrotic fragments were rapidly digested in lysosomes, as shown by acid phosphatase and ruthenium red assays where only the BOECs were labelled. The lytic capacity of the trichomonads was more pronounced in host cell suspensions. SEM of T. foetus pseudocysts (P) obtained directly from fresh preputial secretions. Parasites are seen adhered to the mucus (M). Observe that pseudocysts present rounded form and the flagella are no longer visible. Notice that some pseudocysts have pseudopod-like projections (asterisks) in contact with the mucus. Bar, 2µm. Group publications ( ): 1.Giardia lamblia behavior during encystment: how morphological changes in shape occur. Parasitology International. Parasitol Int Mar;58(1): Hydrogenosomes under microscopy- a review- Marlene Benchimol-Tissue and Cell. Tissue Cell Jun;41(3): Epub 2009 Mar Cytoskeleton in Trichomonads. Trends in Cell & Molecular Biology. Vol.4, pp (2009). 4. Tritrichomonas foetus: budding from multinucleated pseudocysts. Pereira-Neves, Antonio and Benchimol, Marlene-Protist, 2009, 160, Cytopathic effects of Tritrichomonas foetus on bovine oviduct cells. V. Midlej, R. Vilela, A. B. Dias, M. Benchimol. Veterinary Parasitology- Veterinary Parasitology 165 (2009) The protist Trichomonas vaginalis harbors multiple lineages of transcriptionally active Mutator-like elements. Fabrício R. Lopes, Joana C. Silva, Marlene Benchimol, Gustavo G. L. Costa, Gonçalo A. G. Pereira, Claudia M. A. Carareto. BMC Genomics , Cell death induction in Giardia lamblia: effect of beta-lapachone and starvation- Gladys Correa, Ricardo Vilela, Rubem FS Menna-Barreto, Victor Midlej, Marlene Benchimol- Parasitol Int Dec;58(4): Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+. de Meis L, Ketzer LA, da Costa RM, de Andrade IR, Benchimol M. PLoS One Mar 2;5(3):e Methyl jasmonate induces cell death and loss of hydrogenosomal membrane potential in Trichomonas vaginalis" has been accepted for publication in Parasitology International. (2010). Vilela, R. Menna-Barreto, R.F.S and Benchimol, M. Parasitol Intern 59 (3) Desenvolvimento de material multimídia no ensino de Biologia. Marlene Benchimol, Marianna Augusta Ferrari do Outeiro Bernstein, Rodrigo Alcantara de Carvalho e Dirceu Esdras Teixeira. EAD em Foco Trichomonas vaginalis kills and eats evidence for phagocytic activity as a cytopathic effect. Midlej, V., M. Benchimol. Parasitology Jan;137(1):

94 12. Death of neonatal retinal ganglion cells induced by axon damage: caspase dependency and induction of autophagy as a survival mechanism. Cinthya Sternberg, Marlene Benchimol, Rafel Linden. Brazilian Journal of Medical and Biological Research Aceito em julho de Identification of Tritrichomonas foetus pseudocysts in fresh preputial secretion samples from bulls. Antonio Pereira-Neves a, b, Carlos Manuel Campero c, Alfredo Martínez d and Marlene Benchimol a, Veterinary Parasitology. In Press. Books and Book Chapters: 1. BENCHIMOL, M. The Mastigont System In: Structures and Organelles in Pathogenic Protists.1 ed.londres : Springer-Verlag, 2010, v.1, p BENCHIMOL, M. Basic Biology of Giardia lamblia: further studies on median bodies and funis In: Giardia and Criptosporidum: from Molecules to diseases.1 ed. : CABI Publishers, 2009, v.1, p Books organizations: 1. BENCHIMOL, M., BERSTEIN, M. O.- Biologia Celular II. Rio de Janeiro : Fundação Cecierj, 2009, v.1. p.xx. 2. C. Tavares, BENCHIMOL, M.- Botânica II. Rio de Janeiro : CECIERJ, 2009, v Siano Miguel, D. Esdras Teixeira, BENCHIMOL, M.-Dinâmica da Terra. Rio de Janeiro : CEDERJ, 2009, v R. Carvalho, BENCHIMOL, M.- Genética Básica para o Curso de Graduação. Rio de Janeiro : Fundação Cecierj, 2009, v.1. p.xx. 5. BENCHIMOL, M., Agnaldo Esquincalha- Matemática: Pré-Cálculo na era das mídias digitais uma experiência animada. Fundação Cecierj, 2009, v.1. 94

95 AL13 THE Coordinator: Celso B. Sant'Anna Filho, INMETRO Members: Wanderley de Souza Kildare Miranda Gustavo Conde Menezes Daniela Lourenço Danielle Cavalcanti Isabel Port-Carreiro ASSOCIATE LABORATORY OF STRUCTURAL BIOTHECNOLOGY The newly established Biotechnology Laboratory, at National Institute of Metrology, Standardization and Industrial Quality INMETRO, has a group composed by 6 researches, 5 technicians and 6 undergraduate students, which include biologists and physics. The focus of our group is mainly based on bionanometrological and biofuels studies, as described below. Renewable energy sources are developed worldwide, owing to high oil prices and to limit greenhouse gas emissions. Currently, there are many international efforts aimed to finding renewable, sustainable, and environment friendly energy sources to overcome these problems. Biofuels (bioethanol and biodiesel) have attracted considerable attention during the past decade as renewable source fuel with environmental benefits. However, concerns exist about the source of feedstocks, including the impact it may have on biodiversity and land use and competition with food crops. As bioethanol is considered a viable energy source for the future, it is expected to form a sustainable basis, meet socio-economic concerns, providing greater security for energy supply and reduce the environmental impacts associated with fossil fuels. Plant cell wall (PCW) is a high complex structure mainly 95

96 composed of polysaccharides (cellulose and hemicelluloses) and lignin. Lignocellulosic biomass, including sugarcane, has been considered as potential source to second generation biofuel production. The technology used to conversion of fermentable sugar in bioethanol involves pretreatment, which aim to improve digestibility of biomass, such as acidic and thermal degradation. The PCW molecular architecture remains unclear and it has been related to recalcitrance of biomass to deconstruction. A We have applied high resolution microscopy methodologies to have a detailed analysis of sugarcane cell wall architecture, as well as to analyze its deconstruction after pretreatments, focusing on the effect in lignin (Figure 1). Also, using 1D and 2D-PAGE and mass spectrometry, we have also analyzed the protein of PCW sugarcane, to get some information for improving the process of ethanol saccharification (Figure 1d). The feedstocks usually adopted for biodiesel production are vegetable oils. Recently, much attention has been paid to the exploration of microbial oils. The oil productivity of microorganisms greatly exceeds the vegetable productivity and non-arable land is used for production. We isolated some oleaginous yeasts and microalgaes and analyze the oil content using Nile Red staining and Confocal Laser Scanning Microscopy (Figure 2). Other focus of our work is the study of the protozoan members of the Kinetoplastidae family, which are characterized for the presence of specific and unique structures that are involved in different cell activities. The mitochondrial or kinetoplast DNA (kdna) of trypanosomatids and the Paraflagellar Rod (PFR), a complex array of filaments connected to the flagellar axoneme, are two of these structures. Also, in order to obtain information about the process of flagellar cytoskeleton formation, we are investigating the biogenesis of the flagellar structure during the transformation of amastigotes into the promastigotes. To understand the biological significance of the kinetoplast DNA, we are developing a procedure to analyze the intact Figure 1. Sugarcane cell wall observed by different techniques. (a) Confocal laser scanning micrograph; (b) Scanning electron micrograph; (c) Error signal INBEB of Atomic Force INBEB Microscopy ANNUAL (d) 1D-PAGE REPORT of proteins of 96 sugarcane cell wall. B 5 µ Figure 2. Confocal laser scanning micrographs of microalgae M Ankistrodesmus (a) and a yeast (b). Nile red staining shows the lipid bodies (yellow ) inside these microrganisms.

97 isolated kdna networks of trypanosomatids using AFM. The results allow an examination of kdna at high resolution (Figure 3). We were able to identify regions of overlapping kdna molecules and sites where several molecules cross. replicas of quick-frozen, freeze-fractured, deepetched and rotary-replicated cells (Figure 4c) to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme, as well the angles between the intercrossed filaments supporting this idea. Based on the information obtained and using graphic Figure 3. Cross-section analysis of kdna networks showing differences in height measurements that indicate the overlapping of DNA strands. The kdna molecules crossing over up to 3 times in C. fasciculata. Bar - 1µm. The function played by the PFR is not well established. To this, we have started to investigate by atomic force microscopy (Figure 4a-b) and transmission electron microscopy of computation, we proposed an animated model for the PFR structure during flagellar beating, providing a new way to observe the PFR filaments during flagellar beating. A stationary frame of the flagellum in straight state can observed in Figure 4d. Figure 4. (a-b) AFM intermittent contact mode image of a straight state flagellum of Trypanosoma cruzi. (a) Topographic 3D view of part of the flagellum. (b) Phase image of the flagellum showing the lattice organization of the filaments of the PFR (arrows). (c) Deep-etching replica image of PFR filaments (arrows) showing a longitudinal fracture of the intermediate domain. (d) Frame view of PFR animation during flagellar beating. In this straight state, the intercrossed filaments reveal a regular diamond structure. Axoneme light pink; filaments that link the PFR to the axoneme purple; proximal and distal domains of the PFR red; and, the intermediate domain salmon. Bars a-b 200 nm; c 250 nm. 97

98 Group publications ( ): 1. SANT'ANNA, C. ; Nakayasu, E.S.; Pereira, M.G.; Lourenço, D.; DE SOUZA, W.; Almeida, I.C.; Cunha-e-Silva, N.L. Subcellular proteomics of Trypanosoma cruzi reservosomes. Proteomics, v. 9, p , DE SOUZA, W.; SANT'ANNA, C. ; Cunha-e-Silva, N.L. Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa. Progress in Histochemistry and Cytochemistry, v. 44, p , Penha, L. L ; SANT'ANNA, C. ; Mendonca-Previato, L. ; Cunha-e-Silva, N. L ; Previato, J. O ; Lima, A. P. C A. Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain. Glycobiology (Oxford), v. 19, p , Cunha-e-Silva, N. L.; Pereira, M. G. ; SANT'ANNA, C. ; DE SOUZA, W. Reservosomes of Trypanosoma cruzi. In: Wanderley de Souza. (Org.). Structures and Organelles in Pathogenic Protists. 1 ed. : Springer, 2010, v. 1, p Souza, F.S.P.; Rampazzo, R.C.P.; Manhaes, L.; Soares, M.J.; CAVALCANTI, D.P.; Krieger, M.A.; Goldenberg, S.; Fragoso, S.P. Knockout of the gene encoding the kinetoplastassociated protein 3 (KAP3) in Trypanosoma cruzi: effect on kinetoplast organization, cell proliferation and differentiation. Molecular and Biochemical Parasitology, 172: 90-98, CAVALCANTI, D.P.; Shimada, M.K.; Probst, C.M.; Souto-Pádron, T.C.B.S.; DE SOUZA, W.; Goldenberg, S.; Fragoso, S.P.; Motta, M.C.M. Expression and subcellular localization of kinetoplast-associated proteins in the different developmental stages of Trypanosoma cruzi. BMC Microbiology, 9: 120, ROCHA, G.M.; Seabra, S.H.; Miranda, K.; Cunha-e-Silva, N.; Carvalho, T.M.U.; DE SOUZA, W. Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi. Parasitology International 59(4): ROCHA, G.M.; Miranda, K.; Weissmüller, G.; Bisch, P.M.; DE SOUZA, W. Visualization of the flagellar surface of protists by atomic force microscopy. Micron, v. 41, p , ROCHA, G.M.; Teixeira, D.E.; Miranda, K.; Weissmüller, G.; Bisch, P.M.; DE SOUZA, W. Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi. Plos One, v. 5, p. e11407, DE SOUZA, W.; ROCHA, G.M.; Miranda, K.; Bisch, P.M.; Weissmüller, G. AFM as a tool for the study of the ultrastructure of trypanosomatid parasites. In: Pier Carlo Braga; Davide Ricci. (Org.). Atomic Force Microscopy and Its Use in Biomedical Research. New York: Humana Press, Maru, M.M.; Lucchese, M.M.; Legnani, C.; Quirino, W.G.; Balbo, A.; Aranha, I.B.; COSTA, L.T.; Vilani, C.; Sena, L.A.; Damasceno, J.C.; Cruz, T.S.; Lidízio, L.R.; Silva, R.F.; Jorio, A. Achete, C.A. Biodiesel compatibility with carbon steel and HDPE parts. Fuel Proc. Tech. 90, , Jesus, D.M.; COSTA, L.T.; Gonçalves, D.L.; Achete, C.A.; Attias, M.; Moussatché, N.; Damasco, C.R. Cidofovir affects morphogenesis and inhibits genome encapsidation during the replication of vaccinia virus. J of Virology. 83, , Mavropoulos, E.; Costa, A.M.; COSTA, L.T.; Achete, C.A.; Mello, A.; Granjeiro, J.M.; Rossi, A.M. Adsorption and bioactivity studies of albumin onto hydroxyapatite surface. Colloids and Surface B:Biointerfaces. In press. 14. Freitas, M.S.; Follmer, C.; COSTA, L.T.; Vilani, C.; Bianconi, M.L.; Achete, C.A.; Silva, J.L. The interaction between ebola fusion peptide and cellular membranes: The role of lipid rafts in membrane fusion. Aceito para publicação Plos One 98

99 AL14 ASSOCIATE LABORATORY OF STRUCTURAL BIOLOGY. Coordinator: Edilene Oliveira da Silva, Universidade Federal do Pará (UFPA) Main Research: Bioproducts as leishmanicidal agent source Our research focuses on the development of new strategies for treatment of leishmanial infections based on bioproducts research from amazon s biodiversity. The leishmaniasis is an infectious disease caused by various species of the protozoan parasites in the genus Leishmania. Among the various Leishmania species, an important etiological agent is the Leishmania amazonensis which cause human tegumentary leishmaniasis. The chemotherapy is the only effective treatment for this disease. Besides being expensive these drugs are in general toxic and require long-term treatment. Natural products from plants and microorganisms represent an important alternative source of new antileishmanial agents. Physalis angulata is an annual herb distributed in tropical and subtropical regions of the world. In Amazonia, is popularly known as camapu. Extracts from this plant is widely used in popular medicine as analgesic, antirheumatic, antinociceptive and anti-inflammatory. In addition, P. angulata has 99

100 compounds called physalins, which showed antileishmanial activity in vitro and in vivo for cutaneous leishmaniasis. Thus, we consider interesting to analyze the effects of the aqueous extract obtained from roots of P. angulata against promastigotes of L. amazonensis in vitro and its effects on host cell. We have recently demonstrated that P. angulata aqueous extract effectively inhibits growth of promastigotes forms and promotes ultrastructural parasite alterations. Other bioproduct analyzed is the 5-hydroxy-2- hydroxymethyl-γ-pyrone (HMP), which is produced by some species of Aspergillus fungi, has bacteriostatic activity and inhibition effect of the tyrosinase enzyme in the process of melanin biosynthesis. However its antileishmanial activity and the effects on host immune cells are not well known. Macrophages treated with 50 µg/ml of HMP showed increased cytoplasm and spreading ability, followed by cytoskeleton rearrangement, a high number of cytoplasmatic projections and enhancement of the phagocytosis process, besides a greater superoxide production. Moreover, it acted decreasing the growth of promastigotes and intracellular amastigotes. In vivo topical treatment with HMP-ointment promotes healing process and suppressing ulcer dissemination. In addition, many collagen fibers were found in infection sites of HMP-treated animals. These results demonstrated that HMP effectively inhibits the growth of parasites in vitro and in vivo, mainly by the activation of host cell microbicidal response and does not have cytotoxic effects on the host cells. Thus, both HMP and the extract from Physalis angulata could be useful as alternative source for a new antileishmanial agent. FIG. 1. Effects of Physalis angulata extract on growth curve of Leishmania amazonensis promastigotes. CTL: control; GLU: glucantime. 100

101 Figure 2 Skin lesion section of infected animals untreated and treated with HMP-ointment for one month. Light microscopy of lesion site of control (a) showing numerous amastigotes in the vacuoles and HMPtreated animals (d), absence of intracellular amastigotes; H&E. Confocal microscopy of lesion site of control (b), observe few and disorganized collagen fibers and -treated animals (e) with many collagen fibers showing healing process, Sirius red stain. (c) and (f) Transmission electron microscopy of control and HMP-treated animals, respectively. (c) Observe amastigotes parasite in the vacuoles of host cell and few collagen fibers. (f) Observe organized fibers and absence of intracellular amastigotes. 101

102 FIG. 3. Ultrastructural effects of extract from Physalis angulata in promastigotes of Leishmania (L.) amazonensis. a General view of untreated parasite showing the characteristic structure of kinetoplastids. b Promastigotes treated with 50 µg/ml of the extract. Note some vacuoles in the membrane of flagellar pocket (*) and alterations in flagellar membrane (arrow). c-d General view of promastigotes treated with 100 µg/ml of the extract. c Observe the presence of myelin-like figures into the flagellar pocket (arrows) and duplication of kinetoplast (arrowhead). d Note the presence of large number of vesicles inside the flagellar pocket (*) and alterations on forms and size of kinetoplast (arrowheads). N, nucleus; FP, flagellar pocket; K, kinetoplast; F, flagellum; M, mitochondria. Bars represent a 5 µm; b 2 µm; c 5 µm; c inset 2 µm; d 2 µm Group publications ( ): 1. Guimarães, Leda R. C. ; Rodrigues, Ana Paula D. ; Marinho, Patrícia S. B. ; Muller, Adolfo H. ; Guilhon, Giselle M. S. ; Santos, Lourivaldo S. ; Nascimento, José Luiz M. ; Silva, Edilene O.. Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis. Parasitology Research (1987. Print), v. 107, p , Ramos, Patrícia K.S. ; Diniz, José A.P. ; Silva, Edilene O. ; Quaresma, Juarez A.S. ; Saraiva, Elvira M. ; Seabra, Sérgio H. ; Atella, Geórgia C. ; de Souza, Wanderley. Characterization in vivo and in vitro of a strain of Leishmania (Viannia) shawi from the Amazon Region. Parasitology International (Print), v. 58, p , Accepted Manuscript: 3.RODRIGUES, Ana Paula Drummond ; Santos, AS ; ALVES, C. N. ; Carvalho, ASC ; NASCIMENTO, J. L. M. ; SILVA, E. O.. Kojic Acid, a secondary metabolite from Aspergillus ap., acts as an inducer of macrophage activation. Cell Biology International (Print), Patents National: RODRIGUES, Ana Paula Drummond ; SILVA, E. O. ; Santo, A.S. ; ALVES, C. N. ; Carvalho, ASC ; NASCIMENTO, J. L. M.. Use of 5-hydroxy-2- hydroxymethyl-4-pyrona as a macrophage activation agent to combat Cutaneous Leishmaniasis International: RODRIGUES, Ana Paula Drummond ; Silva, E.O. ; Santo, A.S. ; Carvalho, ASC ; ALVES, C. N. ; NASCIMENTO, J. L. M.. Use of 5- hydroxy-2-hydroxymethyl-4-pyrona as a macrophage activation agent to combat Cutaneous Leishmaniasis. No. Reg PCT/BR2009/

103 AL15 ASSOCIATE LABORATORY OF MICROSCOPY CETENE Coordinator: Christina Alves Peixoto, Fundação Oswaldo Cruz and Centro de Tecnologias Estratégicas do Nordeste (FIOCRUZ, CETENE - Pernambuco) Members: Ana Célia Oliveira Santos Karina Alcântara Saraiva Janaina Viana de Melo Diethylcarbamazine is an anti-filaricidal drug that has been used since Despite the fact that this drug has been extensively used, its precise pharmacological action remains unclear. Clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in bronchial asthma, reducing the dosage of corticosteroid and bronchodilators. This drug has an important anti-inflammatory role since it interferes with arachidonic acid metabolism. Recently, work developed in collaboration with our laboratory by Queto et al (2010) demonstrated that DEC effectively prevented airway resistence, Th1/Th2 cytokine production, pulmonary eosinophil accumulation and eosinophilopoiesis by an inos/cd95ldependent mechanism. In the present study entitled ULTRASTRUCTURAL ANALYSIS OF DIETHYLCARBAMAZINE TREATMENT AFTER MONOCROTALINE-INDUCED PULMONARY DYSFUNCTION IN MICE we investigated the efficacy of oral DEC treatment in mice model of pulmonary dysfunction. Mice were submitted to a subcutaneous injection of monocrotaline (MCT) that is a toxin from plants of the Crotalaria species, mainly employed to establish a model of pulmonary dysfunction. 103

