Activities Reports - INCT

Transcrição

ANNUAL REPORT
2009 – 2010
INBEB
Instituto Nacional de Ciência e Tecnologia de
Biologia Estrutural e Bioimagem
SILVA, JERSON LIMA; MEDEI, EMILIANO; VERJOVSKY,
MARINA; TOVAR-MOLL, FERNANDA.
ANNUAL REPORT 2009-2010 – 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, 2010.
160 F. : IL.
ANNUAL REPORT 2009-2010 - UNIVERSIDADE
FEDERAL DO RIO DE JANEIRO, INSTITUTO NACIONAL DE
CIÊNCIA E TECNOLOGIA DE BIOLOGIA ESTRUTURAL E
BIOIMAGEM (INBEB), 2010.
1. 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 2009-2010
– INBEB (INSTITUTO NACIONAL DE CIÊNCIA E
TECNOLOGIA DE BIOLOGIA ESTRUTURAL E BIOIMAGEM
INBEB 2009-2010 INBEB 2009-2010 ANNUAL REPORT
2
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
SUMMARY
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.
Science Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Cooperative and educational activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Perspectives and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
4
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
1
2
3
1. Pará
Universidade Federal do Pará (UFPA).
2. Pernambuco
Universidade Federal de Pernambuco (UFPE, PE);
Centro de Pesquisas Aggeu Magalhães (CPQAG FIOCRUZ);
Centro de Tecnologias Estratégicas do Nordeste
(CETENE).
3. Bahia
Universidade Federal da Bahia (UFBA, BA).
4
5
6
7
4. 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
INBEBCatarina
2009-2010 INBEB 2009-2010 ANNUAL REPORT
Universidade Federal de Santa Catarina (UFSC, SC).
6
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
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
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
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
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
was
microscope
purchased
INBEB
(Figure
with
with
resources
3).
the
Together
electron
microscope (ME 200
KV
with
cryo-
tomography),
which
was
UFRJ
purchased
with
Proinfra
resources, it increased
Figure 3: Environmental Scanning Electron Microscope
our ability to perform
complex microscopy experiments.
11
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
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 2010. 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).
Other
previously
acquired
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
backup
generator
power
supplies
and
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
Anatomic images of high spacial resolution
(0.1 x 0.1 x 1.0 mm3)
RM 7.0T
Paiva et al., 2007
Figure 8: MR Image (7 T) of a Mouse Brain.
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
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 2nd Annual Meeting of INBEB in November
2010. 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
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
2. SCIENCE
HIGHLIGHTS
17
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
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
biophysical
(high
pressure)
and
structural
Using structural and cellular
biology tools, we have found that changes in
biology approaches, we have investigated the
hydration and cavity distribution in the interior
interaction of the prion protein with biological
of the proteins play a key role in different
ligands,
biological processes such as viral membrane
glycosaminoglycans, the co-chaperone hop/STI1
fusion, prion conversion and loss of function of
and copper, and their participation in the
the tumor suppressor protein. In the case of prion
conversion of PrP to the scrapie isoform (Figure
protein, involved in transmissible spongiform
1). Moreover, although several compounds have
encephalopathies, the prion protein becomes less
been evaluated for their ability to inhibit this
hydrated when converts to the aggregated,
conversion, to date there is no effective therapy
scrapie-like isoform and we have demonstrated
for the prevention or reduction of the disease
such
as
nucleic
acids,
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
PrPC 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 (PrPC), an alpha-helical
rich protein, is converted into an infectious
misfolded isoform. This isoform, the scrapie PrP
(PrPSc),
forms
aggregates,
leading
to
neurodegeneration. It has been proposed that the
spontaneous conversion from PrPC to PrPSc 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. PrPC (left)
can misfold into an isoform rich in beta sheet structure capable of
forming toxic and infectious aggregates (PrPSc) (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 PrPSc. PrPC 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 (PrPC) and rPrP (PrPSc-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
inhibition assays. Our results show that two of
amyloid formation might participate in the
the selected compounds can significantly inhibit
malignant process. Aggregation of p53 would act
the aggregation of this prion protein hydrophobic
as a sink to sequester native protein into the
domain, through light scattering, thioflavin-T
inactive conformation, replicating the structural
binding
electron
information, very much like a prion (Figure 2).