104 DEC effectively suppressed the effects of monocrotaline challenge on lung cells: a) optical microscopy demonstrated some alterations after MCT treatment, such as emphysema, and accumulation of inflammatory cells, associated with edema fluid. DEC reversed these effects, showing preserved epithelium; b) Transmission Electron Microscopy revealed some alterations after MCT treatment, such as fluid accumulation in the lung, characteristic of edema, and pneumocytes with dilated mitochondria with degenerated cristae. DEC turn the lung morphology close to the control group; c) Immunohistochemistry and Immunocytochemistry revealed decreased labeling to the inflammatory mediators tested (IL-6, VCAM-1) after DEC treatment. Conclusions: MCT-induced pulmonary dysfunction was attenuated by DEC treatment, probably via inhibition of a number of steps in arachidonic acid pathway, thereby preventing the production of eicosanoids, important inflammatory intermediates. mice. Forty-eight male C57BL/6 mice were separated in groups: control group (C, DEC25 e DEC50) and alcoholic group (CEtOH, EtOH25 e EtOH50). After the induction of alcoholism, mice were submitted 12 days of treatment with DEC solutions in concentrations of 25 and 50mg/kg orally. Biochemical analyses were performed and liver fragments were processed for light microscopy and transmission electron microscopy. The level of AST increased significantly in the control group subjected to alcohol (CEtOH) compared with the control group (C). We observed a significant reduction of serum AST and alcoholic groups DEC-treated EtOH25 and EtOH50 compared with the group that received no treatment CEtOH. The serum ALT, alkaline phosphatase and bilirubin avaliations showed no significant differences among the groups. Histological analysis of CEtOH group showed evident hepatocellular damage. However, in EtOH25 and EtOH50 groups, we observed a reduction of damage caused by the chronic ingestion of ethanol. A B C Figure 1: Ultrastructural analyses of lung tissue. A-Control group. B- MCT-treated group, showing cells in degenerating process. C- 3.DEC treatment reversed these effects, turning the lung morphology close to the control group, showing preserved alveolar epithelium. Another study entitled ANTI- INFLAMMATORY EFFECT OF DIETHYLCARBAMAZINE IN HEPATIC CELLS OF ALCOHOLIC C57BL/6J MICE analyzed the protective effect of diethylcarbamazine in hepatic cells of alcoholic Ultrastructural analysis of hepatocytes EtOH25 and EtOH50 group presented well-preserved organelles. Immunohistochemistry CEtOH group revealed expression of inflammatory markers IL-6, enos, CCR2, VCAM and ICAM, however the EtOH50 group showed no 104

105 immunoreactivity for any of these markers. According to the present results, DEC is a potential drug for the treatment of chronic inflammation induced by alcoholism twenty animals received 25mg/kg body weight of Sildenafil for 4 weeks administered in the drinking water, and the dose was monitored by daily weighing of the water bottle. The control LI LI LI LI V LI V A * * B V C LI Figure 2 - Micrograph of hepatocytes. A- Alcoholic control group (CEtOH), B Alcoholic 25mg/kg DEC-treated group (EtOH25). C- Alcoholic 50mg/kg DEC-treated group (EtOH50). Blood vessel (V), hydropic degeneration (arrow), lipid droplets (Li), necrosis (asterisk), inflammatory infiltrates (star), macrophages (arrowhead), HE staining, Bar = 20μm. Another drug studied by our laboratory group, also composed by twenty animals, is Sildenafil that is an important inhibitor of the phosphodiesterase-5. In fact, Sildenafil is a novel, orally treatment approach for pulmonary hypertension. Its pharmacological action is the inhibition of cgmp-specific phosphodiesterase type 5 (PDE5) enzyme, which is abundant in the pulmonary vasculature. The therapeutic scheme with high daily doses improves the pulmonary vascular haemodynamics and therefore the exercise capacity of patients. Since the expression of PDE5 was detected in Leydig and myoid cells of rat testis, it is necessary to investigate whether compounds that interfere on PDE5 activity could affect the steroidogenesis. In a recent study entitled PHOSPHODIESTERASE TYPE 5 INHIBITOR STIMULATES LEYDIG CELL STEROIDOGENESIS, we reported some ultrastructural alterations in mice Leydig cells and increased testosterone levels after chronic treatment with the phosphodiesterase type 5 inhibitor. One experimental group composed by received only pure water. Leydig cells in control group showed morphological characteristics as areas of cytoplasm rich in anastomosing tubular endoplasmic reticulum, lipid droplets, scattered lysosomes, Golgi, mitochondria with tubular cristae, and rough endoplasmic reticulum. Sildenafil-treated Leydig cells showed some morphological alterations as a vesicular smooth endoplasmic reticulum, large vacuoles in the peripheral cytoplasm, presumably opening into the extracellular space. The mitochondria were enlarged with disarranged or discontinued cristaes, and several vesicles were observed on whole peripheric membranes, probably involved in hormonal secretion. All of these alterations described above are typical of an activated cell. Immunocytochemistry revealed an augmented expression of StAR protein, P450scc and testosterone in isolated Leydig cells, when compared to the controls. The serum testosterone levels were significantly higher in 25mg/kg 105

106 Sildenafil administrated mice when compared to control animals (Mann-Whitney, Z=3.26, p<0.01). The results obtained in the present study are consistent with the hypothesis that the accumulation of cgmp, by PDE5 inhibition, and activation of its dependent pathways could be involved in the androgen biosynthesis stimulation. Important clinical implications of hormonal disorders should be taken into account for patients with pulmonary hypertension. In relation to the effects of chronic treatment of Sildenafil on mice ovary, the work ULTRASTRUCTURAL EFFECTS OF SILDENAFIL TREATMENT IN LUTEAL CELLS showed that Luteal cells from control group showed lipid droplets and mitochondria with tubular cristae. Ribosomes and enlarged smooth endoplasmic reticulum were visible. Luteal cells from Sildenafil-treated group showed numerous large mitochondria, with tubular cristae, and lipid droplets were visible. Several ribosomes were present in cytoplasm, scattered or attached to the endoplasmic reticulum. However, the smooth endoplasmic reticulum was compacted compared to control group. Nevertheless, morphology from control and treated group suggests normal steroidogenesis. Biochemical analyses showed that serum levels of HDL, LDL, VLDL, cholesterol and triglyceride presented no significant differences between groups. Previous studies (Donato et al, 2009) using phosphodiesterase-5 inhibitor Vardenafil showed different results, as a diminished serum level of HDL after treatment. This can be explained by the fact that although Sildenafil and Vardenafil inhibit phophodiesterase-5, they may interact with different cellular mechanisms. In our study, cellular morphology suggests abnormal steroidogenesis in sildenafil-treated group; however, it is necessary to perform a hormonal assay to confirm these results. The therapeutic use of Sildenafil in pulmonary hypertension improves the pulmonary vascular haemodynamics. The treatment involves the administration of high doses daily, and until now, little is known about the action of this scheme on neuronal cells. In the CNS, the cerebellum present constitutively PDE5, and it has been showed that cgmppathways protect oligodendrocytes [1]. Besides, mice lacking inos exhibit more demyelination in demyelinating model [2]. This study entitled SILDENAFIL PDE5 INHIBITOR: THERAPEUTIC OPPORTUNITY FOR MYELIN REPAIR was conducted to investigate the inos role in myelination and astrocytes physiology, and if Sildenafil oral treatment acts in inos-/- mice nervous tissue. Ten B6.129 P2- Nos2 (knockout inos) mice, 21-day-old, were used for each group. One group received 25mg/kg of Sildenafil/8 weeks, administered in the drinking water. The control group received pure water. The genetic background C57BL/6 wild type without treatment was used for comparison. The animals were sacrificed by perfusion and pieces of cerebellum were processed for electron microscopy. The GFAP (glial fibrillary acidic protein) levels were detected by immunohistochemistry. Morphometrical analysis revealed that myelin was thicker in wild type (p < 0.01) and inos-/- Sildenafil (p < 0.001), comparing to inos-/- control; the mean of g-ratio (the ratio of the inner axonal diameter to the total outer diameter), was less in wild type (p < 0.05) and 106

107 inos-/-sildenafil (p < 0.01), than in inos-/- control, indicating thinner myelin in this group. The axons of inos-/-control presented noteworthy ultrastructural alterations in myelin, graded as grade II (diffused local disarrangement of myelin sheath resembling as a collar but preserving structural arrangement), and wild type and inos-/-sildenafil were graded how grade I (well-preserved myelin with few local disarrangement), in a myelin disarrangement scale arbitrarily defined (I-III). The GFAP, an astrocytes activation (reactive gliosis) marker, showed physiological expression in the wild type, however, in inos-/-control, an intense increase in this protein labeling was seen, which returned to physiological levels after Sildenafil treatment. The results obtained in the present study are consistent with the hypothesis that the inducible generated NO absence can downregulate the cgmp signaling, damaging the myelin structure, and causing a clear reactive gliosis, in cerebellum. However, the accumulation of cgmp by PDE5 inhibition could improve the myelination in cerebellum, and extinguish the reactive gliosis. These results point to important potential clinical applications potential of Sildenafil in demyelinating disease, such as multiple sclerosis Figure 3: GFAP immunohistochemistry in paraffin sections of cerebellar cortex. A. Genetic background C57BL/6 showing physiologic GFAP expression. This protein was seen in astrocytic processes (arrows) and cellular bodies (arrow head). B. inos-/-control presented very intense labeling for GFAP in both, processes and cellular bodies of astrocytes (asterisks), indicating reactive gliosis. C. After 8 weeks treatment with Sildenafil, the tissue recovered the normal GFAP expression. This PDE5 inhibitor extinguished the astrocytes reaction in nervous tissue without inos, suggesting that the cgmp accumulation had a protector effect. M Molecular layer; G Granular layer; P Purkinje layer. Bar = 20 μm. 107

108 Group publications ( ): 1. Queto T, Xavier-Elsas P, Gardel MA, Barradas M, Masid D, PEIXOTO, C. A., Vasconcelos ZMF.iNOS/CD95Ldependent Suppression of Pulmonary and Bone-marrow Eosinophilia by Diethylcarbamazine. American Journal of Respiratory and Critical Care Medicine., v.181, p , Couto J.A., SARAIVA, Karina Liddiane Alcântara, Barros C.D., Udrisar D.P., PEIXOTO, A. M. B. C. A., PEIXOTO, C. A., Vieira J.S.B., GALDINO, S. L., PITTA, I. R., Wanderley M.I.Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: in vivo and ex vivo studies. Reproductive Biology and Endocrinology., v.1, p.8-13, SARAIVA, K L A, SILVA, A. K. S. E., Wanderley M.I., Araújo A.A., SOUZA, José Roberto Botelho de, PEIXOTO, C. A.Chronic treatment with sildenafil stimulates Leydig celll and testosterone sphaericus displays cytopathological effects on susceptible and Binary toxin-resistant Culex quinquefasciatus larvae (Epub ahead of print]. secretion. International Journal of Experimental Pathology., v.90, p , DONATO, M. A. M., SARAIVA, K L A, SILVA, A. K. S. E., Wanderley M.I., PEIXOTO, C. A. Follicle development and luteal cell morphology altered by phophodiesterase-5 inhibitor. Micron (Oxford. 1993)., v.40, p , Torres D O C, SANTOS, A. C. O., SILVA, A. K. S. E., Leite J.I.A, SOUZA, José Roberto Botelho de, Beltrão I.C.B., PEIXOTO, C. A. Effect of maternal diet rich in omega-6 and omega-9 fatty acids on the liver of LDL receptor-deficient mouse offspring Accept - Birth Defects Research Part B: Developmental and Reproductive Toxicology - Manuscript BDRB R1. Reproductive Biology and Endocrinology., v.1, p.8-13, MELO, Janaína Viana de, Jones G.W., Berry B, MARQUES, Sílvio Romero, Oliveira C.M.F., Furtado A.F., PEIXOTO, C. A., SILVAFILHA, Maria Helena Neves Lobo Cry48Aa/Cry49Aa toxin from Bacillus Applied and Environmental Microbiology (Print)., v.75, p ,

109 AL16 ASSOCIATE LABORATORY OF MOLECULAR AND CELLULAR CARDIOLOGY Coordinator: Antonio Campos de Carvalho, IBCCF/UFRJ Members: Regina Goldenberg Emiliano Medei Bernardo Rangel Tura Nazareth Novaes Rocha Patricia Cristina da Costa Valdo José Dias da Silva Aldo Rogélis Rodrigues Our group has been working on the isolation and characterization of pluripotent and multipotent human and murine stem cells. We have developed methods for cultivating human mesenchymal stem cells (MSC) from neonatal tissues (amniotic fluid, placenta, Wharton s jelly and the walls from cord artery and vein). These cells have been isolated, characterized immunophenotypically and differentiated into osteoblasts, chondrocytes and adipocytes. Attempts to differentiate these cells into cardiomyocytes have failed up to now. We have also isolated Cardiosphere Derived Cells (CDC), another human cell type, which is supposed to represent cardiac stem cells. These cells have been isolated, expanded in cultured and characterized by flow citometry. Although morphologically similar to MSC from the neonatal tissues, these cells display a distinct phenotype. We are currently testing their potential to differentiate into cardiomyocytes, endothelial and smooth muscle cells. Regarding the pluripotent cells, we have been working to produce induced pluripotent stem cells (ipsc) from neonatal MSC and from MSC derived from menstrual blood. The menstrual blood MSCs have yielded ipsc-like cells after transduction with lentiviral vectors 109

110 containing the Yamanaka factors. In fact we have been able to produce ipsc-like cells using only three factors (Klf4, Sox2, Oct3/4), without the need for c-myc, with great efficiency and in shorter times (first colonies detected at 4 days after transduction). We attribute this improvement to the endogenous expression of some of the pluripotency factors in the MSC from menstrual blood and neonatal tissues. Differentiation of the ipsc-like cells into cell types of ecto, meso and endodermal origin has been shown, and we are currently working on developing teratomas from these cells to prove that they are bona-fide ipsc. We are also working on direct differentiation of human cardiac fibrobasts into cardiomyocytes. This project is at a preliminary stage, where we have cloned 15 transcription factors that play prominent roles during cardiogenesis. We are now starting the preparation of lentiviral vectors containing each of these factors and will transduce fibrobasts containing a cardiac myosin heavy chain promoter linked to a GFP reporter gene to screen for the relevant factors. During the course of this project, Deepak Srivastava, at the Gladstone Institute in California, described the direct differentiation of mouse dermal and cardiac fibrobasts into cardiomyocytes, demonstrating the feasibility of this project. All our in vitro experiments are geared towards generating cells that can be used for cardiac repair. In our in vivo experiments we are using four animal disease models of cardiovascular alterations: a myocardial infarction model, a chronic chagasic cardiomyopathy model, a pulmonary hypertension model, and an induced diabetes model. These models are well characterized in our lab and checked by electro and echocardiography. In testing the validity of cell therapies in each of these models we are using different cell types (MSC, CDC, ESC) that are injected intravenously or in some instances by echo-guided intracardiac injection. The injected cells are transduced with luciferase and serially tracked using a bioluminescence apparatus. Cardiac function is serially evaluated by ECG and echocardiography, and after animal sacrifice histopathology is performed. In the diabetes model we also measure vascular reactivity in isolated aorta. Shortly we will be able to record MRI images of the Isolation of bone marrow mononuclear cells using the Ficoll gradient method. 110

111 hearts and vasculature of these animals, using the multi-imaging facility that was created by our Institute. We have also actively participated in clinical trials performed in patients with Chagasic and dilated cardiopathies and in stroke patients, using bone marrow derived mononuclear cells. Flow citometry of human MSC in third passage. Left column represents unlabeled cells (green). Right column shows MSCs positive for CD73 and CD90 and negative for CD45 and CD34 (markers of hematopoietic lineage). 111

112 Image shows the needle used to inject cells directly into the myocardium of an infarcted mouse heart, using the high resolution echocardiogram. Distribution of luciferase transduced mesenchymal stem cells, after retroocular injection (see inset), in the streptozotocin diabetes rat model, using the bioluminescence imaging modality. 112

113 Group publications ( ): 1. Ribeiro, V. P. ; Maia, A. C. V. ; Werneck-De- Castro, João Pedro S. ; Oliveira, Patricia Fidelis ; Goldenberg, R. C. S. ; Campos De Carvalho, A. C.. Human Umbilical Cord Blood Cells In Infarcted Rats. Brazilian Journal Of Medical And Biological Research, V. 43, P , Louzada, Ruy A. N. ; Oliveira, Patricia F. ; Cavalcanti-De-Albuquerque, Joao Paulo A. ; Cunha- Carvalho, Leandro ; Baldanza, Marcelo R. ; Kasai- Brunswick, Taís H. ; Goldenberg, Regina C. S. ; Campos De Carvalho, Antonio ; Werneck-De-Castro, Joao P. S.. Granulocyte-Colony Stimulating Factor Treatment Of Chronic Myocardial Infarction. Cardiovascular Drugs And Therapy, V. 2, Epub, Nihei, Oscar K ; Fonseca, Paula C ; Rubim, Nara M ; Bonavita, Andre G ; Lyra, Jurandy Spo ; Neves- Dos-Santos, Sandra ; Campos De Carvalho, Antonio C ; Spray, David C ; Savino, Wilson ; Alves, Luiz A ; Campos De Caravalho Antonio C.. Modulatory Effects Of Camp And Pkc Activation On Gap Junctional Intercellular Communication Among Thymic Epithelial Cells. Bmc Cell Biology (Online), V. 11, P. 3, Lachtermacher, S. ; Esporcatte, B. L. B. ; Montalvao, F. ; Costa, P. C. Dos S. ; Rodrigues, D. C. ; Belem, L. ; Rabischosffky, A. ; Faria Neto Hcc ; Vasconcellos, R. ; Iacobas, Dumitru A. ; Iacobas S ; Dohmann, H. F. ; Spray, D. C. ; Goldenberg, R. C. S. ; Campos De Carvalho A. C.. Cardiac Gene Expression And Systemic Cytokine Profile Are Complementary In A Murine Model Of Post Ischemic Heart Failure.. Brazilian Journal Of Medical And Biological Research (Impresso), V. 43, P , Lessa A S ; Paredes, Bruno Dias ; Dias, J. V. ; Carvalho, A. B. ; Quintanilha, L. F. ; Takyia, Cristina Maeda ; Tura, Bernardo R. ; Razende, G. F. Da M. ; Campos De Carvalho A. C. ; Resende, C. M. C. ; Coeli Dos Santos Goldenberg, Regina. Ultrasound Imaging In An Experimental Model Of Fatty Liver And Cirrhosis In Rats. BMC Veterinary Research, V. 6, P. 6, Barbosa Da Fonseca, Lea Mirian ; Xavier, Sérgio S. ; De Castro, Paulo Henrique Rosado ; Lima, Ronaldo S.L. ; Gutfilen, Bianca ; Goldenberg, Regina C.S. ; Maiolino, Angelo ; Chagas, Claudia L.R. ; Pedrosa, Roberto C. ; De Carvalho, Antonio Carlos Campos. Biodistribution Of Bone Marrow Mononuclear Cells In Chronic Chagasic Cardiomyopathy After Intracoronary Injection. International Journal Of Cardiology (Print), V. 3, Epub, Ribeiro, Antonio Luiz P. ; De Carvalho, Antonio Carlos Campos ; Lombardi, Federico ; Talvani, André ; Teixeira, Mauro Martins ; Rocha, Manoel Otávio Costa. In Vivo Inhibitory Effect Of Anti-Muscarinic Autoantibodies On The Parasympathetic Function In Chagas Disease. International Journal Of Cardiology (Print), V. 1, P , Lima L P; Cruz Ff; Fujisaki Lc; Oliveira Gp ; Samary Cs ; Ornellas Ds ; Maron-Gutierrez T ; Rocha, Nazareth De Novaes ; Goldenberg, Rcs; Garcia Csnb ; Marcos Marcelo Morales ; Capelozzi Vl ; Abreu Mg ; Pelosi P ; Rocco, P. Hypervolemia Induces And Potentiates Lung Damage After Recruitment Maneuver In A Model Of Sepsis-Induced Acute Lung Injury. Critical Care (London), V. 14, P , Da Fonseca, L. M. B.; Gutfilen, Bianca; Batistela, V.; Castro, P. H. R.; Goldenberg, Rcs; Kasai- Brunswick, T. H.; Chagas, C. L. R. ; Wajnberg, E ; Maiolino A ; Xavier Ss ; Andre, C ; Mendez-Otero, R. ; De Freitas, G. R. Migration And Homing Of Bone Marrow Mononuclear Cells In Chronic Ischemic Stroke After Intra- Arterial Injection. Experimental Neurology, V. 221, P , Medei, Emiliano ; Marocolo, Moacir ; Rodrigues, Deivid De Carvalho ; Arantes, Paulo Cesar ; Takiya, Christina Maeda ; Silva, Juliana ; Rondinelli, Edson ; Goldenberg, Regina Dos Santos ; Campos De Carvalho, Antonio Carlos ; Nascimento, José Hamilton Matheus. Chronic Treatment With Anabolic Steroids Induces Ventricular Repolarization Disturbances: Cellular, Ionic And Molecular Mechanism. Journal Of Molecular And Cellular Cardiology, P. 1-11, Carvalho, Adriana Bastos ; Campos De Carvalho, Antonio Carlos. Heart Regeneration: Past, Present And Future. World Journal Of Cardiology, V. 2, P , Esporcatte, B. L. B. ; Rocha, N. N. ; Mello Db ; Asensi K D ; Lachtermacher, S. ; Goldenberg, R. C. S. ; Campos De Carvalho A. C.. Ecocardiograma De Alta Resolução E O Modelo De Infarto Do Miocárdio Em Camundongos. Revista Brasileira De Ecocardiografia, V. 23, P , Martino, H. F. ; Oliveira, P. S. ; Souza, F. C. ; Costa, P. C. Dos S. ; Assuncao, E. ; Villela, R. ; Gaze, M. ; Weitzel, L. H. ; Oliveira Jr, A. ; Muccillo, F. B. ; Arvelo, S. N. S. ; Guimarães, T. C. F. ; Tura, B. R. ; Campos De Carvalho A. C.. Cell Therapy In Dilated Cardiomyopathy: A Safety And Feasibility Study.. Brazilian Journal Of Medical And Biological Research V. 43, P , Azevedo-Pereira, R.L ; Medei, E. ; Mendez- Otero R ; Marcondes J ; Alves-Leon S.V. "Isolation Ofneurosphere-Like Bodies From An Adult Patient With Refractory Temporal Lobeepilepsy..". Arquivos De Neuro- Psiquiatria Epub, Medei, E. ; Lima-Leopoldo Ap ; Pereira Jr Pp ; Campos D ; Nascimento Jhm ; Raimundo J ; Sudo Rt ; Zapata-Sudo G ; Bruder-Nascimento T ; Cordellini S ; Cicogna Ac. Could An Unsaturated High-Fat Acid Diet Impair The Cardiovascular System?. Canadian Journal Of Cardiology, Epub Pereira Jr Pp ; Moacir Jr, M. ; Rodrigues F ; Medei, E. ; Nascimento Jhm. Noninvasive Method For Electrocardiogram Recording In Conscious Rats: Feasibility For Heart Rate Variability Analysis. Anais Da Academia Brasileira De Ciências (Impresso), V. 82, P , Matavel, A. ; Medei, E. ; Lopes Cmb. Pka And Pkc Partially Rescue Long Qt Type 1 Phenotype By Restoring Channel-Pip2 Interactions. Channels, V. 223, P , Tanowitz, Herbert B. ; Machado, Fabiana S. ; Jelicks, Linda A. ; Shirani, Jamshid ; Campos De Carvalho A. C. ; Spray, David C. ; Factor, Stephen M. ; Kirchhoff, Louis V. ; Weiss, Louis M.. Perspectives On Trypanosoma Cruzi Induced Heart Disease (Chagas Disease). Progress In Cardiovascular Diseases, V. 51, P , Jasmin,. ; Spray, David Conover ; Campos De Carvalho, Antonio Carlos ; Mendez-Otero, Rosalia. Chemical Induction Of Cardiac Differentiation In P19 Embryonal Carcinoma Stem Cells. Stem Cells And Development 19(3): , Carvalho, Antonio Carlos C. ; Goldenberg, Regina Coeli S. ; Tuche, Fábio Antonio A. ; Dohmann, Hans Fernando R.. Bases Da Terapia Celular Em Cardiologia. Revista Brasileira De Hematologia E Hemoterapia, P ,

114 21. Goldenberg, Regina Coeli Dos Santos ; Iacobas, Dumitru A. ; Iacobas, Sanda ; Rocha, Leonardo Lima ; Da Silva De Azevedo Fortes, Fabio ; Vairo, Leandro ; Nagajyothi, Fnu ; Campos De Carvalho, Antonio Carlos ; Tanowitz, Herbert B. ; Spray, David C.. Transcriptomic Alterations In Trypanosoma Cruzi-Infected Cardiac Myocytes. Microbes And Infection, V. 11, P , Mannheimer, Elida Gripp ; Quintanilha, Luiz Fernando ; Carvalho, Adriana Bastos ; Paredes, Bruno Diaz ; Gonã Alves De Carvalho, Felipe ; Takyia, Cristina Maeda ; Resende, Cã Lia Maria Coelho ; Ferreira Da Motta Rezende, Guilherme ; Campos De Carvalho A. C. ; Schanaider, Alberto ; Coeli Dos Santos Goldenberg, Regina. Bone Marrow Cells Obtained From Cirrhotic Rats Do Not Improve Function Or Reduce Fibrosis In A Chronic Liver Disease Model. Clinical Transplantation, V. 1, P. Epub, Silva, Henrique B. ; Medei, Emiliano ; Rodrigues, Deivid C. ; Rondinelli, Edson ; Almeida, Norma A.S. ; Goldenberg, Regina C.S. ; De Carvalho, Antonio C. Campos ; Nascimento, Josã H.M.. Voltage-Dependent Calcium And Chloride Currents In S17 Bone Marrow Stromal Cell Line. Journal Of Cellular Physiology (Print), V. 5, P. 1-12, Campos De Carvalho A. C.. Physiology In Brazil: Past And Present. Journal Of The Physiological Society Of Japan, V. 71, P , Campos De Carvalho, Antonio ; Goldenberg, Regina C. S. ; Jelicks, Linda A. ; Soares, Milena B. P. ; Dos Santos, Ricardo Ribeiro ; Spray, David C. ; Tanowitz, Herbert B.. Cell Therapy In Chagas Disease. Interdisciplinary Perspectives On Infectious Diseases, V. 2009, P. 1-7, Barbosa Da Fonseca, L. M.; Battistella, V. De Freitas, G. R.; Gutfilen, B.; Goldenberg, Rcs; Maiolino, A. ; Wajnberg, E. ; Rosado De Castro, P. H. ; Mendez-Otero, R. ; Andre, C.. Early Tissue Distribution Of Bone Marrow Mononuclear Cells After Intra-Arterial Delivery In A Patient With Chronic Stroke. Circulation (New York), V. 120, P , Costa A ; Torres L ; Medei, E. ; Nascimento Jhm ; Bassani J ; Oshiro M ; Ferreira A ; Tucci P. The Negative Inotropic Action Of Canrenone Is Mediated By L- Type Calcium Current Blockade And Reduced Intracellular Calcium Transients. British Journal Of Pharmacology, V. 158, P , Py M ; Maciel L ; Pedrosa Rc ; Nascimento Jhm ; Medei, E.. The Presence Of Antiautonomic Membrane Receptor Antibodies Do Not Correlate With Brain Lesions In Chagas Disease. Arquivos De Neuro- Psiquiatria (Impresso), V. 67, P , Dias Da Silva, V. J. ; Miranda, R. ; Oliveira, L. ; Alves, C. H. F. R. ; Gils, G. H. F. V. ; Porta, A. ; Montano, N.. Heart Rate And Arterial Pressure Variability And Baroreflex Sensitivity In Ovariectomized Spontaneously Hypertensive Rats. Life Sciences (1973), V. 84, P , Goncalves, J. G. F. ; Dias Da Silva, V. J. ; Borges, M. C. C. ; Prata, A. ; Correia, D.. Mortality Indicators In Chronic Chagas Disease Patients In Endemic Area.. International Journal Of Cardiology, V. In, P. 1-20, Dias Da Silva, V. J. ; Machado, M. P. R. ; Voltarelli, J. C.. Current Status Of Cell Therapy For Systemic Arterial Hypertension. Expert Review Of Cardiovascular Therapy, V. 7, P , Goncalves, J. G. F. ; Dias Da Silva, V. J. ; Correia, D.. Predicting Prognosis In Patients With Chagas Disease: Why Are The Results Of Various Studies So Different? Author's Reply.. International Journal Of Cardiology, V. 135, P. 1-2, Soares, M. B. P.; Lima, RS; Rocha, Leonardo L; Vasconcelos, J F; Rogatto, S R ; Santos, Ricardo Ribeiro dos; Iacobas, Sanda ; Goldenberg, RCS; Iacobas, Dumitru Andrei; Tanowitz, Herbert B; Campos-de-Carvalho, AC; Spray, David C. Gene expression changes associated with myocarditis and fibrosis in hearts of mice with chronic chagasic cardiomyopathy. The Journal of Infectious Diseases, v. 202, p , Lachtermacher, S.; Esporcatte, B. L. B. ; Montalvao, F. ; Costa, P. C. dos S. ; Rodrugues, D. C. ; Belem, L. ; Rabischosffky, A. ; Faria Neto HCC ; Vasconcellos, R. ; Iacobas, Dumitru A. ; Iacobas S ; Dohmann, H. F. ; Spray, D. C.; Goldenberg, RCS.; Campos de Carvalho, AC. Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post ischemic heart failure. Brazilian Journal of Medical and Biological Research, v. 43, p ,

115 AL17 ASSOCIATE LABORATORY OF ION TRANSPORT PHYSIOLOGY IN HEALTH AND DISEASE Coordinator: Adalberto Vieyra, IBCCF/UFRJ Members: Celso Caruso Neves Marcelo Einicker Lamas Jennifer Lowe Lucienne Lara Morcillo Elaine Gomes Quintana JR Meyer Fernandes Luiz Roberto Ferreira Aloa Machado de Souza Ana Durce Paixão Common interests in the field of ionactivated ATPases led 3 different research groups involved in 5 main research lines to combine their expertise, forming the Laboratory Ion transport physiology in health and disease of the National Institute of Science and Technology for Structural Biology and Bioimaging. The main results obtained in the present period of evaluation are described in the different topics below. In addition 10 selected publications are cited along the topics and listed at the end of this report. 1) Cell therapy in nephropathies. In the period we demonstrated that: (i) bone marrow-derived mononuclear cells (BMMC) are recruited by injured kidneys in a murine model of chronic nephropathy (unilateral ureteral obstruction - UUO) and (ii) they play a significant role in tissue recovery (Fig. 1; [1]). Clearly, the fibrotic process that accompanies all chronic renal lesions was almost completely blunted when BMMC were infused in a single dose via the cava vein. The regression of fibrosis is accompanied by a strong stimulus of tubule cells proliferation and a significant inhibition of apoptosis with a clear participation of resident adult progenitor cells. 115

116 Figure 1. Left: -smooth muscle actin ( -SMA) immunoexpression is partially blocked by BMMC in UUO. Myofibroblast immunolocalization and surface density of -SMA in kidney medulla were evaluated. (A) Representative UUO image. (B) Representative UUO + BMMC image. (C) Graphic representation (means SEM of -SMA surface density in the experimental conditions indicated on the abscissa. *P < vs SHAM-operated group. # P < 0.05 vs UUO. Bar: 100 m. Right: BMMC reduce collagen deposition in UUO. Picro-Sirius staining of kidney sections and evaluation of collagen surface density were carried out. (A) Representative image of UUO medulla. (B) Representative image of UOO + BMMC medulla. (C) Graphic representation (means SEM) of collagen surface density in the experimental conditions indicated on the abscissa. *P < vs SHAM and SHAM + BMMC. # P < vs UUO. Bar: 100 m. Taken from [1]. In the period, we focused primarily on the mechanisms behind the overall impact of cell therapy on structure and function. These mechanistic aspects are currently neglected in most studies in the field of cell therapy. One of our major accomplishments was to demonstrate that bioactive lipids play a pivotal role in tissue recovery with a huge effect on the molecular machinery that is responsible for the fine tuning of cytosolic calcium the plasma membrane Ca 2+ -ATPase. Therefore, the bioactive lipids act in the mechanism involved in triggering signals for cell life and death, which are vital for either tissue injury or recovery (Fig. 2; [2]). Importantly, BMMC infusion shifted the bioactive renal lipids from a pattern of lesion to one of regeneration. We also found that BMMC act at the level of mitochondrial respiration; our experiments show that they completely restore viability and growth of ATP-depleted renal cells (in a model of acute renal ischemia) (Fig. 3). The mechanism underlying this mitochondrial rescue is exerted directly at the levels of electron and proton fluxes (unpublished observations). Figure 2. UUO alters Ca 2+ -ATPase in basolateral membranes of proximal tubule cells in ipsilateral and contralateral kidneys, but BMMC infusion maintains control levels. (A) Representative immunoblotting for Ca 2+ - ATPase (5F10 antibody). (B) Densitometric representation of the Ca 2+ -ATPase, calculated by the ratio between the band intensity from (A) and the corresponding band on the nitrocellulose membrane stained with Rouge Ponceau. Empty bars: shamoperated group; grey bars: UUO group; filled bars: UUO + BMMC group. Results are means SEM of three experiments with different membrane preparations. *P < 0.05 and **P < 0.01 compared to control. Abbreviations are the same meaning as in Fig. 1. Taken from [2]. 116

117 A B CTR ATPDP ATPDP/MSC * Figure 3. Mesenchymal cells completely recover viability of renal tubule cells after ATP depletion. Proximal tubule cells (LLC-PK lineage) were incubated for 2 h in DMEM, in the presence of antimycin A (10 M) to block mitochondrial respiration and, therefore, ATP synthesis. Then the cells were washed and re-incubated in DMEM (5% CO 2 and 95% air at 37 0 C) for 6 h without or with mesenchymal cells (MSC) in a Millicell system. Cell death was evaluated by propidium iodide incorporation. (A) Light microscopy of renal cells (upper panels) and fluorescence microscopy of propidium iodide positive cells (lower panels). (B) Graphic representation of propidium iodide positive cells (number per field, means SEM) in the three conditions. Control (no depletion, CTR), ATPdepleted (ATPDP) and ATP-depleted cells co-cultured with MSC..(ADPDP/MSC). 2) Molecular and cellular alterations in renal function due to undernutrition. Laboratory N o 17 has a branch at Federal University of Pernambuco, where pioneer studies showing that programming by perinatal undernutrition or chronic undernutrition leads to severe cardiovascular and renal dysfunctions in adult life have been developed over the last decades. In the period covered by this report we published together 4 studies where we demonstrated for the first time that renal lesions occur at the level of sodiumtransporting ATPases and the signaling cascades involved in their regulation. The main finding was to demonstrate that undernutrition leads to a constitutive up- or down-regulation of key protein kinases that participate in the regulation of these ATPases by angiotensin II (Ang II) and derived peptides. Most importantly, we demonstrated that prevention of deleterious effects of early undernutrition can be obtained at the level of gene transcription and protein expression [3]. 3) Cellular and molecular basis for the action of hormones and autacoids in renal tissue. Clinical, epidemiological and experimental data confirm the critical role of the kidneys in the long-term regulation of systemic blood pressure and in essential hypertension, which is strictly correlated to the ability of the kidneys to fine-tune the level of sodium excretion. Generation of hypertension appears to be correlated, at least in part, with high levels of Ang II in the renal cortex. In this context two main findings emerged from the studies carried out in this period. First, we found that high levels of Ang II in the kidney are associated with the generation of Ang-(3-4), a peptide that antagonizes the influence of Ang II on cellular calcium via stimulation of the plasma membrane Ca 2+ -ATPase. We described the interconnected enzymatic pathways of Ang-(3-4) formation in renal cortex and its effects on calcium homeostasis (Figs. 4,5; [4,5]). Second, we demonstrated that Ang II type 1 receptor is involved in the regulation of renal Na + -ATPase in spontaneously hypertensive rats (SHR), and long-term treatment with an antagonist of this receptor (losartan) blocks the onset of hypertension in adult rats (Fig. 6; [6]). The results indicate a clear correlation between AT 1 receptor activation in SHR and increased 117

118 Na + -ATPase activity and open new possibilities towards the understanding of the physiopathological mechanisms involved in the increased Na + reabsorption in proximal tubules found in essential hypertension. Taken as a whole these data and those described above [4,5] can be integrated in a picture that helps in the comprehension of the physiopathology of altered Na + reabsorption in a disease that is prevalent in Brazil and worldwide. Figure 4. Proposed pathways for Ang-(3-4) formation from Ang II in kidney basolateral membranes. The scheme shows the intermediates and the enzymes characterized in [4] and [5]. Peptidases and their abbreviations are: Plummer s sensitive carboxypeptidase (PsCP), prolyl carboxypeptidase (PCP), angiotensin-converting enzyme (ACE), carboxypeptidase (CP), aminopeptidase A (APA), aminopeptidase N (APN), dipeptidyl aminopeptidase (DPP). The circled letters A, B and C denote the pathways that have Ang-(1-7) (A,B) or Ang III (C) as key intermediates. Taken from [4]. 4) Alterations of copper transporters in neglected diseases. Progress in understanding the basis for altered copper homeostasis in the neglected Menkes and Wilson diseases can be summarized as follows: (i) we succeeded for the first time in precise measuring of copper affinity in the femtomolar range ( M) for Figure 5. Femtomolar Ang-(3-4) reactivates the Ang II-inhibited Ca 2+ -ATPase after binding to AT 2 receptors. (A) Basolateral membranes preincubated or not with M Ang II, as shown, were assayed for Ca 2+ -ATPase activity as described under Materials and Methods in [5], in the presence or absence of Ang-(3-4) and the AT 2 receptor antagonist PD in the combinations and concentrations shown on the abscissa. (B) Ca 2+ -ATPase activity was measured with the combination of M Ang II and increasing concentrations of Ang-(3-4) as shown. pa 1/2 ~15.5 was calculated by hand (arrow). Open circle: control without peptide additions. Data bars and points indicate means SEM of at least six determinations with different membrane preparations. *P < 0.05 with respect to control without additions. Taken from [5]. ATP7B, the liver or Wilson Cu(I)-ATPase, with the use of a bathocuproine disulfonate/copper buffer [6]; (ii) we demonstrated that physiological activation of cyclic AMPdependent protein kinase (PKA) modulates ATP7B (Fig. 6; [7]); (iii) we described the serines of highest scores for phosphorylation by PKA in eukaryotes (Ccc2 in Saccharomyces cerevisiae; ATP7A and ATP7B in humans) that appear to be involved in the regulation of copper-transporting ATPases (Fig. 7). 118