microscopy. Moreover, we verified that these
The search for molecules that preclude the
aminoquinolines are not toxic to neuroblastoma
formation of the misfolded conformation, which
cells in culture. Therefore, such aminoquinolines
may ultimately lead to the prevention of tumor
might be considered as candidates for the further
development, is a major goal in cancer research.
development of therapeutics to prevent the
We have used aptameric nucleic acids as an
development of prion diseases. We also found
alternative to prevent aggregation and to rescue
that murine recombinant PrP 23-231 interacts
activity (Figure 2). A more stable variant of p53
with low-molecular-weight heparin (LMWHep)
would shift the equilibrium toward the soluble
at pH 7.4 and 5.5. The soluble complex of
and active form of the protein. Because nucleic
LMWHep-rPrP
aggregation
acids are susceptible to various modifications
induced by RNA, which raises the possibility to
(such as phosphorothioate), which alter in vivo
use glycosaminoglycans in anti-scrapie therapy.
processing
assays
and
is
transmission
resistant
to
without
affecting
target
discrimination, our study paves the way for the
2. Structure and function of the tumor
suppressor protein p53 and its association
with
malignant
mutation
is
the
tumors.
most
application of aptameric nucleic acids in cancer.
To
search
for
correlation
TP53
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
conformation
the
altered
results
in
p53
protein
aggregation.
Evidence
that
the
three
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
as measured by fluorescence: p53 (blue circles), consensus-bound p53 (red
aggregates is quite striking, making binding
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
21model 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.
between p53 aggregation and malignancy, we
3.
Targeting
Viruses
of
Medical
analyzed 88 biopsies from patients residing in
Importance. In the last two years, our group has
the metropolitan area of Rio de Janeiro, and
used as study models several animal and human
performed TP53 mutation screening using direct
viruses of great medical importance, such as
sequencing of exons 5 to 10. Seventeen
Hepatitis C (HCV), Yellow Fever (YFV),
mutations were detected, 12 of them were of
Dengue (DENV), Mayaro (MAYV), Ebola and
missense type, 2 nonsenses, 2 deletions and 1
Influenza viruses, since there is an urgent need to
insertion. The presence of TP53 mutation was
develop new approaches to prevent and treat the
highly
tumor
diseases caused by these viruses. The overall
aggressiveness of IDC cases, indicated here by
proposal of our group is to study the problem of
Elston Grade III (p < 0.0001). Paraffin embedded
macromolecular recognition, using different
breast cancer tissues were analyzed for the
variables
presence
through
temperature and chemical denaturants, and
immunofluorescence co-localization assay, using
employing various structural and spectroscopic
anti-aggregate primary antibody A11, and anti-
methods such as fluorescence spectroscopy,
p53. Our results show that mutant p53 co-
nuclear magnetic resonance, circular dichroism,
localizes with amyloid-like protein aggregates,
calorimetry, molecular dynamics simulations,
depending on mutation type, suggesting that
and new approaches and technologies of
mutant p53 may form aggregates in breast cancer
microscopy and fluorescence spectroscopy, such
cells, in vivo (Figure 3).
as multiphoton microscopy and fluorescence
statistically
of
p53
associated
aggregates
to
as
high
hydrostatic
pressure,
correlation
spectroscopy,
field
a
pioneered
by us in the state
of Rio de Janeiro.
The
use
pressure
of
to
inactivate
non-
enveloped
and
enveloped viruses
is being evaluated
to be used in the
preparation
of
vaccines, such as
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.
against
Yellow
Fever,
Dengue,
Foot-and-Mouth
disease virus and
22
Influenza (Figure 4). Most of the studies have the
explore the structural biology of HCV particles
Figure
4Pressure
inactivation
of
picornaviruses.