119 Figure 6. The AT 1 receptor is involved in the activation of Na + -ATPase in adult SHR. WKY-V are Wistar Kyoto rats that received vehicle (water) for 10 weeks; SHR-V are SHR treated only with vehicle; SHR-L up to 10 wks are SHR treated with losartan for 6 weeks and then vehicle for 4 weeks; and SHR-L up to 14 wks are rats treated with losartan for 10 weeks (n = 6 per group). (A) Mean arterial blood pressure (MAP) measured weekly in all groups. (B) Renal cortex Na + -ATPase activity in the animal groups shown in panel A. Results is expressed as % of the control (means SEM). *P < 0.05 with respect to control. Figure 7. Modulation of Cu(I)-ATPase activity by PKA. Cu(I)-ATPase activity was measured in the presence of forskolin (FSK), camp, PKA - catalytic subunit (PKA, 0.25 ml assay), cholera toxin (CTX), or the inhibitor of PKA (the PKAi (5-24) peptide) in the general assay conditions described in [7]. The letters above the bars indicate statistically significant differences (P < 0.05). Inset: representative immunoblotting of PKA -catalytic subunit in the membranes probed anti-pka antibody. Taken from [7]. Figure 8. Comparison of Ccc2, ATP7A and ATP7B primary structures and location of a conserved PKA phosphorylation site. Primary sequences of Ccc2 wt and both human copper ATPases aligned in the neighborhood of the corresponding serine residue (Ser 258 in Ccc2, Ser 653 in ATP7B and ATP7A; highlighted in black) located at the boundary between the end of the N-terminal region and the first transmembrane segment. The potential sites for PKA were predicted using the pkaps tool (Neuberger, G., Schneider, G., and Eisenhaber, F. (2007) Biol. Direct 2, 1-23 (unpublished results from this laboratory). 5) ATPase- and phosphatase-based chemotherapy for parasitic diseases. In this field we have been able to shed light on the role of cell surface ATPases and phosphatases (ecto-atpases and ectophosphatases) in the oral manifestations of Human Immunodeficiency Virus (HIV) and in the mechanisms associated with opportunistic colonization by Candida sp. in HIV and in other immunocompromised individuals. We showed that C. albicans from HIV+ individuals has an ecto-phosphatase significantly higher than isolates from HIV- subjects (Fig. 8A; [8]) and, most important, yeast expressing higher levels of 119

120 surface phosphatase activity showed greater adhesion to epithelial cells, thus favoring the early steps for host cell invasion (Fig. 8B; [8]). In this regard we also demonstrated that expression of a Mg 2+ -stimulated ecto-atpase is increased in C. parapsilosis, which is the second most common species found in the bloodstream of seriously infected nosocomial patients (Fig. 9; [9]). Again, a direct relationship between ecto- ATPase activity and adhesion was observed. More important, we provided evidence showing that inhibitors of these ecto-atpases and ectophosphatases can be envisioned as a new potential class of specific antimycotic agents. Inhibition of enzyme activity resulted in decreased levels of yeast adhesion (Fig. 8B; [8]). A Finally, it is important to mention that a clear relationship also exists between these surface activities and physiological adaptative responses of the microorganisms during their life cycle. Nutrients acquisition, which is vital for parasite life (growth and development and, in many cases, host invasion), evidence a central strategy for acquire essential substances via surface ATPases and phosphatases [9,10]. 6) Integrated conclusion. The association established in the Laboratory 17 of the National Institute of Structural Biology and Bioimaging has succeeded during these 2 years in allowing the comprehension at least in part of the mechanisms involved in the pathogenesis of a diverse spectrum of prevalent and serious diseases. B Figure 9. (A) Phosphatase activity of Candida albicans isolates from HIV+ (n = 20) and HIV- (n = 15) subjects. Mean values of and picomoles of 4- MU/h/10 7 cells for HIV+ and HIV- group, respectively (Mann-Whitney test (P < 0.05). Note: o 2, o 10, * 26 : outliner ectophosphatase activity. (B) Adhesion of Candida albicans strains (CAS, PRI, ACS-C, RAS-C) to epithelial cells is correlated with ectophosphatase activity. Isolates presenting higher levels of enzyme activity (inset in panel B) are associated more efficiently with host cells. Pretreatment of fungi with the irreversible phosphatase inhibitor orthovanadate resulted in decreased levels of association with epithelial cells (P <0.01 for HIV+ strains; P <0.05 for HIV-). Taken from [8]. 120

121 Figure 9. Expression of Mg 2+ - stimulated ecto-atpase by different isolates of Candida parapsilosis. Enzyme activities in strains H297 and RFO (from the bloodstream of an infected individual and from the oral cavity of a human patient, respectively) were significantly higher (*P <0.05) than that observed in strain CCT3834. Taken from [9]. Group publications ( ): 1. Hilário-Souza E, Valverde RHF, Britto-Borges T, Vieyra A, Lowe J. Golgi membranes from liver express and ATP with femtomolar copper affinity, inhibited by camp-dependent protein kinase. Int J Biochem Cell Biol Accepted for publication. 2. Diogo Vives, Sílvia Farage, Rafael Motta, Aníbal G. Lopes, Caruso-Neves, C. Atrial natriuretic peptides and urodilatin modulate proximal tubule Na+-ATPase activity through activation of the NPR- A/cGMP/PKG pathway. Peptides (New York, N.Y. 1980)., v.31, p , Gabriel, J.S. Silva, A.E. Kummerle, R.T. Sudo, Landgraf, S.S., Caruso-Neves, C., C.A.M. Fraga, E.J. Barreto, Gisele Zapata-Sudo LASSBio-294, A Compound with inotropic and Lusitropic activity, decreases cardiac remodeling and improves Ca2+ influx into sarcoplasmic reticulum after myocardial infarction. American Journal of Hypertension., v.23, p , MADEIRA, E. P. Q., LARA, L. S., WENGERT, M., Landgraf, S.S., SOARES, J. D. L., Gisele Zapata-Sudo, Roberto T Sudo, Christina Maeda Takiya, Elaine Gomes-Quintana, Aníbal G. Lopes, Caruso-Neves, C.Na+-ATPase in spontaneous hypertensive rats: Possible AT1 receptor target in the development of hypertension. Biochimica et Biophysica Acta. Biomembranes., v.1798, p , LARA, L. S., Diogo Vives, Juliana S. Correa, Flavia P. Cardozo, Maria Fernanda Marques- Fernades, Aníbal G. Lopes, Caruso-Neves, C. PKAmediated effect of MAS receptor in counteracting angiotensin II-stimulated renal Na+-ATPase. Archives of Biochemistry and Biophysics (Print)., v.496, p , SOUZA, A. M., CARVALHO, T. L. G., LARA, L. S., QUINTANA, E. G., Aníbal G. Lopes, Caruso-Neves, C. The stimulatory effect of angiotensin II on Na+-ATPase activity involves sequential activation of phospholipases and sustained PKC activity. Biochimica et Biophysica Acta. Biomembranes., v.1798, p , Ecto-nucleoside triphosphate diphosphohydrolase activities in trypanosomatids: possible roles in infection, virulence and purine recycling. The Open Parasitology Journal, 2010, in press. 8. Russo-Abrahão T, Cosentino-Gomes D, Daflon-Yunes N, Meyer-Fernandes JR. Giardia duodenalis: Biochemical characterization of an ecto-5'- nucleotidase activity. Exp Parasitol. 2010, in press. 9. Portela MB, Kneipp LF, Ribeiro de Souza IP, Holandino C, Alviano CS, Meyer-Fernandes JR, de Araújo Soares RM. Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates. Oral Dis. 2010, 16: Almeida-Amaral EE, Cardoso VC, Francioli FG, Meyer-Fernandes JR. Leishmania amazonensis: heme stimulates (Na(+)+K(+))ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways. Exp Parasitol. 2010, 124: Dick CF, Dos-Santos AL, Fonsecade-Souza AL, Rocha-Ferreira J, Meyer-Fernandes JR. Trypanosoma rangeli: differential expression of ectophosphatase activities in response to inorganic phosphate starvation. Exp Parasitol. 2010, 124: Silva LA, Vieira-Filho LD, Barreto IS, Cabral EV, Vieyra A, Paixão AD. Prenatal Undernutrition Changes Renovascular Responses of Nimesulide in Rat Kidneys. Basic Clin Pharmacol Toxicol p. n/a. 13. Kiffer-Moreira T, Fernandes Sampaio ME, Alviano DS, Axelband F, Cesar GV, Cosentino-Gomes D, Rodrigues ML, Nimrichter L, Vieyra A, Alviano CS, Meyer-Fernandes JR. Biochemical characterization of an ecto-atp diphosphohydrolase activity in Candida parapsilosis and its possible role in adenosine acquisition and pathogenesis. FEMS Yeast Res : Vieira FS, Corrêa G, Einicker-Lamas M, Coutinho-Silva R. Host cell lipid rafts: a safe door to microorganisms?. Biol Cell : Mazzoli-Rocha F, Fernandes S, Einicker-Lamas M, Zin WA. Roles of oxidative stress in signaling and inflammation induced by particulate matter. Cell Biol Toxicol : Axelband F., Dias J., Ferrão F., Einicker-Lamas M. Nongenomic signaling pathways triggered by thyroid hormones and their metabolite 3- iodothyronamine on the cardiovascular system. J. Cell Physiol. DOI: /jcp (versão eletrônica) Verdoorn KS, Lindoso RS, Lowe J, Lara LS, Vieyra A, Einicker-Lamas M. Bone marrow mononuclear cells shift bioactive lipid pattern in injured kidney towards tissue repair in rats with unilateral ureteral obstruction. Nephrol Dial Transplant p. n/a. 18. Cabral LM, Wengert M, Almeida FG, Caruso-Neves C, Vieyra A, Einicker- Lamas M. Ceramideactivated protein kinases A and C zeta inhibit kidney proximal tubule cell Na+-ATPase. Arch Biochem Biophys :

122 19. Barreira AL, Takiya CM, Castiglione RC, Maron-Gutierrez T, Barbosa CM, Ornellas DS, Verdoorn KS, Pascarelli BM, Borojevic R, Einicker-Lamas M, Leite M Jr, Morales MM, Vieyra A. Bone marrow mononuclear cells attenuate interstitial fibrosis and stimulate the repair of tubular epithelial cells after unilateral ureteral obstruction. Cell Physiol Biochem : Axelband F, Dias J, Miranda F, Ferrão FM, Barros NM, Carmona AK, Lara LS, Vieyra A. A scrutiny of the biochemical pathways from Ang II to Ang-(3-4) in renal basolateral membranes. Regul Pept : Cardoso HD, Cabral EV, Vieira-Filho LD, Vieyra A, Paixão AD. Fetal development and renal function in adult rats prenatally subjected to sodium overload. Pediatr Nephrol : Vieira-Filho LD, Lara LS, Silva PA, Luzardo R, Einicker-Lamas M, Cardoso HD, Paixão AD, Vieyra A. Placental oxidative stress in malnourished rats and changes in kidney proximal tubule sodium ATPases in offspring. Clin Exp Pharmacol Physiol : Costa-Silva JH, Silva PA, Pedi N, Luzardo R, Einicker-Lamas M, Lara LS, Bezerra AM, Castro-Chaves C, Vieyra A. Chronic undernutrition alters renal active Na+ transport in young rats: potential hidden basis for pathophysiological alterations in adulthood? Eur J Nutr : Axelband F, Assunção-Miranda I, de Paula IR, Ferrão FM, Dias J, Miranda A, Miranda F, Lara LS, Vieyra A. Ang-(3-4) suppresses inhibition of renal plasma membrane calcium pump by Ang II. Regul Pept : Lindoso RS, Verdoorn KS, Einicker- Lamas M. Renal recovery after injury: the role of Pax-2. Nephrol Dial Transplant : Amazonas JN, Cosentino-Gomes D, Werneck-Lacerda A, Pinheiro AA, Lanfredi-Rangel A, De Souza W, Meyer-Fernandes JR.Giardia lamblia: Characterization of ecto-phosphatase activities. Exp Parasitol. 2009, 121: Mariano AC, Santos R, Gonzalez MS, Feder D, Machado EA, Pascarelli B, Gondim KC, Meyer- Fernandes JR. Synthesis and mobilization of glycogen and trehalose in adult male Rhodnius prolixus. Arch Insect Biochem Physiol : Dutra PM, Vieira DP, Meyer- Fernandes JR, Silva-Neto MA, Lopes AH. Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF). Acta Trop. 2009, 111: Cosentino-Gomes D, Russo-Abrahão T, Fonseca-de-Souza AL, Ferreira CR, Galina A, Meyer- Fernandes JR.Modulation of Trypanosoma rangeli ectophosphatase activity by hydrogen peroxide.free Radic Biol Med : Sodré CL, Moreira BL, Meyer- Fernandes JR, Dutra PM, Lopes AH, Scofano HM, Barrabin H.Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes. Parasitology. 2009, 136: Fonseca-de-Souza AL, Dick CF, dos Santos AL, Fonseca FV, Meyer-Fernandes JR. Trypanosoma rangeli: a possible role for ecto-phosphatase activity on cell proliferation.exp Parasitol. 2009, 122: Moreira OC, Rios PF, Esteves FF, Meyer-Fernandes JR, Barrabin H. CrATP as a new inhibitor of ecto-atpases of trypanosomatids. Parasitology. 2009,136: Leite MS, Thomaz R, Oliveira JH, Oliveira PL, Meyer-Fernandes JR. Trypanosoma brucei brucei: effects of ferrous iron and heme on ecto-nucleoside triphosphate diphosphohydrolase activity. Exp Parasitol. 2009,121: Caruso-Neves, C., Wengert, Mira, Pinheiro, Ana Acacia de Sá, Landgraf, Sharon Schilling, Paes-de-Carvalho, Roberto, Leão-Ferreira, Luiz Roberto, Caruso-Neves, Celso Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membraneassociated protein kinase A. Archives of Biochemistry and Biophysics (Print)., v.486, p.44-50, Assaife-Lopes, Natália, Wengert, Mira, Ana Acacia S. Pinheiro, LEAO-FERREIRA, L. R., Caruso- Neves, C. Inhibition of renal Na+ATPase activity by inosine is mediated by A1 receptor-induced inhibition of the camp signaling pathway. Archives of Biochemistry and Biophysics (Print)., v.489, p.76-81, SARAIVA, V. B., WENGERT, M., QUINTANA, E. G., HEISE, N., Caruso-Neves, C. Na+-ATPase and protein kinase C are targets to 1-Ohexadecylphosphocoline (miltefosine) in Trypanosoma cruzi.. Archives of Biochemistry and Biophysics., v.481, p.65-71, REYES, M.M. GALINDO, L. GARCÍA, D. SEGURA-PEÑA, Caruso-Neves, C., A. EBLEN-ZAJJUR, MARIN, R., PROVERBIO, F.Ouabaininsensitive, Na+-stimulated ATPase of several rat tissues: activity during a 24 h period.. Physiological Research (Print)., v.58, p ,

123 AL18 ASSOCIATE LABORATORY OF IMMUNOLOGY Coordinator: Júlio Scharfstein, IBCCF /UFRJ Member: Nils Erik Svensjo Rationale and brief summary of results During the past year, the research activities conducted in our lab have demonstrated that the discrete interstial edema resulting from the early-stage activation of innate sentinel cells by certain types of pathogens (Trypanosoma cruzi, the intracellular parasitic protozoa that causes Chagas heart disease and Porphyromomas gingivalis, a gram negative bacteria implicated in periodontitis) is a prerequisite for the extravascular activation of proteolytic cascades, such as the kinin and complement system. Chagas disease: mechanisms underlying infection-associated vasculopathy. In a recent study (Andrade et al., submitted to publication), we obtained evidence that T. cruzi trypomastigotes evokes neutrophil-dependent edema via mechanisms involving the participation of TLR2, CXCR2, bradykinin B2 receptors (B2KR), with the additional cooperation of two subtypes of endothelin receptors (ETBR and ETBR). Although the role of the endothelin pathway is not clearly defined, preliminary experiments suggest that the parasites may trigger the release of endothelins from mast cells via 123

124 activation of TLR2. In the settings of natural chagasic infection, it is well accepted that trypomastigotes are occasionally released from infected cardiovascular cells. Viewed from the perspective of pathogenesis, a major challenge in this project is to determine if trypomastigotes also activate the cardiac microcirculation via the same mechanism, perhaps evoking interstitial edema in the chagasic heart. As a first step towards addressing this question, we recently used the IVIS technology (Andrade et al., abstract presented at the II Annual INBEB Meeting) to determine the temporal course of the microvascular reactions elicited by trypomastigotes. So far performed in infected paw tissues, our preliminary studies (Fig.1) revealed that trypomastigotes induce potent microvascular responses, which peak between min. Consistent with our previous findings, the microvascular reactogenicity of trypomastigotes was drastically reduced, either by BK2R antagonist (HOE-140) or ETR antagonists (bosentan) (Fig.1). A minor and transient microvascular reaction was seen in the sites of PBS injection. Remarkably, these effects were completely blocked in mice pretreated with HOE-140 or bosentan, suggesting that the PBS Bosentan-treated TCT PBS TCT Dm28c TCT T=0 T= 30min T= 60min T= 90min T= 120min T= 150min T= 180min Fig.1. IVIS profiles showing that the vascular reactogenicity of T. cruzi trypomastigotes is blunted by bosentan (ETAR/ETBR antagonists). Similar data was obtained in mice pretreated with HOE-140. Observation: note that the early inhibitory effect of the GPCR antagonists is profound, albeit transient. A delayed vascular response is observed in the paw of drug-treated mice, presumably reflecting persistent presence of the parasites in the infection site. 124

125 needle puncture slightly activates the endothelin/kinin pathway, but the reaction is rapidly resolved. Immunological studies on ACE inhibitors, kinins and innovative vaccination strategies. In previous studies, we have shown that lymphoid-resident dendritic cells sense the endogenous peptides (such as kinins) liberated in extravascular tissues. Acting as a prototypic endogenous adjuvant, bradykinin (BK) activates their cognate G-protein coupled receptors expressed by lymphoid-resident dendritic cells, converting these specialized antigen-presenting cells into inducers of T cell effector development. By the time that we submitted our workplan to INBEB, we were unaware that of recently published peptidome data showing that BK is present in the prenodal (human) lymph. This interesting finding implies that kininogens (kinin precursor molecules circulating in the plasma, but also produced in various tissues) undergo processing in interstitial spaces, even in healthy individuals (steady state). Once collected by efferent lymphatics, kinins-along with C5a and a wide range of self-peptides originally produced in extravascular tissues (Clement et al., 2010), are likely transported the DC-rich cortical areas of the node via specialized conduits. In the absence of infection, it is very unlikely that the low endogenous levels of kinins released from kininogens ever reach the activation threshold required to convert immature (tolerogenic) DCs into immunogenic antigen-presenting cells (APCs). However, considering that interstitial edema is a common sequel of accidental tissue injury (sterile trauma), it is reasonable to predict that the innate immune system has evolved safeguards to prevent excessive exposure of tolerogenic DCs to proinflammatory peptides produced under such trivial circumstances. During infection, however, the peptidase barrier may be surpassed, or not, depending on the phenotypic profile and intensity of innate response ignited by any given pathogen. The first precedent that transient blockade of ACE leads to a robust upregulation of Th1 responses via the BK2R pathway was obtained in a subcutaneous model of T. cruzi infection (Monteiro et al., 2006). However, In the case of mucosal infection by P.gingivalis, the synergism between TLR2 ligands and kinin-releasing proteases was sufficiently strong to induce Th1 and/or Th17 responses via the BK2R pathway, irrespective of ACE blockade (Monteiro et al., 2009). Preliminary studies conducted in our lab (supported by the GCE program of Gates foundation/phase I) suggest that it may be possible to harness CD8+ T-dependent protective immunity against intracellular pathogens such as T. cruzi through the administration of a single dose of captopril, prior to vaccination and booster. 125

126 Group publications ( ): 1. Monteiro AC, Scovino A, Raposo S, Gaze VM, Cruz C, Svensjö E, Narciso MS, Colombo AP, Pesquero JB, Feres-Filho E, Nguyen KA, Sroka A, Potempa J, Scharfstein J. Kinin danger signals proteolytically released by gingipain induce Fimbriaespecific IFN-gamma- and IL-17-producing T cells in mice infected intramucosally with Porphyromonas gingivalis. J Immunol. 2009; 183(6): Schmitz V, Svensjö E, Serra RR, Teixeira MM, Scharfstein J. Proteolytic generation of kinins in tissues infected by Trypanosoma cruzi depends on CXC chemokine secretion by macrophages activated via Toll-like 2 receptors. J Leukoc Biol. 2009; 85(6): Grab DJ, Garcia-Garcia JC, Nikolskaia OV, Kim YV, Brown A, Pardo CA, Zhang Y, Becker KG, Wilson BA, de A Lima AP, Scharfstein J, Dumler JS. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells. PLoS Negl Trop Dis. 2009; 3(7):e Svensjö E, Saraiva EM, Bozza MT, Oliveira SM, Lerner EA, Scharfstein J. Salivary gland homogenates of Lutzomyia longipalpis and its vasodilatory peptide maxadilan cause plasma leakage via PAC1 receptor activation. J Vasc Res. 2009; 46(5): Villalta F, Scharfstein J, Ashton AW, Tyler KM, Guan F, Mukherjee S, Lima MF, Alvarez S, Weiss LM, Huang H, Machado FS, Tanowitz HB. Perspectives on the Trypanosoma cruzi-host cell receptor interactions. Parasitol Res. 2009; 104(6): Review. 6. Scharfstein J, Gomes Jde A, Correa-Oliveira R. Back to the future in Chagas disease: from animal models to patient cohort studies, progress in immunopathogenesis research. Mem Inst Oswaldo Cruz. 2009;104 Suppl 1: Review. 7. Coelho Dos Santos JS, Menezes CA, Villani FN, Magalhães LM, Scharfstein J, Gollob KJ, Dutra WO.Captopril increases the intensity of monocyte infection by Trypanosoma cruzi and induces human T helper type 17 cells. Clin Exp Immunol Oct 21. doi: /j x. [Epub ahead of print] 126

127 AL19 ASSOCIATE LABORATORY OF CELLULAR AND MOLECULAR NEUROLOGY Coordinator: Rosalia Mendez Otero, IBCCF/UFRJ Members: Arthur Giraldi Guimarães Bianca Gutfilen Gabriel Rodriguez Freitas Lea Miriam Fonseca Guilherme Rezende Rogério Panizzutti Joaquim F. M. da Silva Main Research Lines and Objectives: The main lines of research in our group aim to establish animal models of neurological diseases which will allow us to test the safety and efficacy of therapy with stem cells, steps necessary for clinical studies with stem cells in neurological patient. The isolation and characterization of the stem cells to be used in the therapies is also an important component of our research. It is also important to be able to label the cells in order to investigate the migration and homing of these cells after transplantation into the animal models and patients. In this respect, we have investigated labeling techniques which could be used both in pre-clinical and clinical studies. During the period covered by this report (January Nov 2010) we were able to conclude some of the goals of our proposal and the main results of each specific objective will be summarized below: Specific Objectives/Goals: progress reached in this period Evaluate the effectiveness of stem cell therapy with multipotent (mesenchymal cells, endothelial progenitors and neural stem cells) 127

128 and pluripotent (embryonic and inducible) in animal models of neurological diseases: We have investigated the functional benefit of cell therapy with multipotent stem cells in several models of neurological disorders obtained from bone marrow and umbilical cord blood. We were able to establish animal models of optic nerve lesion (a model of lesion to the central nervous system), stroke, Huntington disease, ALS (amyotrophic lateral sclerosis), hipoxiaischemic encephalopathy and peripheral nerve lesion. In some of the models we showed that that cell therapy with the mononuclear fraction or with mesenchymal stem cells reduces the functional deficits generated by the lesion to the nervous system. In addition, we have investigated the cellular and molecular mechanisms involved in this improvement and demonstrated that multipotent stem cells released factors that resulted in neuroprotection and also reduced the response of the reactive microglia. Some of the results were published during this period ( Zaveruchado-Valle et al., 2010; Pimentel-Coelho et al., 2010a, b; de Vasconcelos dos Santos et al., 2010; Giraldi-Guimaraes et al., 2009; Ribeiro- Resende et al., 2009) and some of them are still in the process of submission or analysis. It is important to mention that the results from the pre-clinical studies allowed us to propose a Phase I clinical study to evaluate the safety of cell therapies with multipotent stem cells from the bone marrow in patients with ischemic stroke. Some of the results from the clinical trial were also published during this period (Fig. 1) (Barbosa da Fonseca et al., 2009; 2010). Test the labeling of different types of stem and progenitor cells with superparamagnetic iron oxide nanoparticles (SPION) in vitro and invivo; Establish protocols for incorporation of nanoparticles by different types of stem cells through reaction for detection of SPIO; Investigate the effects of incorporated SPIONs on the proliferation, differentiation and cell death in vitro and in vivo; Develop new coatings to increase the capacity of incorporation of nanoparticles by cells and/or by specific sub-population: Fig 1: Left panel: Anterior whole-body scans performed 2 (A) and 24 hours (B) after infusion in the territory of the MCA show the distribution of BMMCs labeled with 99m Tc. Uptake in the left brain hemisphere is well visualized. The remaining activity was distributed mainly to the liver and spleen. Upper panel: Computed tomography showing ischemic lesion in the left MCA territory. B, Brain perfusion 99Tc ECD SPECT showing left hypoperfusion. C, 99m Tc BMMC brain SPECT revealing accumulation of the BMMCs in the left hemisphere 2 hours after cell transplantation. In the clinical studies, we have used stem cells labeled with 99m Technetium in order to analyze the migration and homing of the 128

129 transplanted cells to the lesioned region in the patients (Fig 1). However, the half-life of this radioactive compound is of approximately 6 hs which gives us only a maximum of 24 hs to visualize migration and homing of the injected cells. To solve this problem we have investigated the possibility of labeling different stem cells (pluri and multipotent) with commercial available SPIONs and also with SPIONs specially generated by our group. We were able to establish protocols for each cell type and for the different SPIONs (Fig 2). Using these protocols we have also tested the proliferation, viability and differentiation capacity of the labeled cells and concluded that the incorporation of SPIONs does not affect any of these cellular functions. The results of these studies were submitted for publication (Jasmin et al., 2010, submitted). Evaluate the safety and effectiveness of different types of labeled cells in different animal models of disease (nervous system, heart and kidney) with respect to toxicity and limit of detection; Verify whether the transplanted cells migrate to the lesion sites through the same reactions as well as monitor their destination by MRI at different times after the transplant; Evaluate the possible role of labeled cells in animal models of cell therapy: These are ongoing projects and we are still in the process of doing the experiments and analyzing the results. We have however preliminary results with respect to toxicity and limit of detection. We have found that we can detect mesenchymal stem cells labeled with SPIONs using a 1.5 T RMI (Fig 3). The cells were injected stereotaxically into the striatum of an adult rat and the signal was still present 60 days after the injection. We suggest that with the 7 T equipment (that has just become available) it will be possible to detect even a smaller number of labeled cells. Fig 2 : Bone marrow mesenchymal cells incorporate SPIONs as revealed by immunostaining (A), Prussian blue histochemistry (B, C). Figure 3: MR images from a rat brain (coronal sections) illustrating the injection site. Bone marrow mesenchymal cells (5x10 5 ) were labeled with SPIONs (Feridex ) and injected stereotactically into the striatum. The image was obtained on a 1.5 T MR scanner (Magnetom Avanto, Siemens, Germany) in collaboration with Dr Emerson Gasparetto (Hospital Universitario Clementino Fraga Filho/UFRJ). 129

130 Group publications ( ): 1: Zaverucha-do-Valle C, Gubert F, Bargas-Rega M, Coronel JL, Mesentier-Louro LA, Mencalha A, Abdelhay E, Santiago MF, Mendez-Otero R. Bone-marrow mononuclearcells increase retinal ganglion-cell survival and axon regeneration in the adult rat. Cell Transplant Aug 18. [Epub ahead of print] PubMed PMID: : Jasmin, Spray DC, Campos de Carvalho AC, Mendez-Otero R. Chemical induction of cardiac differentiation in p19 embryonal carcinoma stem cells. Stem Cells Dev.2010 Mar;19(3): PubMed PMID: : Pimentel-Coelho PM, Mendez-Otero R. Cell therapy for neonatal hypoxic-ischemic encephalopathy. Stem Cells Dev Mar;19(3): Review. PubMed PMID: : Barbosa da Fonseca LM, Gutfilen B, Rosado de Castro PH, Battistella V, Goldenberg RC, Kasai- Brunswick T, Chagas CL, Wajnberg E, Maiolino A, Salles Xavier S, Andre C, Mendez-Otero R, de Freitas GR. Migration and homing of bone-marrow mononuclear cells in chronic ischemic stroke after intra-arterialinjection. Exp Neurol Jan;221(1): Epub 2009 Oct 22. PubMed PMID: : de Vasconcelos Dos Santos A, da Costa Reis J, Diaz Paredes B, Moraes L,Jasmin, Giraldi-Guimarães A, Mendez-Otero R. Therapeutic window for treatment of cortical ischemia with bone marrow-derived cells in rats. Brain Res Jan 8;1306: Epub 2009 Sep 30. PubMed PMID: : Barbosa da Fonseca LM, Battistella V, de Freitas GR, Gutfilen B, Dos Santos Goldenberg RC, Maiolino A, Wajnberg E, Rosado de Castro PH, Mendez- Otero R, Andre C. Early tissue distribution of bone marrow mononuclear cells afterintra-arterial delivery in a patient with chronic stroke. Circulation Aug11;120(6): PubMed PMID: : Barnabe GF, Schwindt TT, Calcagnotto ME, Motta FL, Martinez G Jr, de Oliveira AC, Keim LM, D'Almeida V, Mendez-Otero R, Mello LE. Chemicallyinduced RATmesenchymal stem cells adopt molecular properties of neuronal-like cells but donot have basic neuronal functional properties. PLoS One. 2009;4(4):e5222. Epub2009 Apr 16. PubMed PMID: ; PubMed Central PMCID: PMC : Giraldi-Guimarães A, Rezende-Lima M, Bruno FP, Mendez-Otero R. Treatment with bone marrow mononuclear cells induces functional recovery and decreasesneurodegeneration after sensorimotor cortical ischemia in rats. Brain Res. 2009Feb 9. [Epub ahead of print] PubMed PMID: : Pimentel-Coelho PM, Magalhães ES, Lopes LM, deazevedo LC, Santiago MF,Mendez-Otero R. Human cord blood transplantation in a neonatal rat model ofhypoxic-ischemic brain damage: functional outcome related to neuroprotection inthe striatum. Stem Cells Dev Mar;19(3): PubMed PMID: : de Bittencourt-Navarrete RE, do Nascimento IC, Santiago MF, Mendez-Otero R.NMDA receptor blockade alters the intracellular distribution of neuronal nitricoxide synthase in the superficial layers of the rat superior colliculus. Braz JMed Biol Res Feb;42(2): PubMed PMID: : Ribeiro-Resende VT, Pimentel-Coelho PM, Mesentier-Louro LA, Mendez RM,Mello-Silva JP, Cabralda-Silva MC, de Mello FG, de Melo Reis RA, Mendez- Otero R.Trophic activity derived from bone marrow mononuclear cells increases peripheral nerve regeneration by acting on both neuronal and glial cell populations. Neuroscience Mar 17;159(2): Epub 2009 Jan 7. PubMed PMID: : Gubert F, Zaverucha-do- Valle C, Pimentel-Coelho PM, Mendez-Otero R, Santiago MF. Radial glia-like cells persist in the adult rat brain. Brain Res Mar3;1258: Epub 2008 Dec 24. PubMed PMID: : Moraes L, de Moraes Mello LE, Shimabukuro MK, de Castro Batista CM,Mendez-Otero R. Lack of association between PSA-NCAM expression and migration in the rostral migratory stream of a Huntington's disease transgenic mouse model.neuropathology Apr;29(2): Epub 2008 Aug 14. PubMed PMID: : Ribeiro-Resende VT, Ribeiro-Guimaraes ML, Lemes RM, Nascimento IC, Alves L,Mendez-Otero R, Pessolani MC, Lara FA. Involvement of 9-O-acetyl GD3 Ganglioside in Mycobacterium Leprae Infection of Schwann Cells. J Biol Chem Aug 25.[Epub ahead of print] PubMed PMID: : Mendez-Otero, R., Giraldi-Guimarães, A., Pimentel-Coelho, P. M., de Freitas G. R.Terapia celular no acidente vascular cerebral. Revista Brasileira de Hematologia e Hemoterapia., v.31, p , : Calcagnotto ME, Ruiz LP, Blanco MM, Santos-Junior JG, Valente MF, Patti C,Frussa-Filho R, Santiago MF, Zipancic I, Alvarez-Dolado M, Mello LE, LongoBM. (2010). Effect of neuronal precursor cells derived from medialganglionic eminence in an acute epileptic seizure model. Epilepsia.51: : Njaine B, Martins RA, Santiago MF, Linden R and Silveira MS. (2010).Pituitary adenylyl cyclaseactivating polypeptide controls the proliferationof retinal progenitor cells through downregulation of cyclin D1. (2010). EurJ Neurosci 32(3): :Prota LF, Lassance RM, Maron-Gutierrez T, Castiglione RC, Garcia CS, Santana MC, Souza-Menezes J, Abreu SC, Samoto V, Santiago MF, Capelozzi VL, Takiya CM, Rocco PR, Morales MM. (2010) Bone Marrow Mononuclear Cell Therapy led to alveolar-capillary membrane repair improving lung mechanics in endotoxininduced Acute Lung Injury. Cell Transplant. May 4. [Epub ahead ofprint] 19: Oliveira AC, Alencar B, Tzelepis F, Klezewsky W, Silva RN, Neves FS,Cavalcanti GS, Nunes MP, Santiago MF, Nóbrega A, Rodrigues MM and Bellio M.(2010). Innate Immunity in Tlr4-/- Mice but Preserved CD8+ T Cell Responsesagainst Trypanosoma cruzi in Tlr2-, Tlr4-, Tlr9- or Myd88-Deficient Mice. PLoS Path 6(4):e : Santiago MF, Alcami P, Striedinger KM, Spray DC, Scemes E. (2010). The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation. J Biol Chem. 285(16): : Motta LS, Ramos IB, Gomes FM, de Souza W, Champagne DE, Santiago MF, Docampo R, Miranda K, Machado EA. (2009). Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplanetaamericana. Insect Biochem Mol Biol.39(3):

131 AL20 ASSOCIATE LABORATORY OF INFLAMMATION AND METABOLISM Coordinator: Fernando Augusto Bozza, IPEC/FOC Members: Alysson Roncally Carvalho Antonio Giannella Neto and Frederico Caetano Jandre Marcus F. Oliveira and Aurélio Vicente Graça-Souza Frederico Caetano Jandre Aurélio Vicente Graça-Souza In this brief report, we will address the main research areas of the Laboratory of Inflammation and Metabolism from the INCT of Structural Biology and Bioimaging, Lab 20. In summary, two are the main research areas in our group. Despite both of them deal with applications of biomedical imaging in different problems and fields of research, the main focus is on the metabolism repercussions of a given inflammatory process. In the first, the uptake pattern of [18]fluorodeoxyglucose (18FDG) in the lungs at the very early stage of acute lung injury is the main field of interest. In the second one, the application of biomedical imaging in the field o neuroinflammation and aging is the main topic. Each area will be briefly described as follows. 18FDG uptake in early acute lung injury Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are syndromes of acute respiratory failure that result in acute pulmonary edema and inflammation. ALI and ARDS are a major problem in critically ill patients because their high incidence and, despite advances in 131

132 supportive therapy, their mortality remains unacceptably elevated. The diagnosis of ALI and ARDS is based on clinical, radiological and gas exchange parameters, but those are late events occurring after molecular signaling and fluid accumulation in the lung. Traditional methods of Another important research field of the Laboratory of Inflammation and Metabolism is the application on biomedical images in the study of neuroinflammation, aging and dementia. For decades, magnetic resonance imaging (MRI) has provided non-invasive assessment of many imaging (chest x-rays and computed neurological disorders, as well as acute tomography) have small sensitivity and neuroinflammatory diseases. MRI allows specificity in the early diagnosis. Positron macrostructural, cellular and metabolic emission tomography (PET) with measurements. T1-weighted gradient echo [18]fluorodeoxyglucose (18FDG) has been images show gray and white matter considered a noninvasive and highly sensitive abnormalities. T2-weighted spin echo images imaging technique that can be used to quantify highlight fluid from neuroinflammation. pulmonary inflammation. In the Laboratory of Advanced diffusion-weighted imaging (DWI) Inflammation and Metabolism we are interested shows demyelination, axonal damage and in describing the pattern of 18F-FDG uptake in the lung parenchyma of experimental models of ALI in rats and mice (Figure 1). Additionally, we are interested in the early uptake pattern and in the kinetics of glucose incorporation by the lung remyelination. Phosphorous MR spectroscopy (31P-MRS) provides simultaneous in vivo bioenergetic assessments, such as ATP, PCr (phosphocreatine) and Pi (inorganic phosphate). Intracellular ph can be assessed by the chemical parenchyma of rodents with ALI, as well as in shift of Pi relative to PCr. Longitudinal the molecular mechanisms responsible for such event. assessments may reflect changes in energy metabolism and mitochondrial function due to MicroPET/CT imaging Lung uptake of FDG- 18 F after LPS in rats Control HU = HU = Bq/ml = 43.1 Bq/ml = 83.4 HU = (+/ ) 6 hours HU = (+/- 53.8) 24 hours Bq/ml = Imaging and Neuroinflammation Bq/ml = hours pathological processes. More sophisticated MRI techniques may have applicability to severe neuroinflammatory conditions, such as sepsis, and measurements of reactive oxygen species (ROS). Perivascular edema, and spectroscopic abnormalities can be demonstrated by MRI and 1H- MRS in a mouse sepsis model (Bozza et al., JCBFM 2010). The detection of oxidative damage and reactive oxygen species (ROS) by MR techniques is now possible through the use of 132

133 Gd-based spin trapping contrast agents that track the formation of protein radicals. It is unknow whether characteristic MR abnormalities define subjects with age-associated chronic low-level neuroinflammation, or whether disease activity may be assessed by either traditional or novel MR techniques. Positron emission tomography (PET) is used for evaluation of dementia, seizures, and for assessment of recurrent tumor versus radiation necrosis, and may be applicable to neuroinflammatory disorders. Activated microglia, monocytes and macrophages show an increase in expression of peripheral benzodiazepine receptors (PBR). PBR binding ligands, such as [(11)C]PK11195 are currently under development and investigation and may play a future role in assessing neurodegenerative where inflammation plays a role. 18F fluorodeoxyglucose (FDG) PET shows increased uptake in acute cerebral inflammation, and decreased uptake in the late stages of the diseases. Increased uptake of glucose analogs is a very early event (< 6h) in experimental sepsis, possibly from excitotoxity or activation of microglial orastrocytes (Figure 2). Decreased FDG uptake in neocortical regions of the brain 24h after endotoxin, and could be due to neuronal injury or dysfunction. FDG PET may provide assessment of both the acute and chronic phases of neuroinflammation. In AD, decreased uptake of FDG occurs in the parieto-temporal, cingulate, and medial temporal cortices. MR findings of AD show early hippocampal and medial temporal volume loss. The discrepancy between FDG PET and MRI findings may be due to technical limitations of PET in measuring small structures. Many patients with mild cognitive impairment (MCI) show similar, but less-severe, regional hypometabolism. This might lead to the speculation that pre-clinical AD can be diagnosed by FDG PET, but this has not been supported by longitudinal studies. MRI and FDG PET may provide useful indicators that MCI has progressed into frank AD, particularly when longitudinal studies can be performed. However, the utility of MRI or FDG PET for characterizing MCI and chronic neuroinflammation has not been studied. The brains of AD patients reveal betaamyloid plaques and neurofibrillary tangles, which contain beta-amyloid peptides (Abeta) and highly phosphorylated tau proteins. Abeta deposition leads to an increase in beta-amyloid plaques, the initial neuropathological change in AD. Radiotracers for in vivo imaging betaamyloid in brain is an important focus of research development. The most widely used and studied of these agents is N-methyl-[(11)C]2-(4'- methylaminophenyl)-6-hydroxybenzothiazole ([(11)C]PIB). (11C)PIB positron emission tomography (PET) has been validated as showing increased binding in subjects with AD, compared to normals. (11)C]PIB binding also occur in subjects with mild cognitive impairment (MCI) and also some elderly normal patients without neurocognitive effects. Abeta deposition, the primary pathological feature of AD, has also been shown occur in response to lipopolysaccharide-induced neuroinflammation in animal models. (11C)PIB uptake may, in some cases, indicate a population of individuals with ongoing neuroinflammation due to chronic or recurrent low level systemic inflammation. However, this has not been studied. 133