(A)
Structure
of
human
rhinovirus 14 (HRV14). (B)
Scheme
of
pressure
inactivation of HRV14.
participation
of
State
enabling a better understanding of the infection
Company in FIOCRUZ. As a result of interaction
cycle of this virus and thereby helping in the
with Biomanguinhos, we filed a patent entitled
development of new antiviral drugs. We have
―Method for Stabilized Vaccine Production‖.
analyzed the structure of a HCV fusion peptide
The present invention proposes to solve the
candidate (HCV421-445) and its interaction with
problem of lack of stability of polio vaccine
micelles and lipid membranes, besides the viral
strains (serotypes 1, 2 and 3) present in the
capsid assembly in vitro. Our results show that
attenuated
of
the peptide induces lipid vesicle aggregation and
hydrostatic pressure makes it possible to obtain
interacts with micelles in a process enthalpically
the stability of the three strains, indicating that
driven involving Trp residues. The peptide also
the addition of any chemical agent would
becomes structured, suggesting its participation
become totally unnecessary. The results were
in the entry process of HCV. We also show that
also published in the journal "Vaccine".
the assembly process is highly cooperative and
vaccine
Biomanguinhos,
(Sabin).
The
a
use
The Hepatitis C Virus, the leading cause
driven by the neutralization of basic residues.
of liver disease, chronically affects 3% of word
MAYV, YFV and DENV are viruses
population. HCV entry and assembly are
transmissible by mosquitoes, responsible for
complex
still
causing diseases of major global impact. The
unknown. So, the main aim of our work is to
principal objective of this work is to evaluate
processes
with many steps
23
virus-cell
interaction,
investigating
the
importance of cholesterol (Chol) and the fusion
induction of apoptosis by YFV occurs in a p53dependent manner.
peptide for infection, the participation of heparan
Influenza viruses are important threats to
sulfate (HS) for virus entry and the cellular
human healthy. Among different types, we work
mechanisms involved in the apoptosis process.
with H3N8, an avian Influenza virus, which was
Since there is no treatment for the infection with
originally isolated from birds, later found in
these viruses, the discovery of effective drugs to
horses. We study the stability, inactivation and
control these infections becomes an important
immunogenic capacity of H3N8 by using high
strategy and has an extreme scientific interest.
hydrostatic pressure (HHP) as main tool. Our
We have evaluated the importance of Chol and
present goal is to assess the immunogenic
its dependence to the membrane fusion process
capacity of these pressurized particles in murine
of DENV-2. We found that depletion of viral
model. The initial results are very promissing,
Chol promoted a significant reduction of
showing good humoral and mucosal answer. All
approximately 3 logs in viral titer.
our data reinforce the potential of HHP in the
It is known that membrane fusion occurs
development of vaccines against avian influenza
by exposure of a hydrophobic segment (FLA98-
with low cost, safety and promissing good
110),
immune response.
the fusion peptide (FP), present in the
glycoprotein
two
The challenge of elucidating the
conserved FPs, we found that the interaction
mechanisms of entry and assembly/disassembly
involved Trp residues independently of the pH
of the virus and cellular response involved in
and partially dependent on negative charge. We
infection may represent an advance in the
have also developed studies on the interaction of
rational development of vaccines and new
the Ebola fusion domain with target membranes
inhibitors based on structure, mode of interaction
by
and function of viral proteins. The group of Dr.
NMR,
of
flaviviruses.
atomic
force
Using
microscope
and
calorimetry.
Davis Ferreira has worked on the antiviral
Apoptosis is a response involved in
properties of new molecules against Flavi and
cytopathogenicity during infection by DENV and
Alphaviruses. More than twenty new molecules
YFV. Our results show that DENV and YFV
were tested, some with promising antiviral
induce high rate of cell death only after 120 h
activity. The group is also evaluating the profile
and 72 h of infection, respectively. The process
of vertebrate and invertebrate cells infected with
of programmed cell death was demonstrated by
Dengue
virus
by
Proteomics
different methods. We evaluated the loss of
Glycoproteomics,
in
collaboration.
integrity of 
collaboration
m
and the importance of opening
with
North
Carolina
and
In
State
the channel VDAC, suggesting the involvement
University, novel studies have been performed
of mitochondrial pathway in the apoptotic
on the structure of Dengue virus using cryo-
process. Our studies also demonstrate that the
electron microscopy.
24
Group publications (2009-2010):
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: 271-279.
2. Silva, JL, Gomes, MPB, Vieira, TCRG and
Cordeiro, Y (2010) Functional and Pathological Roles of
Prion-Nucleic Acid Interactions. Frontiers in Bioscience,
15: 132-150.
3. 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: 54685473.
4. 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: 2857–2866.