134 Figure 2 (upper panels): Upper left: FDG PET (GE Advance clinical PET scanner, resolution 4mm). Upper right: quantitative assessment of FDG uptake in ex-vivo samples of brain. Lower left: Digital fluorescence autoradiolography (BAS-3000, resolution 40 um) of NBDG. Lower right: Phosphor imager (BAS-5000) digital autoradiography (resolution 25 um) of 14C-2DG. Images show a similar pattern, with increased cortical uptake of glucose analogs at early time points post LPS, which decreases by 24h. Group publications ( ): 1. Bozza, Fernando A ; Salluh, Jorge I.F.. An urban perspective on sepsis in developing countries. Lancet Infectious Diseases, v. 10, p , Larsen, R. ; Gozzelino, R. ; Jeney, V. ; Tokaji, L. ; Bozza, Fernando A.; Japiassu, A. M. ; Bonaparte, D. ; Cavalcante, M. M. ; Chora, A. ; Ferreira, A. ; Marguti, I. ; Cardoso, S. ; Sepulveda, N. ; Smith, A. ; Soares, M. P.. A Central Role for Free Heme in the Pathogenesis of Severe Sepsis. Science Translational Medicine, v. 2, p. 51ra71-51ra71, Bozza, Fernando A ; Carnevale, Renata ; Japiassú, André Miguel ; Castro-Faria-Neto, Hugo Caire ; Angus, Derek C. ; Salluh, Jorge I. F.. EARLY FLUID RESUSCITATION IN SEPSIS. Shock (Augusta, Ga.), v. 34, p , Salluh, Jorge I.F. ; Soares, Márcio ; Coelho, Luis M. ; Bozza, Fernando A. ; Verdeal, Juan Carlos R. ; Castro-Faria-Neto, Hugo C. ; Silva, José Roberto Lapa e ; Bozza, Patrícia T. ; Póvoa, Pedro. Impact of systemic corticosteroids on the clinical course and outcomes of patients with severe community-acquired pneumonia: A cohort study. Journal of Critical Care, p. xx, Wang, Li-Ming ; Becker, J. Sabine ; Wu, Qi ; Oliveira, Marcus F. ; Bozza, Fernando A. ; Schwager, Andrea L. ; Hoffman, John M. ; Morton, Kathryn A.. Bioimaging of copper alterations in the aging mouse brain by autoradiography, laser ablation inductively coupled plasma mass spectrometry and immunohistochemistry. Metallomics, v. 2, p , Salluh, Jorge I. F. ; Bozza, Fernando A.; Japiassu AM ; Castro-Faria-Neto, Hugo C. ; Bozza, Patricia T ; Póvoa, Pedro. Corticosteroids in Sepsis: Pathophysiological Rationale and the Selection of Patients. Endocrine, Metabolic and Immune Disorders. Drug Targets, v. 00, p , Reis, Patricia A. ; Comim, Clarissa M. ; Hermani, Fernanda ; Silva, Bruno ; Barichello, Tatiana ; Portella, Aline C. ; Gomes, Flavia C. A. ; Sab, Ive M. ; Frutuoso, Valber S. ; Oliveira, Marcus F. ; Bozza, Patricia T. ; Bozza, Fernando A. ; Dal-Pizzol, Felipe ; Zimmerman, Guy A. ; Quevedo, João ; Castro-Faria-Neto, Hugo C.. Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral Malaria, and Is Reduced by Additive Antioxidant Therapy. PLoS Pathogens, v. 6, p. e , Salluh, Jorge I.F. ; Shinotsuka, Cássia Righy ; Soares, Márcio ; Bozza, Fernando A. ; Lapa e Silva, José Roberto ; Tura, Bernardo Rangel ; Bozza, Patrícia T. ; Vidal, Carolina Garcia. Cortisol levels and adrenal response in severe community-acquired pneumonia: A systematic review of the literature. Journal of Critical Care, p , Japiassú AM,; Amancio, R. ; Mesquita, EC ; Medeiros, DM ; Bernal, HB ; Nunes, EP ; LUZ, PM ; Grinsztejn, B ; Bozza, F. A.. Sepsis is a major determinant of outcome in critically ill HIV/AIDS patients. Critical Care (London), v. 14, p. 152, Stiebler, Renata ; Timm, Bruno L. ; Oliveira, Pedro L. ; Hearne, Giovanni R. ; Egan, Timothy J. ; Oliveira, Marcus F.. On the physico-chemical and physiological requirements of hemozoin formation promoted by perimicrovillar membranes in Rhodnius prolixus midgut. Insect Biochemistry and Molecular Biology, p , Stiebler, R. ; Hoang A.N. ; Egan, T. J. ; Wright D.W. ; Oliveira, M. F.. Increase on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro. Plos One, v. 5, p. e12694-e12694, Caiaffa, C.D. ; Stiebler, R; Oliveira, M.F. ; Lara, F.A. ; Paiva-Silva, G.O. ; Oliveira, P.L.. Sn-

135 protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut. Insect Biochemistry and Molecular Biology, p. 1-8, Gama de Abreu, Marcelo ; Cuevas, Maximiliano ; Spieth, Peter M ; Carvalho, Alysson R ; Hietschold, Volker ; Stroszczynski, Christian ; Wiedemann, Barbel ; Koch, Thea ; Pelosi, Paolo ; Koch, Edmund. Regional lung aeration and ventilation during pressure support and biphasic positive airway pressure ventilation in experimental lung injury. Critical Care (London), v. 14, p. R34, Peter M; Carvalho, Alysson R; Pelosi, Paolo; Koch, Thea; Gama de Abreu, Marcelo. Pressure support improves oxygenation and lung protection compared to pressure controlled ventilation and is further improved by random variation of pressure support. Crit Care Med, In Press 15. Beda, Alessandro ; Jandre, Frederico C. ; Giannella-Neto, Antonio. A Numerical Model of the Respiratory Modulation of Pulmonary Shunt and PaO2 Oscillations for Acute Lung Injury. Annals of Biomedical Engineering, v. 38, p , Santos, E. L. ; Novaes,J.S. ; Reis, V.M. ; Giannella Neto, A.. Low Sampling Rates Bias Outcomes from the Wingate Test. International Journal of Sports Medicine, v. 31, p. 1-6, Giannella Neto, A. ; Motta Ribeiro, GC ; Santos, E. L. ; Soares, J. H. N. ; Leão Nunes, MV ; Jandre, F. C.. Control of positive end-expiratory pressure (PEEP) for small animal ventilators. BioMedical Engineering Online, v. 9, p. 36, London NR, Zhu W, Bozza FA, Smith MC, Greif DM, Sorensen LK, Chen L, KaminohY, Chan AC, Passi SF, Day CW, Barnard DL, Zimmerman GA, Krasnow MA, Li DY. Targeting Robo4-dependent slit signaling to survive the cytokine storm in sepsis and influenza. Sci Transl Med Mar 17;2(23):23ra Rodrigues RS, Carvalho AR, Morton KA, Bozza FA. (18)-F-fluorodeoxyglucose positron emission tomography/computed tomography study in acute lung injury/acute respiratory distress syndrome. Crit Care Med Jan;38(1): Albuquerque LM, Trugilho MR, Chapeaurouge A, Jurgilas PB, Bozza PT, Bozza FA, Perales J, Neves-Ferreira AG. Two-dimensional difference gel electrophoresis(dige) analysis of plasmas from dengue fever patients. J Proteome Res Dec;8(12): Bozza FA, Garteiser P, Oliveira MF, Doblas S, Cranford R, Saunders D, Jones I, Towner RA, Castro-Faria-Neto HC. Sepsis-associated encephalopathy: a magnetic resonance imaging and spectroscopy study. J Cereb Blood Flow Metab Feb;30(2): Soares M, Caruso P, Silva E, Teles JM, Lobo SM, Friedman G, Dal Pizzol F, Mello PV, Bozza FA, Silva UV, Torelly AP, Knibel MF, Rezende E, Netto JJ, Piras C, Castro A, Ferreira BS, Réa-Neto A, Olmedo PB, Salluh JI; Brazilian Research in Intensive Care Network (BRICNet). Characteristics and outcomes of patients with cancer requiring admission to intensive care units: a prospective multicenter study. Crit Care Med Jan;38(1): Assunção-Miranda I, Amaral FA, Bozza FA, Fagundes CT, Sousa LP, Souza DG, Pacheco P, Barbosa-Lima G, Gomes RN, Bozza PT, Da Poian AT, Teixeira MM, Bozza MT. Contribution of macrophage migration inhibitory factor to the pathogenesis of dengue virus infection. FASEB J Jan;24(1): Japiassú AM, Salluh JI, Bozza PT, Bozza FA, Castro-Faria-Neto HC. Revisiting steroid treatment for septic shock: molecular actions and clinical effects a review. Mem Inst Oswaldo Cruz Jul;104(4): Review. 25. Rodrigues RS, Bozza FA, Christian PE, Hoffman JM, Butterfield RI, Christensen CR, Heilbrun M, Wiggins RH 3rd, Hunt JP, Bentz BG, Hitchcock YJ, Morton KA. Comparison of whole-body PET/CT, dedicated highresolution head and neck PET/CT, and contrast-enhanced CT in preoperative staging of clinically M0 squamous cell carcinoma of the head and neck. J Nucl Med Aug;50(8): Salluh JI, Dal-Pizzol F, Mello PV, Friedman G, Silva E, Teles JM, Lobo SM, Bozza FA, Soares M; Brazilian Research in Intensive Care Network. Delirium recognition and sedation practices in critically ill patients: a survey on the attitudes of 1015 Brazilian critical care physicians. J Crit Care Dec;24(4): Maracajá-Neto LF, Verçosa N, Roncally AC, Giannella A, Bozza FA, Lessa MA. Beneficial effects of high positive end-expiratory pressure in lung respiratory mechanics during laparoscopic surgery. Acta Anaesthesiol Scand Feb;53(2): Bozza FA, Shah AM, Wey rich AS, Zimmerman GA. Amicus or adversary: plateletsin lung biology, acute injury, and inflammation. Am J Respir Cell Mol Biol Feb;40(2): Carvalho, Alysson R. ; Spieth, Peter M. ; Pelosi, Paolo ; Beda, Alessandro ; Lopes, Agnaldo J. ; Neykova, Boriana ; Heller, Axel R. ; Koch, Thea ; de Abreu, M G. Pressure SupportVentilation and Biphasic Positive Airway Pressure Improve Oxygenation by Redistribution of Pulmonary Blood Flow. Anesthesia and Analgesia, v. 109, p , de Abreu, M G; Cuevas, Maximiliano ; Spieth, Peter M ; Carvalho, Alysson R ; Hietschold, Volker ; Stroszczynski, Christian ; Wiedemann, Barbel ; Koch, Thea ; Pelosi, Paolo ; Koch, Edmund. Regional lung aeration and ventilation during pressure support and biphasic positive airway pressure ventilation in experimental lung injury. Critical Care (London), v. 14, p. R34, Spieth, P. M ; Carvalho, A. R ; Pelosi, P. ; Hoehn, C. ; Meissner, C. ; Kasper, M. ; Hubler, M. ; von Neindorff, M. ; Dassow, C. ; Barrenschee, M. ; Uhlig, S. ; Koch, T. ; de Abreu, M G. Variable Tidal Volumes Improve Lung Protective Ventilation Strategies in Experimental Lung Injury. American Journal of Respiratory and Critical Care Medicine,, Spieth, Peter M. ; Carvalho, Alysson R. ; Güldner, Andreas ; Pelosi, Paolo ; Kirichuk, Oleg ; Koch, Thea ; de Abreu, M G. Effects of Different Levels of Pressure Support Variability in Experimental Lung Injury. Anesthesiology (Philadelphia), v. PAP, p. 342, Beda, Alessandro ; Jandre, Frederico C. ; Giannella-Neto, Antonio. A Numerical Model of the Respiratory Modulation of Pulmonary Shunt and PaO2 Oscillations for Acute Lung Injury. Annals of Biomedical Engineering, v. 38, p , Carvalho, N C ; Beda, A ; de Abreu, M G ; Spieth, P M ; Granja-Filho, P ; Jandre, F C ; Giannella- Neto, A. Comparison of objective methods to classify the pattern of respiratory sinus arrhythmia during mechanical ventilation and paced spontaneous breathing. Physiological Measurement, v. 30, p , Lara, F. A. ; Kahn S ; Fonseca A ; Bahia C ; Pinho J ; Graca-Souza, A. V. ; Houzel J.C. ; Oliveira, Pedro L. ; Neto V. M ; Oliveira, M. F.. On the fate of extracellular hemoglobin and heme in brain. Journal of Cerebral Blood Flow and Metabolism, v. 29, p ,

136 36. Menna-Barreto, Rubem F.S. ; Goncalves, Renata L.S. ; Costa, Elaine M. ; Silva, Raphael S.F. ; Pinto, Antonio V. ; Oliveira, Marcus F. ; de Castro, Solange L.. The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are mediated by mitochondrial dysfunction. Free Radical Biology & Medicine, v. 47, p , Soares, J. B. R. C. ; Menezes, D. ; Vannier, M. A. ; Ferreira-Pereira, A. ; Almeida G.T. ; Venancio TM ; Verjovski-Almeida S ; Zishiri VK ; Kuter D ; Hunter R ; Egan, T. J. ; Oliveira M.F.. Interference with hemozoin formation represents an important mechanism of schistosomicidal action of antimalarial quinoline methanols. PLoS Neglected Tropical Diseases, v. 3, p. e477-e493, Gonçalves, Renata L. S. ; Machado, Ana Carolina L. ; Paiva-Silva, Gabriela O. ; Sorgine, Marcos H. F. ; Momoli, Marisa M. ; Oliveira, Jose Henrique M. ; Vannier-Santos, Marcos A. ; Galina, Antonio ; Oliveira, Pedro L. ; Oliveira, Marcus F.. Blood-Feeding Induces Reversible Functional Changes in Flight Muscle Mitochondria of Aedes aegypti Mosquito. Plos One, v. 4, p. e7854,

137 3. COOPERATIVE AND EDUCATIONAL ACTIVITIES 137

138 During the the first year of the development, we created a network of interactions among the different Associated Laboratories (ALs) and the Multiuser Facilities. The interactions have increased mainly at the level of exchange between students and researchers. This is evidenced in publications that are listed above, where we can observe the co-authoring of several articles by the INBEB participants, including students from the different groups. The homepage of the INBEB was an important tool in bringing the groups together. We posted on the homepage information about courses, lectures, discussions, data, and listings of activities of interest to group members. We had a monthly lecture program at the INBEB (Rio de Janeiro) with participation of members of different ALs. In addition, we also coordinated specific topic meetings and roundtables. 138

139 Member interactions Overall, the ALs have interacted significantly, with much exchange between students seeking new skills and expertise to apply to their studies. These exchanges have led to better understanding of their research problems. part of a collaborative effort. AL7, PI Prof. Carlos Ramos: At the laboratory at UFRJ as AL4, PI Prof. Russolina Zingali: The group of Dr. Wanderley de Souza - analysis of the microorganism Giardia; The group of Prof. Marlene Benchimol - identification of proteins isolated from Trichomonas by immunoprecipitation and mass spectrometry; The group of Prof. Adalberto Vieyra - analysis of the phosphorylation profile of renal cells that were in contact with mesenchymal cells; The group of Prof. Debora Foguel - study of proteome and immunomapping of the venom from B. jararacussu, and Micrurus altirostris; The group of Dr. Fabio Almeida - analysis of the mass of synthetic peptides. The group of Dr Paul Bisch - proteomic study of liver cells infected with Dengue virus. The group of Dr. Robson - study of breast cancer cells and their interaction with the hemostatic system. Mass spectrometry - mass analysis of natural and recombinant proteins in order to confirm the sequence of several groups of proteins. INTERACTION WITH OTHER INSTITUTIONS AND/OR BUSINESSES: An interaction with the Instituto Vital Brazil to study the venoms and anti-venoms was undertaken. Interaction with Hygeia to produce anti-thrombotic substances. 139

140 AL-11, PI Prof. Thais Souto Padrón: Interaction with other groups within the INCT, especially the group of Prof. Wanderley de Souza was emphasized. This collaboration has naturally matured over many years due to the common projects, common interests even on different projects, and natural collaboration that occurs between laboratories that possess similar equipment. COOPERATIVE ACTIVITIES THAT INVOLVED INCTS AND OTHER INSTITUTIONS (E.G., COMPANIES, NGOS, AND GOVERNMENTAL INSTITUTIONS) An integration panel was held in Rio de Janeiro where three related INCTs met on December 3-4, 2009: the INBEB, the INOFAR National Institute of Science and Technology for Drugs and Medicines) coordinated by Prof. Dr. Eliezer Barreiro (UFRJ) and the INBEQMeDI (National Institute of Science and Technology of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases), coordinated by Prof. Dr. Glaucius Oliva (IFSC-USP). These three INCTs perform high-quality research on infectious and degenerative diseases with the aim of developing new drugs. The meeting brought together about 50 researchers from the three INCTs. At the meeting, the main research areas that had been developed by each INCT were addressed. Additionally, an overall global vision was created, and new strategies for working towards the proposed goals were defined. These goals can be summarized as the development of new drugs to fight various diseases, especially those designated as "neglected" diseases including malaria, leishmaniasis, leptospirosis, schistosomiasis, and Chagas disease. The Second Annual Meeting of INBEB occurred on November 8-10, 2010 (see II Annual Meeting Abstract). The meeting had oral presentations from members of the 20 Associated Laboratories and 192 poster presentations by undergraduate, graduate and postdoctoral trainees. The meeting had an external evaluation by three distinguished external scientists that will make a report with suggestions and criticisms. 140

141 National and international events Presentation of papers, courses, seminars, lectures, and round table discussions a) In Rio de Janeiro, with the direct support of the INBEB, the Second South American Workshop on Advanced Fluorescence Techniques: Spectroscopy of the Microscope" was organized. The workshop consisted of a five-day course that included theoretical and practical classes. These classes addressed various aspects related to the acquisition and computerized processing of data from fluorescence spectroscopy experiments by both traditional methods, such as measurement by cuvette, and new state-of-the-art techniques, such as microscopy techniques, including fluorescence correlation spectroscopy, imaging correlation spectroscopy, and images of fluorescence lifetime, including measurements in living cells. These techniques involve detecting data from a single molecule - i.e., "single molecule spectroscopy". Currently, this area of spectroscopy is rapidly expanding. The use of these techniques is growing and becoming more important, and there are an increasing number of studies that benefit from the use of such approaches in the current literature. These new techniques were recently made available in Rio de Janeiro through the Unit for Multi-User Fluorescence Correlation Spectroscopy, which was installed at the Institute of Medical Biochemistry, UFRJ, and contains a portion of the array of equipment that is present at the INBEB. The course was held as a satellite event at the VII Ibero-American Congress of Biophysics in 2009, and its objective was to teach the students the various aspects involved in fluorescence spectroscopy. It also included actual hands-on user sessions. 141