5. Cano-Estrada A, Vázquez-Acevedo M,
Villavicencio-Queijeiro A, Figueroa-Martínez F, MirandaAstudillo 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. 2010 1797(8):1439-48.
6. 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: 580-584.
7. 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: 99-108.
8. 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: 21050861).
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:
21056685).
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-1kinase association within the membrane of human
erythrocytes. Biochimie. 92(5):538-44. Epub 2010 Feb 6.
11. 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):576-8.
PubMed PMID: 20202456.
12. 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):4308-4316. Epub 2009
Aug 24.
16. Silva JL, Oliveira AC. (2009). Science and
technology to combat dengue virus. An Acad Bras Cienc.
81(4):631-632.
17. Silva, JL and Foguel, D (2009) Hydration,
cavities and volume in protein folding, aggregation and
amyloid assembly. Phys. Biol. 6: 15002 (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:
6126-6135.
19. 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: 6811-23.
20. 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: 5-6.
21. 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): 135-139.
22. 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: 162-168.
23. 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:
272–279.
24. 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: 255– 259.
25. 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): 5332-7.
25
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. 1-12.
2. 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. 13-26.
3. 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: 7615617; Issue date: Nov 10, 2009
2. 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).
WO2009111849 (A1)
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
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 βsheets 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 80years old and is characterized by heavy
Fig
1:
TTR
quaternary structure.
Each monomer is
shown in a different
color.
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
(CNSA). Among the variants of TTR, V30M
Five decades of evidence support the
and L55P are the most important because of
interaction of TTR with Zn2+ in the biological
their high frequency of occurrence and the
milieu
aggressiveness of the symptoms they evoke.
accompanying
but
nothing
is
structural
known
of
the
correlates.
The
2+
in the
individuals
are
A25T, on the other hand, is one of the few
observation that concentrations of Zn
TTR mutations associated with a rare type of
plasma
amyloidosis that is restricted to the CNS and is
approximately 12-15 μM, while the Zn2+:TTR
characterized by amyloid fibril deposition in
apparent dissociation constant (Kdapp) is 1 μM,
leptomeningeal and subarachnoid vessels.
suggests that TTR may circulate as a complex
T119M is a non amyloidogenic variant
with Zn2+ in plasma. High concentrations of
alleviating the symptoms of the V30M
Zn2+ and Cu2+ can trigger TTR amyloid
mutation.
formation in vitro, and chelating agents disrupt
of
healthy
Several hypotheses have been proposed
these amyloid structures. Recently, through the
to explain the amyloidogenic properties of
use of X-ray crystallography and NMR we
TTR. The most accepted one presupposes the
have shown that TTR presents three canonical
dissociation of the tetramers into a monomeric,
Zn2+-binding sites. Zn2+ binding induces
partially folded state, which is aggregation
structural perturbations that lead to TTR
prone. Our group has characterized an altered
aggregation and decreases its affinity for
tetramer of TTR (T4*, pink circles) which is
retinol binding protein one of its partner in the
aggregation prone (Fig. 2).
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.
Trapping the monomer of T119M for its use
as a therapeutic strategy against TTR
related amyloidoses:
Nowadays, FAP patients are treated
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:
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
Searching for T4* in a close to physiological
condition:
prevent TTR dissociation and aggregation
through
tetramer
stabilization
in
vitro.
28
Recently, our group has shown that a
The variant A25T of TTR is involved in
combination of high hydrostatic pressure
leptomeningeal
(HHP) with subdenaturing concentrations of
characterized by amyloid fibril deposition in
urea facilitates the dissociation of T119M. At 1
leptomeningeal and subarachnoid vessels.
o
Very little data are available in the literature
T119M irreversibly dissociates and denatures
regarding
in less than 30 min at 3,000 bar in a
pathways of this specific variant of TTR. Our
concentration-dependent
These
group has determined its x-ray structure in the
unfolded monomers (here called MT119M,U)
apo form and when bound to thyroxine - a
remain in solution as long as urea (4 M) is kept
TTR natural ligand-, and to flufenamic acid
in the buffer, but upon dilution of the urea (0.4
(FA), a NSAID. Different from what has been
M) at pH 7.0, they refold initially into folded
observed with other TTR variants, the structure
monomers (MT119M, F) and then slowly (hours)
of A25T showed profound alterations in
into native tetramers. T119M monomers
relation to the wt-TTR structure, not only in
presented a similar thermodynamic stability to
the vicinity of position 25, but spread
that of the wt monomer, suggesting that most
throughout the whole tetrameric structure. The
of the differences in stability between the
two ligands were able to correct part of the
tetramers of T119M and wt TTR can be
structural alterations caused by the mutation.