142 The lectures were accompanied by hands-on sessions utilizing cutting-edge equipment like the spectrofluorometer and multiphoton microscopes that were available at the Center for Health Sciences of the UFRJ and the INCT II building. Despite the fact that the practical classes were restricted to a group of 22 students due to space limitations, the theoretical part included lectures that were open to the public and could accommodate all interested parties who felt that they might benefit from the techniques that were presented. There were 141 trainees and participants, proving that the course achieved its goal of spreading knowledge on these state of the art techniques that are now available in the INCT, for the first time in Brazil. The classes were taught by renowned professors/researchers who specialize in the use of fluorescence, both in spectroscopy and microscopy, and work at research centers of excellence in the United States and Argentina. The professors at the Instituto de Bioquímica Médica (Institute for Medical Biochemistry), UFRJ, and the members of INBEB who organized the event also taught several classes. The event was very successful at increasing the knowledge of these techniques, especially among undergraduate and graduate students, as well as various doctors and professors from the Centro de Ciências da Saúde (Center for Health Sciences), UFRJ. The program reached students from other graduate programs as well as students and professors from other state universities across the country, such as São Paulo and Pernambuco, and institutions in other Latin American countries, including Argentina and Chile. This event was just one of several initiatives that were planned within the framework of the Multi-User Units and the INBEB to train new researchers proficient in the area. b) The INBEB also contributed to the organization of the VII Iberoamerican Congress of Biophysics in 2009 in Buzios, Rio de Janeiro, between September 30-October 3, 2009, at the Atlantic Hotel in Buzios. The Congress brought together the Annual Meeting of the Biophysical Society in Latin America and the Iberian Peninsula in one gathering throughout the Community of Biophysics of these two regions. The Congress was attended by 600 participants and provided a special forum for interaction, discussion, scientific collaborations and assembly strategies for regional development of biophysics and other related areas. Many INBEB researchers participated in 142

143 the Congress by giving lectures. There was also a great participation of students from several Latin American countries. c) Participation of members of the INBEB in the First Course of Biophysics for Graduated Latin American Students in Buzios (5-9 October 2009). d) The ALs members participated in several national and international scientific meetings presenting lectures. e) Opening of the National Center for Bioimaging II (CENABIO II) followed by a scientific Round Table. The inauguration of the second building of the INBEB was held on May 5, The program consisted of an opening lecture in the morning held by Prof. Jerson Silva, a round table of various authorities, and an afternoon round table presenting different aspects of the use of small animal imaging techniques that are available in the INBEB such as MRI, PET-SPECT-CT, high resolution ultrasound equipment and bioluminescence. Among the authorities, we were pleased to have the Minister for Science and Technology, Dr. Sergio Rezende; Dr. Reinaldo Guimarães, Secretary of Science and Technology and Strategic Inputs of the Ministry of Health; Dr. Carlos Alberto Aragao, President of CNPq; Jorge Guimaraes, President of CAPES; Dr. Ricardo Gattass - Superintendent of FINEP; Dr. Luis Edmundo, State Secretary of Science and Technology; Dr. Ruy Garcia Marques, President of FAPERJ, among other authorities. At this inauguration, the video produced by INBEB Influenza was presented. f) Two other events also deserve to be mentioned, both organized by the group of Dr. Wanderley de Souza and Dr. Marlene Benchimol. The first was the organization, for the first time in South America, of the XIII International Congress of Protistology (August 23-28) in Buzios, Rio de Janeiro, which was attended by 600 researchers, most of them from abroad. The congress was preceeded by 2 pre-conference courses, a special symposium that set a standard for the nomenclature of isolates of Trypanosoma cruzi, 4 plenary lectures and 15 symposia. The second is the organization of the School for Advanced Studies in Cell Biology of Protists (October) in Rio de Janeiro and Buzios. This course was sponsored by CAPES and by several other graduate courses from our University as well as by the course on Cell Biology and Life Sciences, State University of North. 143

144 144

145 Training and teaching human resources The researchers of INBEB are involved in different graduate programs, most of them with a grade 6 or 7 by Capes. There were at least 78 completed Master Dissertations and 43 PhD Theses, as shown below: Theses Completed ( ): AL 1: Master Guilherme Augusto Piedade de Oliveira. Aspectos Clínicos e Termodinâmicos da Leucemia Mielóide Crônica (LMC) Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Jerson Lima da Silva. Carlos Alberto Marques Carvalho. Rastreamento das proteínas de Envelope do Vírus Mayaro durante os Eventos Iniciais de Infecção Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Andre Marco de Oliveira Gomes. Claudia Bustamante Levy. Análise de mutações no gene TP53 em casos de câncer de mama e estudo da proteína p53 mutante: aspectos fisiopatológicos do tumor Dissertação (Biologia Humana e Experimental) - Universidade do Estado do Rio de Janeiro Marina Silva Rodrigues. Analise de polimorfismos genéticos de TP53 e XRCC1 e sua associação com as caracteristicas de casos de câncer de mama Dissertação (Mestrado em Fisiopatologia Clínica e Experimental) - Universidade do Estado do Rio de Janeiro, Fundação Carlos Chagas Filho de Amparo à Pesq. do Estado do Rio de Janeiro. Advisor: Cláudia Vitória de Moura Gallo. Pedro Nicolau Neto. Estudo da metilação de genes associados ao Cancer de Mama Dissertação (Mestrado em Biologia (Biociências Nucleares)) - Universidade do Estado do Rio de Janeiro, Fundação Carlos Chagas Filho de Amparo à Pesq. do Estado do Rio de Janeiro. Advisor: Cláudia Vitória de Moura Gallo. Doctoral Ana Paula Dinis Ano Bom. Caracterização da estabilidade, atividade e agregação do domínio central da proteína supressora de tumor p53 em diferentes phs Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro. Advisor: Jerson Lima da Silva. Tuane Cristine Ramos Gonçalves Vieira. Aspectos estruturais da interação da proteína do prion com heparina Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Jerson Lima da Silva. Ygara da Silva Mendes. Biologia Estrutural de Flavivírus: Propriedades Biofísicas da Interação de 145

146 Peptídeos de Fusão com Membranas Biomiméticas e Implicações para o Desenvolvimento de uma Vacina Inativada por Alta Pressão Hidrostática Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Andrea Cheble de Oliveira. Ivanildo Pedro de Souza Jr.. Estudo da Interação entre alfavírus e microdomínios de membrana Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Andre Marco de Oliveira Gomes. Theo Luiz Ferraz de Souza. Aspectos estruturais, dinâmicos e termodinâmicos envolvidos na montagem in vitro do capsídeo do vírus da hepatite C e na inibição da proteína inibidora de apoptose XIAP, revelados por análises espectroscópicas e calorimétricas Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. AL 2: Master Ricardo Sant Anna de Oliveira. Pequenas moléculas como inibidores de agregação da proteína amiloidogênica transtirretina (TTR) Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Debora Foguel. Cícero Figueiredo-Freitas. Formação de S- nitrosomiosina sob condições fisiológicas Dissertação (Mestrado em Quimica Biologica) - Instituto de Bioquimica Medica - UFRJ, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Martha Meriwether Sorenson. Luciana Elena de Souza Fraga Machado. Efeito do fenol na interação acto-s Dissertação (Mestrado em Quimica Biologica) - Instituto de Bioquimica Medica - UFRJ, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Martha Meriwether Sorenson. Leandro Teixeira Oliveira. O acúmulo do peptídeo beta amilóide no espaço intraneuronal e a relação com uma proteína motora associada à actina Dissertação (Mestrado em Quimica Biologica) - Instituto de Bioquimica Medica - UFRJ, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Martha Meriwether Sorenson. Maria Thereza Cargnelutti do Carmo. Estudos funcionais e cristalográficos da interação de análogos de suramina com alfa-trombina humana Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Luis Mauricio Trambaioli da Rocha e Lima. Doctoral Fernando Lucas Palhano. Príon de levedura Sup35 e proteína humana transtirretina: Bioquímica, biologia estrutural e celular de duas proteínas amiloidogênicas Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Debora Foguel. Leonardo de Castro Palmieri. Complexo transtirretina humana e zinco: caracterização estrutural, funcional e termodinâmica Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Debora Foguel. Renato Fernandes de Paulo. Melanofilina, prefoldina 4 e miosina Va: Parceria para o correto enovelamento durante o transporte vesicular Tese (Doutorado em Quimica Biologica) - Instituto de Bioquimica Medica - UFRJ, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Martha Meriwether Sorenson. AL 3: Master José Eduardo da Silva Rabelo. Caracterização funcional e termodinâmica de proteínas de alta mobilidade de Aedes aegypti (AeHMGB1) Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Instituto de biofísica Carlos Chagas Filho, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Ronaldo da Silva Mohana Borges. Marcela da Silva Rosa. Identificação de novos alvos moleculares para diagnóstico e prognóstico da dengue através de técnicas proteômicas Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Ronaldo da Silva Mohana Borges. Doctoral Francisco Gomes Neto. Utilizacao de modelos explicitos de membrana para calculos de estrutura de proteinas soluveis que se associam a membrana a partir de dados de ressonancia magnetica nuclear Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Fabio Ceneviva Lacerda Almeida. Guilherme Razzera Maciel. Estudos Estruturais dos enovelamentos proteicos de defensinas e globinas atraves de RMN Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Ana Paula Canedo Valente. AL 4: Master Ricardo de Araujo Teixeira. B. jaracacussu imunoma e proteoma para desenvolvimento de kit diagnóstico Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advidor: Russolina Benedeta Zingali Saulo Martins Vieira. Análise da Atividade da Trombina com Substratos Fluorogênicos Baseados no Receptor PAR Dissertação (Mestrado em Quimica Biologica) - Instituto de Bioquímica Médica /CCS / UFRJ,. Advisor: Russolina Benedeta Zingali. Douglas Bayer Vieira. Análise proteômica do baço de galus galus infectado ou não com Eimeria tenella (coccidiose) Dissertação (Mestrado em Bioquímica e Fisiologia) - Universidade Federal de Pernambuco,. Advisor: Russolina Benedeta Zingali. Reinaldo Ramos. A pós-graduação brasileira e os ventos de bolonha: uma discussão qualitativa Dissertação (Mestrado em Quimica Biologica) - Instituto de Bioquímica Médica /CCS / UFRJ, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Russolina Benedeta Zingali. Morgana Guimarães Soares. Efeito do anticoagulante Ixolaris no crescimento tumoral de gliomas humanos implantados em encéfalo de roedores Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional 146

147 de Desenvolvimento Científico e Tecnológico. Advisor: Robson de Queiroz Monteiro. Aline da Costa Cruz. Diagnóstico sorológico da infecção pulmonar por Pseudomonas aeruginosa em crianças com Fibrose Cística Dissertação (Mestrado em Microbiologia) - Universidade do Estado do Rio de Janeiro, Fundação Carlos Chagas Filho de Amparo à Pesq. do Estado do Rio de Janeiro. Co-Advisor: Bianca Cruz Neves. Andreia da Silva de Oliveira. Conclusão em 16/03/2010. Mestrado em Química Biológica da Universidade Federal do Rio de Janeiro. Efeito antitumoral do Ixolaris, um potente inibidor da coagulação sanguínea, sobre o melanoma murino B16F10. (Orientador). Bolsista CNPq. Angélica Dutra de Oliveira. Conclusão em 31/05/2010. Mestrado em Química Biológica da Universidade Federal do Rio de Janeiro. Mecanismos procoagulantes envolvidos na ativação de receptores PAR em linhagens de glioblastoma humano. (Orientador). Bolsista CNPq. Doctoral Flavia Serra Frattani ferreira. Caracterização farmacológica e mecanística do perfil anti-hemostático de derivados acilhidrazônicos Tese (Doutorado em Quimica Biologica) - Instituto de Bioquímica Médica /CCS / UFRJ, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Orientador: Russolina Benedeta Zingali. Douglas Siqueira de Almeida Chaves. O potencial terapêutico da salsa (Petroselinum crispum), um alimento funcional, na prevenção da trombose Tese (Doutorado em Química de Produtos Naturais) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. (Coorientador). Silas Rodrigues Pessini. Analise proteômica de folhas de papaia infectado pelo Virus da meleira Tese (Doutorado em Quimica Biologica) - Instituto de Bioquímica Médica /CCS / UFRJ, Conselho Nacional de Desenvolvimento Científico e Tecnológico. (Orientador). Luciana Wermelinger Serrão. Estudo do efeito de desintegrinas e ecotina na hemostase Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico.Orientador: Russolina Benedeta Zingali. Sheila Albert dos Reis. Mecanismos relacionados ao disparo de apopose durante a infecção pelos vírus Cantagalo e vaccinia-ioc e estudo dos genes virais envolvidos no processo Tese (Doutorado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Clarissa Rosa de Almeida Damaso. AL 5: Master Jacqueline Santos Cruz. Isolamento e Identificação de Metabólitos Bioativos de Penicillium waksmanii Zalessky Dissertação (Mestrado em Química) - Instituto Militar de Engenharia, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Orientador: Jose Daniel Figueroa Villar Marcelle Souza Ferreira. Síntese de 2,4- diaminopirimidina-5-carboxialdeído para Preparação de Novos Compostos Heterocíclicos como Potenciais Antimalariais Dissertação (Mestrado em Química) - Instituto Militar de Engenharia, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Orientador: Jose Daniel Figueroa Villar Pedro Henrique Monteiro Torres. Estudo do Fragmento N-terminal da Endostatina por Modelagem e Dinâmica Molecular Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Orientador: Pedro Geraldo Pascutti Maurício Garcia de Souza Costa. Estudo Computacional de Interações entre a Heparina e Proteínas Envolvidas na Angiogênese Tumoral Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Pedro Geraldo Pascutti. Reinaldo de Oliveira Júnior. Dissociação molecular sob alta pressão hidrostática por simulação computacional Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Pedro Geraldo Pascutti. Tácio Vinício Amorim Fernandes. Estudo do Enovelamento de Proteínas por Dinâmica Molecular e Generalized Simulated Annealing Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Pedro Geraldo Pascutti. Doctoral Tatiana Santana Ribeiro. Síntese e Avaliação de Oximas como Antídotos para Intoxicação com Organofosforados Neurotóxicos Tese (Doutorado em Química) - Instituto Militar de Engenharia, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Jose Daniel Figueroa Villar. Magdalena Nascimento Rennó. Avaliação de Inibidores da Nucleosídeo Hidrolase de Leishmania donovani Tese (Doutorado em Química) - Instituto Militar de Engenharia, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Jose Daniel Figueroa Villar. Arlan da Silva Golçalves. Estudo da Reativação da Acetilcolinesterase Humana Inibida pelo Organofosforado Tabun, Através de Métodos Híbridos Cássico Quanto-Mecânicos Tese (Doutorado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Pedro Geraldo Pascutti. Paulo Ricardo Batista. Estudo da flexibilidade da Protease do HIV-1 por Modelagem e Dinâmica Molecular, Análise dos modos normais e dos modos consensus Tese (Doutorado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Pedro Geraldo Pascutti. Gabriel Limaverde Soares Costa Sousa. Estudo Estrutural das Proteínas Antiangiogênicas Endostatina e Anastelina e Potenciais Implicações na Terapia contra o Câncer Tese (Doutorado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Pedro Geraldo Pascutti Diego Enry Barreto Gomes. Modelos de Confinamento para Organofósforo Hidrolase em Nanoestruturas Tese (Doutorado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Orientador: Pedro Geraldo Pascutti 147

148 Rafael de Cássio Bernardi. Estrutura e Dinâmica de Anestésicos Locais em Biomembranas Tese (Doutorado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Orientador: Pedro Geraldo Pascutti AL 6: Master Angela Menegatti. Análise proteômica de micoplasmas Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Santa Catarina, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Hernan Francisco Terenzi. Carolina Tavares. Proteômica de micoplasmas Dissertação (Mestrado em Pós Graduação Bioquímica) - Universidade Federal de Santa Catarina, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Hernan Francisco Terenzi. Franciele Luanne Fischer. Nucleases Químicas Dissertação (Mestrado em Quimica) - Universidade Federal de Santa Catarina,. Advisor: Hernan Francisco Terenzi. Jean Borges Bertoldo. Caracterização bioquímica de uma lipase recombinante de S. xylosus Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Santa Catarina,. Advisor: Hernan Francisco Terenzi. Doctoral Claus Troger Pich. Interação de complexos metálicos com ácidos nucléicos f. Tese (Doutorado em Biotecnologia) - Universidade Federal de Santa Catarina,. Advisor: Hernan Francisco Terenzi. AL 7: Master Paula Fernanda Lacarini Borin. Caracterização estrutural de uma proteína hipotética (XACb0033) da bactéria Xanthomonas axonopodis pv. citri Dissertação (Mestrado em Química) - Universidade Estadual de Campinas,. Advisor: Ljubica Tasic. Doctoral AL 8: Master Sabrina Gondim Ribeiro Mota. Ensaio in vitro e análise quimiométrica de inibidores da enzima lanosterol 14alfa-desmetilase de Moliniophora perniciosa de moniliophtora perniciosa Dissertação (Mestrado em Biotecnologia) - Universidade Estadual de Feira de Santana, Fundação de Amparo à Pesquisa do Estado da Bahia. Advisor: Marcelo Santos Castilho. Alessandra Gomes Marques Pacheco Advisor: Marcelo Santos Castilho. AL 9: Master Paulo Roberto Gonçalves de Freitas Junior. Efeitos da Miltefosina na Proliferação, Ultraestrutura e Biossíntese Fosfolipídica de Crithidia deanei, um tripanosomatídeo que contém endosimbionte Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Maria Cristina Machado Motta. Iamara da Silva Andrade. Caracterização de uma Proteína do endossimbionte de Crithidia deanei semelhante à porinas bacterianas: a origem procariota da membrane externa Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Maria Cristina Machado Motta. AL 10: Master Patrícia Machado de Barros. Gerência da Execução de Workflows Científicos de Bioinformática em Ambientes Distribuídos Dissertação (Mestrado em Engenharia de Sistemas e Computação) - Universidade Federal do Rio de Janeiro. Co-Advisor: Paulo Mascarello Bisch. AL 11: Master Roberta Ferreira Cura das Neves intitulada Dinâmica de componentes da superfície celular de tripomastigotas do Trypanosoma cruzi pertencentes à cepa Y ao clone CL-Brener do Curso de Pós-graduação em Microbiologia, IMPPG, UFRJ (bolsa CAPES). Destino: Programa de Doutorado em Ciências (Microbiologia) Thaís Souza Silveira intitulada Predação e digestão da bactéria magnetotática Candidatus Magnetoglobus multicellularis pelo ciliado Euplotes vannus do Curso de Pós-graduação em Microbiologia, IMPPG, UFRJ (bolsa CAPES). Destino: Programa de Doutorado em Ciências (Microbiologia) Anne Cristine Silva Fernandes intitulada Papel da fosfolipase A2 cálcio independente nas vias endocítica e exocítica de Leishmania amazonensis do Curso de Pósgraduação em Microbiologia, IMPPG, UFRJ (bolsa CNPq). Destino: Programa de Doutorado em Ciências (Microbiologia). Fernando Pereira de Almeida. Estudo do comportamento da bactéria magnetotáctica Candidatus Magnetoglobus multicellularis sob campo magnético aplicado Dissertação (Mestrado em Ciências (Microbiologia)) - Universidade Federal do Rio de Janeiro,. Co-Advisor: Ulysses Garcia Casado Lins. Tais Hanae Kasai Brunswick. Caracterização morfofuncional das células de medula óssea de pacientes submetidos à terapia celular Dissertação (Mestrado em Ciencias Biologicas) - Instituto de Biofisica Carlos Chagas Filho. Advisor: Antonio Carlos Campos de Carvalho. Thaís Souza Silveira. Predação e digestão da bactéria magnetotática Candidatus Magnetoglobus multicellularis pelo ciliado Euplotes vannus Dissertação (Mestrado em Ciências (Microbiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Ulysses Garcia Casado Lins. Doctoral Tatiana Pinotti do Curso de Pós-graduação em Microbiologia, intitulada Diversidade de leveduras endofíticas em plantas de agricultura orgânica (Seropédica RJ) e sua produção de micocinas e proteases do Curso de Pós-graduação em Microbiologia, UFRJ (bolsa CAPES parcial) ; Camila Marques Adade intitulada Avaliação dos efeitos do veneno (total e frações) da serpente Crotalus viridis viridis sobre a morfologia, proliferação e infectividade do Trypanosoma cruzi do Curso de Pósgraduação em Microbiologia, UFRJ (bolsa CAPES/CNPq). Fernanda de Ávila Abreu. Bactérias magnetotáticas em ambientes extremos Tese (Doutorado em Ciências (Microbiologia)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de 148