attributed to the inter-subunit contacts inside
Combining an array of techniques, we
the tetramers and not to the intra subunit
analyzed A25T aggregation kinetics in buffer
contacts of the monomers. Our data also show
at pH 5.0, 6.0 and 7.0, as well as at
that T119M monomers can be successfully
physiological environments such as human
incorporated into amyloidogenic tetramers,
plasma and CSF. AFM revealed that, at these
even when the exchange is performed in a
pH values, A25T formed aggregates with
more physiological environment, such as the
different morphologies and susceptibility to
plasma; these monomers render the resultant
proteinase K digestion. A25T was also able to
heterotetramers less amyloidogenic. The data
aggregate massively at CSF and in plasma
presented here are relevant for understanding
forming in the former case amyloid fibrils with
the T119M folding and association reactions
a peculiar morphology. FA and T4 blocked
and provide a protocol for producing T119M
completely the aggregation of A25T in CSF.
monomers that function as inhibitors of TTR
Through the use of high hydrostatic pressure,
aggregation when incorporated in tetramers;
we showed that A25T is 3.1 kcal/mol less
this protocol may be a new strategy for treating
stable than L55P. We also showed that
TTR diseases.
amyloid formation in plasma and CSF arrested
C (pH 5.0), in the presence of 4 M urea,
fashion.
the
amyloidosis,
folding
and
which
is
aggregation
several protein components present in these
Structural determination of A25T, the most
complex milieu.
amyloidogenic variant of TTR:
29
Cardiomiopathies
caused
by
Defective
Muscle Proteins (Sorenson’s laboratory):
human
cardiac
TnC
(E59D/D75Y)
successfully demonstrated that there are ~25%
The main focus of the muscle research
fewer sites available for cross-bridges to bind
group of AL2 is on defective proteins that lead
to the thin filament when the double mutant is
to cardiomyopathies, as well as associated
used to reconstitute the filaments in vitro.
mechanisms of action of normal contractile
These
and regulatory proteins.
fluorescently labeled thin filaments with
A
project
involving
human
results,
which
involved
titrating
cardiac
myosin heads, explain why there is a reduction
troponin C (TnC) mutants that are associated
in isometric force when skinned myocytes
with hypertrophic cardiomyopathies has led to
contain the double mutant (Dweck et al, 2010).
a characterization of Ca2+ off-rates from the
An
additional
project
led
to
regulatory Ca-binding sites I and II of TnC.
characterization of the importance of redox
Increasing complexity of the reconstituted
state for a cysteine residue located in the
system (from isolated TnC to the whole
central helix of skeletal muscle TnC. When
troponin complex to filaments reconstituted
this cysteine is fully reduced, TnC´s affinity
with actin and troponin to filaments containing
for the thin filament is greater than when it is
actin, troponin and myosin cross-bridges
oxidized. This means that redox state may
([subfragment
progressively
modify the ability of TnC to transmit messages
closer approximations of the increase in Ca2+
along its axis from the thin filament (bearing
sensitivity observed with intact skinned fibers
cross-bridges) up through the C-domain to the
containing
N-domain, where Ca regulation occurs (Pinto
1])
these
produced
mutants.
For
these
experiments our fast-kinetics capabilities were
et al, 2010).
combined with the fluorescently labeled
human cardiac mutants and other myofibrillar
Biological and Medicinal Chemistry of
proteins produced by our collaborators at Univ.