149 Desenvolvimento Científico e Tecnológico. Advisor: Ulysses Garcia Casado Lins. Júlia Peixoto de Albuquerque. Caracterização morfológica da população de bactéias oxidantes de enxofre dos gêneros Beggiatoa Tese (Doutorado em Ciências (Microbiologia)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Ulysses Garcia Casado Lins. Destino: Programa de Pós-Doutorado FIOCRUZ AL 12: Master Ivone Rosa de Andrade. Caracterização de proteínas do Complexo de Golgi de Tritrichomonas foetus Dissertação (Mestrado em Ciências Morfológicas) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Marlene Benchimol. Antonio Pereira das Neves-Neto Victor do Valle Midlej Ricardo Vilela AL 13: This new group exists for only two years and is now forming the first s students. AL 14: Master Davi Marcos de Souza Oliveira. Leihmaniose visceral no município de Barcarena-PA: inluência das ações antrópicas e urbanização do vetor Dissertação (Mestrado em Biologia de agentes Infecciosos e Parasitários) - Universidade Federal do Pará,. Advisor: Edilene Oliveira da Silva. Ana Paula Drummond Rodrigues AL 15: Master Amanda Karolina Soares Silva. Avaliação do tratamento com pioglitazona sobre o endotélio de camundongos C57BL6/J submetidos a dietas com diferentes tipos de ácidos graxos Dissertação (Ciências Biológicas) - Universidade Federal de Pernambuco Sura Wanessa Santos Rocha. Efeito da dietilcarbamazina (DEC) sobre hepatócitos de camundongos normais, desnutridos e expostos ao etanol Dissertação (Ciências Biológicas) - Universidade Federal de Pernambuco Juliana Falcão de Araújo Lima. Identificação de diferenças entre cepas de Mycobacterium spp utilizando métodos moleculares para o alvo 16S e 23S Dissertação (Ciências Biológicas) - Universidade Federal de Pernambuco Mariana Aragão Matos Donato. Avaliação dos efeitos do inibidor de fosfodiesterase-5 (sildenafil) sobre a ovogenese de camundongos Dissertação (Ciências Biológicas) - Universidade Federal de Pernambuco Bruna Santos da Silva. Caracterização dos efeitos da Dietilcarbamazina (DEC) sobre a Ovogênese de Camundongos Dissertação (Ciências Biológicas) - Universidade Federal de Pernambuco Tiago Bento de Oliveira. Novas tiazolidinadionas: síntese, caracterização estrutural, modelagem molecular e avaliação da atividade antiinflamatória Dissertação (Ciências Farmacêuticas) - Universidade Federal de Pernambuco (co-advisor) Doctoral Dilênia de Oliveira Cipriano Torres. Influência da dieta materena sobre o processo inflamatório, estresse oxidativoe disfunção endotelial em filhotes de camundongos Tese (Ciências Biológicas) - Universidade Federal de Pernambuco Karina Lidianne Alcântara Saraiva. tratamento de camundongos pré-púberes e adultos com inibidor da fosfodiesterase-5 e avaliação de seus efeitos sobre a a espermatogênese Tese (Saúde Pública) - Centro de Pesquisas Aggeu Magalhães AL 16: Master Luiza de Lima e Silva Bagno. Estudo da função cardíaca no transplante de células progenitoras de tecido adiposo em ratos com infarto cicatrizado Dissertação (Mestrado em Ciencias Biologicas) - Instituto de Biofisica Carlos Chagas Filho, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Antonio Carlos Campos de Carvalho. Leandro Vairo. Células Tronco Embrionárias: Eletrofisiologia, Acoplamento juncional e Potencial cardiogênico Dissertação (Mestrado em Fisiologia) - Instituto de biofísica Carlos Chagas Filho, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Regina Coeli dos Santos Goldenberg. Débora Bastos Mello. Interação do Trypanosoma cruzi com Células Progenitoras Cardíacas: Uma análise da Apoptose e do Ciclo Celular Dissertação (Mestrado em Ciências Biológicas (Fisiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Regina Coeli dos Santos Goldenberg. Ricardo Alexandre de Morais Brandólis. Modulação autonômica cardiovascular no modelo experimental de obesidade induzida por glutamato monossódico em ratos Dissertação (Mestrado em Patologia) - Universidade Federal do Triângulo Mineiro,. Advisor: Valdo Jose Dias da Silva. Estela de Oliveira Lima. Células Progenitoras Hemetopoiéticas no Modelo Experimental de Doença de Chagas em Camundongos Dissertação (Mestrado em Medicina Tropical e Infectologia) - Universidade Federal do Triângulo Mineiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Valdo Jose Dias da Silva. Doctoral Ricardo Luiz de Azevedo Pereira. Avaliação do inibidor de cisteína proteases E64 na diferenciação de células-tronco embrionárias em células neurais Tese (Doutorado em Fisiologia) - Instituto de Biofisica Carlos Chagas Filho, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Antonio Carlos Campos de Carvalho. Elizabete Nogueira Januário. Efeitos da Estimulação Colinérgica com Brometo de Piridostigmina sobre a Modulação Autonômica Cardiovascular em Ratos com Insuficiência Cardíaca decorrente do Infarto do Miocárdio Tese (Doutorado em Patologia) - Universidade Federal do Triângulo Mineiro, Advisor: Valdo Jose Dias da Silva. Maria de Lourdes Borges. Efeitos Crônicos da Amiodarona sobre os Reflexos Cardiovasculares em Ratos Normotensos e Espontaneamente Hipertensos Tese (Doutorado em Patologia) - Universidade Federal do Triângulo Mineiro,. Advisor: Valdo Jose Dias da Silva. AL 17: Master Ricardo Luiz Luzardo Filho. Mecanismos moleculares das consequências da programação metabólica sobre os transportadores renais de Na Dissertação 149

150 (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Adalberto Ramon Vieyra. Sharon Landgraf Schlup. Mecanismos moleculares envolvidos no desenvolvimento da Hipertensão primária Dissertação (Mestrado em Ciências Biológicas (Fisiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Celso Caruso Neves. Rafael Soares Lindoso. Respostas desencadeadas nas células renais em decorrência da co-cultura com células derivadas da medula óssea Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Marcelo Einicker Lamas. Claudia Fernanda Dick. Influência do fosfato inorgânico extracelular nas atividades ecto-enzimáticas de Trypanosoma rangeli Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Jose Roberto Meyer Fernandes. - Karine da Silva Verdoorn (2009) Fernanda Magalhães Ferrão (2010) Vanessa da Silva Baldez (2010). Luzia da Silva Sampaio (2010) Doctoral Tina Kiffer Moreiira. Ecto-fosfatase e ecto- ATPdifosfohidrolase em Candida parapsilosis: caracterização bioquímica e possível envolvimento na virulência e aquisição de adenosina Tese (Doutorado em Química Biológica) - Instituto de Bioquímica Médica, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Jose Roberto Meyer Fernandes. AL 18: Master Aline Miranda Scovine. Inflamação gengival causada pela bactéria PORPHYROMONAS GINGIVALIS: O papel do receptor TLR2 e do sistema cininas nos mecanismos de indução de imunidade adaptativa Dissertação (Mestrado em Ciencia Biológicas) - Instituto de biofísica Carlos Chagas Filho, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Julio Scharfstein. Larissa Nogueira de Almeida. Doctoral Ilka Maria Bakker Coelho de Abreu. Mecanismo de Invasão Tissular no Câncer de prostata Tese (Ciências Biológicas) - Instituto de Biofísica Carlos Chagas Filho UFRJ (Co-orientação com a Profa Christiane Bandeira de Mello) AL 19: Master Mariana Godoy. Efeitos da restrição proteica sobre a neurogenese de ratos Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Fundação Carlos Chagas Filho de Amparo à Pesq. do Estado do Rio de Janeiro. Co-Advisor: Rosalia Mendez-Otero. Michelle Bargas Rega. Analise da expressao de gangliosideos em modelo animal de lesao cerebelar Dissertação (Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro- Valeria Battistella Amado dos Santos. Seguranca do transplante autologo de celulas mononucleares da medula ossea em pacientes com acidente vascular cerebral isquemico subagudo Dissertação (Mestrado em Clínica Médica) - Universidade Federal do Rio de Janeiro,. Co-Advisor: Rosalia Mendez-Otero. Rafael de Castro Martins. Valor do PET/CT como preditor de câncer em nódulos pulmonares Dissertação (Mestrado em Medicina (Radiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Léa Mirian Barbosa da Fonseca. Maria Verônica Fonseca Torres de Oliveira. Desenvolvimento da marcação da doxorrubicina com Tc- 99m - Estudos " in vitro' e " in vivo" Dissertação (Mestrado em Medicina (Radiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Bianca Gutfilen. Andréia de Vasconcelos dos Santos. Janela terapêutica para administração de células da medula óssea, em um modelo pré-clinico de acidente vascular encefálico isquêmico Dissertação (Mestrado em Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Co-Advisor: Arthur Giraldi Guimarães. Doctoral Camila Zaverucha do Valle. Potencial Terapeutico das celulas de medula ossea na regeneracao do nervo optico Tese (Doutorado em Ciências Biológicas (Fisiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Rosalia Mendez-Otero. Pedro Moreno Pimentel Coelho. Avaliação do papel de células da fração mononuclear do sangue do cordão umbilical humano no sistema nervoso central de ratos submetidos a hipóxia-isquemia-neonatal Tese (Ciências Morfológicas) - Universidade Federal do Rio de Janeiro Fernanda de Mello e Souza Valente Gubert. Terapia celular após isquemia cerebral modula diferenciação de células do tipo glia radial em ratos adultos Tese (Ciencias Biologicas - Fisiologia) - Universidade Federal do Rio de Janeiro. Ricardo Luiz de Azevedo Pereira. Avaliação do inibidor de cisteína proteases E64 na diferenciação de células-tronco embrionárias em células neurais Tese (Ciências Biológicas (Biofísica)) - Universidade Federal do Rio de Janeiro. Flávia Maria de Souza Clímaco. Identificação do linfonodo sentinela no câncer de mama com injeção profunda de radiofármaco Tese (Doutorado em Medicina (Radiologia)) - Universidade Federal do Rio de Janeiro,. Advisor: Léa Mirian Barbosa da Fonseca. Maria Carolina Pinheiro Pessoa. Avaliação da Neurotransmissão Adrenérgica Cardíaca com Metaiodobenzilguanidina-123I na doença de Chagas Tese (Doutorado em Medicina (Radiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Léa Mirian Barbosa da Fonseca. AL 20: Master Joao Paulo Costa Pinho. Estudos sobre as funções mitocondriais no músculo de vôo do inseto Rhodnius prolixus. Início: Dissertação (Mestrado em quimica biologica) - Universidade federal do rio de janeiro. (Advisor Fernando Bozza). Natália Vasconcelos Casquilho. POTENCIAL TERAPÊUTICO DE LASSBio-596 VIA ORAL EM 150

151 CAMUNDONGOS INTOXICADOS POR MICROCISTINA-LR. Início: Dissertação (Mestrado em Ciências Biológicas (Fisiologia)) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. (Advisor Fernando Bozza). Patrícia Duque Estrada Jacintho. Efeito da pressão positiva expiratória nas vias aéreas na arritmia sinusal respiratória Dissertação (Mestrado em Engenharia Biomédica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Alysson Roncally Silva Carvalho. Ana Caroline de Paiva Gandara. Caracterização da produção de espécies reativas no trato digestivo do barbeiro Rhodnius prolixus Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Co-Advisor: Marcus Fernandes de Oliveira. Camila Alves Fernandes. Caracterização da Pressão Arterial de Oxigênio em Ventilação Mecânica: Influência da Pressão Positiva Expiratória Final, Volume Corrente e Freqüência Respiratória Dissertação (Mestrado em Engenharia Biomédica) - Universidade Federal do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico. Advisor: Frederico Caetano Jandre de Assis Tavares. Patricia Vieira de Souza Rocha. Efeitos dos atrasos, filtros e modelos de estimação sobre um índice de distensão pulmonar de pacientes ventilados mecanicamente Dissertação (Mestrado em Engenharia Biomédica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Frederico Caetano Jandre de Assis Tavares. Kathleen Silva Gonçalves. Secreção de metaloproteinase-9 (MMP-9) de matriz em macrófagos murinos induzida por heme: Envolvimento do estresse oxidativo e possíveis implicações no processo inflamatório Dissertação (Mestrado em Química Biológica) - Universidade Federal do Rio de Janeiro,. Advisor: Aurélio Vicente Graça de Souza. Doctoral Andre Miguel Japiassu. Resposta Inflamatória e Disfunção Mitocondrial em Pacientes com Choque Séptico Tese (Doutorado em Biologia Celular e Molecular) - Instituto Oswaldo Cruz. Advisor: Fernando Augusto Bozza. Rosana Souza Rodrigues. Imagem Molecular da Sindrome de Angústia Respiratória Aguda: Estudo Clínico e Experimental Tese (Doutorado em Medicina (Radiologia)) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Fernando Augusto Bozza. Christine Cruz Oliveira. Degradação de heme e inflamação: Estudos sobre o papel inibitório da biliverdina na migração de leucócitos Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Advisor: Aurélio Vicente Graça de Souza. Diego S Menezes. Modulação do metabolismo redox em tripanossomatideos como novas ferramentas quimioterápicas. Início: Tese (Doutorado em Biotecnologia) - Instituto Gonçalo Moniz. (Co-Advisor Oliveira MF). Joana da Costa Pinto d Avila. Estudos sobre as disfuncoes mitocondriais em modelos de sepse Tese (Doutorado em Química Biológica) - Universidade Federal do Rio de Janeiro. Advisor: Marcus Fernandes de Oliveira. 151

152 152

153 Science Education and Outreach Activities The National Institute of Science and Technology for Structural Biology and Bioimaging (INBEB) have different education programs related to the basic primary school. These programs involve not only the students, but also, the teachers of the basic school. In this regard, the main objective is to contribute to update teachers knowledge in different scientific topics. Video entitled "Influenza: Knowing the image and structure of viruses" This video, which was made by the INBEB, sought to disclose to the public how research about the influenza virus is conducted. This included research on infection on the common flu, swine flu (A H1N1), and avian influenza. This was the first popular science video that was produced by the INBEB. The story is about a sixth year student from the Colegio Pedro II, João Paulo, who needs to perform a science project at home. He turns to the researchers at the UFRJ, who are affiliated with INBEB, for more information about the influenza virus. Due to the boy s curiosity, various aspects of biomedical research related to structural biology and bioimaging are discussed. Influenza: Knowing the image and structure of the virus is a video story with both fictional and real characters. We wanted a child to participate in the video, and we remembered that João Paulo had been highlighted in A Vacation Course for Science, which is taught by some teachers at our institute. With a duration of 33 minutes, in addition to presenting accurate scientific information about influenza viruses, the video also seeks to increase children s interest in science. The video was launched on November 25, 2009 at the Forum for Science and Culture at the Universidade Federal do Rio de Janeiro. AL07, Responsible Prof. Carlos Ramos: A lecture was given, followed by a visit to UNICAMP, by high school students from the region of Campinas, Brazil. A book Chapter dedicated to Science education: Ronaldo A. Pilli & Carlos H. I. Ramos (2010). Chapter: Ser Humano e saúde. 153

154 Book: Fundamentos de Ciências II. Curso de Especialização em Ensino de Ciências e Matemática da UNICAMP. AL16, Responsible Prof. Antonio Carlos C. Carvalho: Creation of a video about INCT. He organized lectures that were delivered at the Fourth International Symposium on Advanced Therapies and Stem Cells (Recife), a CABI course on "Stem cells: from bench to bedside" (Buenos Aires), and the International Symposium on Stem Cell Research (Buenos Aires). He was involved in the development of a web page for the National Network of Cell Therapy (www.rntc.org.br). Conferences at the XXIV Annual Meeting of the Federação de Sociedades de Biologia Experimental (Federation of Experimental Biology Societies), FESBE, the Institute of Biological Sciences UFRJ and FIOCRUZ - RJ and the XXXIII International Congress of Protistology / XXV Annual Meeting of the Brazilian Society of Protozoology / XXXVI Annual Meeting on Basic Research in Chagas Disease, the round table discussion on cell therapy in the treatment of Chagas Disease. Additionally, a talk entitled "Stem Cell Therapy for Systemic Arterial Hypertension" was presented at the Fourth "Frontiers of Physiological Sciences" Thematic Symposium of the Department of Physiology and Biophysics-ICB/USP in August 2009, São Paulo, SP. AL11, Responsible Prof. Thais Souto Padrón: In November 2009, a course was offered for students in public schools in which she demonstrated methods that are used to isolate and characterize microorganisms and the importance of microorganisms in industry, medicine, and other fields. This course was based upon the standards set by the Institute of Microbiology for medical, pharmacy, biology, nursing, nutrition, and dentistry courses as well as the microbiology baccalaureate course. 154

155 INBEB has consolidated the group on scientific public diffusion and education. In 2010, the group of scientific public diffusion and education had the first approved grant from FAPERJ (coordinated by Prof. Emiliano Medei). The Project entitled Encontro marcado: estudantes do ensino médio e cientistas debatem células-tronco ao vivo e em vídeo (or Meeting scheduled: high school students and scientists debate stem cells in vivo and in video") was one of the 45 approved grants (among 130 applications to FAPERJ). In this project, a multidisciplinary group of scientists and students (most of them working on stem cells) as well as journalists aimed to establish interaction with high school and basic students through lectures in the schools, visits to the labs, and the development of short courses in the research laboratories of UFRJ. The meetings aimed to establish debates with the students and their teachers. All of the activities are iterative and aim to identify the questions and doubts of the young students. All activities are filmed, and the videos are used for discussion with the target students as well as by the researchers to plan future activities. The filmographic material may also be used for producing specialized videos for Science Dissemination. Thus, the audiovisual material will try to address scientific and ethical issues of interest to the target students and the themes that are most relevant to the reality of these young people. These initiatives aim to refresh and stimulate the critical thinking skills of our youth, bring them closer to the university and to stimulate their interest in scientific knowledge. The goal is to show them, through knowledge on stem cells, that biomedical sciences go far beyond names and technical terms found in textbooks. The 1st Meeting of INBEB with the middle school was a success. The team from INBEB developed three activities with the students of the State School José Martins da Costa. State school José Martins da Costa 155

156 This school is located about 180km from the Federal University of Rio de Janeiro, in São Pedro da Serra, Municipio de Friburgo Rio de Janeiro. The first day of activity occurred on September 27, when Prof. Emiliano Medei from the Institute of Biophysics and his students Barbara Guerra, Josuel Lessa (both trained in biology) and Luciana Brandão Nascimento (graduate of performing arts) visited the school. The group spoke to young people about stem cells, their uses and applications in various areas of scientific research in Brazil, such as in the use of transgenic animals for research and development of new therapies for various human and animal diseases. After the first meeting between researchers and students in the school, they were invited to visit the University at July 10 and September 11. The students had presentations and discussions with researchers from the INBEB. Then they were shown around four laboratories: The National Center of Bioimaging (Cenabio), the National Center for Nuclear Magnetic Resonance Jiri Jonas (CNRM), the Cardiac Electrophysiology Laboratory and the Laboratory of Molecular and Cellular Cardiology. Then the students were able to closely monitor how the research projects are conducted within the laboratories. Students at CNRMN Laboratory Professor Emiliano Medei at the school 156

157 All of them made a lot of question to the researchers in every Laboratory they have visited, as: The hormones that act in the cardiovascular system could help to grow the rat moustache? In addition, the questions made by the student in the school and in the University will help us to develop an educational audiovisual material (video), as we made previously in the Video entitled "Influenza: Knowing the image and structure of viruses". The activities taken together help to stimulate students' critical thinking regarding the scientific topics covered and at the end of each day's activities, the group asked the students to answer evaluation questionnaires. Thus, the students feedback is one of the more important parameters to evaluate our goals. So far, all students positively evaluated the meetings, which considered the important and interesting topics discussed. Most had heard about stem cells, but knew little about them (66%), had never personally known a scientist (66%), and had never visited a university (68%) or a research lab (77% ). In addition, 71% said the activity at the school made them feel better about science. 157

158 On the other hand, the results obtained with the present education program will be carefully analysed and spread in the form of scientific publications and the presentation in scientific events. Students and the INBEB team in front of CENABIO s biulding. 158

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