Infectious
of Miami and our doctoral student DP
(Mauricio’s Laboratory):
Reynaldo
performed
the
stopped-flow
and
Laboratory
Degenerative
for
Diseases
Pharmaceutical
experiments (Pinto et al, 2010). The data show
Biotechnology at the School of Pharmacy at
that cross-bridges contribute significantly to
UFRJ:
the cardiomyopathic phenotype, even though
Diabetes and amyloidosis – amylin is a
the mutation occurs in TnC and not in myosin.
peptide secreted by islet beta cells in
These same mutants (A8V, C84Y, D145E and
conjunction with insulin, involved in the
E134D) are under analysis in Rio for changes
regulation of several metabolic properties.
in the C-domain of TnC, a region normally
Therapeutic use of amylin is limited by its low
associated only with anchoring the protein on
water solubility and propensity to form
the thin filament.
amyloid deposits. We have developed a
A second project aimed at characterizing a
nanostructured confined polymeric system
dilated cardiomyopathy double mutation of
(NPhIAPP) from which amylin is sustained
30
and
controlled
Pharmacological
natural occurrence may be involved in
subcutaneous
physiological modulation of thrombin, acting
administration of NPhIAPP is able to reduce
as a molecular switch between a procoagulant
glycemia in mices for long periods. We believe
and inhibitory pathways.
evaluation
released.
shows
that
that amylin nanoconfinement in varying
Nucleic acid interference – nucleic acid
matrices is a promising approach for the
recognition is a key regulatory during all
therapeutic replacement of basal levels of
stages
amylin.
processes. This fenomena is usually performed
Coagulopathies – blood haemosthasis
by
of
large
life
and
molecular
physiopathological
protein
assemblies,
control affects several diseases. On of the most
comprising a nucleic acid binding and
important enzymes in the blood cascade is
recognition domain and one or more protein
thrombin. One of our projects focuses in the
regions responsible for interaction with other
characterization of thrombin ligands as lead
molecular partners. Besides the dimension of
compounds in the treatment of Coagulopathies.
the nucleic acid binding proteins, only a small
We have recently characterized the mechanism
segment is usually responsible for binding and
of interaction between thrombin and suramin, a
recognition. Moreover, specific recognition is
naphtyl sulfonated urea. Suramin interaction
in part driven by a recognition code between
with thrombin is mediated through anion
nucleic acid base and aminoacid side chain.
exosite II, in a dual mode: activation and
We have designed small peptides that target
inhibition. Crystallographic analysis revealed
cognate DNA sequences according to the
thrombin dimerization mediated by suramin,
recognition code, which proved to be highly
which was confirmed by small angle X-ray
selective for the specific DNA sequences.
analysis. A larger investigation with a series of
Further optimization in peptide stability and
suramin analogues of varying sizes and
derivatization are currently in development in
substituents in the methyl group of suramin
order
indicated
component
selectivity. The simplicity and easy of peptide
correlates with a dimerization event, whilst the
design brings us on step toward the specific
activation component is linked to direct
nucleic
binding of suramin to monomeric thrombin.
compatible biopharmaceuticals for the use in
We believe that other sulfonated compounds of
varying biotechnological needs.
that
the
inhibitory
1.Pinto JR, Reynaldo DP, Parvatiyar MS, Dweck
D, Liang JS, Jones MA, Sorenson MM, Potter JD (2010)
Strong crossbridges potentiate the Ca2+ 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
to
optimize
acid
binding
targeting
with
affinity
and
biologically
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):17371-17379.
4.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.
2010 Sep 29. doi:10.1016/j.abb.2010.09.022
5. The binding of synthetic triiodo l-thyronine
analogs to human transthyretin: Molecular basis of
cooperative and non-cooperative ligand recognition.
31
Trivella DB, Sairre MI, Foguel D, Lima LM, Polikarpov
I. J Struct Biol. 2010 ahead of print]PMID: 20937391
6. 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):31731-41.
7. 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):1917-24.
8. 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;11:22.
9. 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. 2010 170(3):522-31.
10. 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, MirandaAstudillo H, Cordeiro Y, Mignaco JA, Foguel D, Cardol P,
Lapaille M, Remacle C, Wilkens S, González-Halphen D.
Biochim Biophys Acta. 2010, 1797(8):1439-48.
11. Immunome and venome of Bothrops
jararacussu: a proteomic approach to study the molecular
immunology of snake toxins. Correa-Netto C, TeixeiraAraujo R, Aguiar AS, Melgarejo AR, De-Simone SG,
Soares MR, Foguel D, Zingali RB. Toxicon. 2010,
15;55(7):1222-35.
12. 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):100-10.
13. 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):271-9.
14. 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):4308-16.
15. 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):6811-23.
16. 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):1-13.
17. 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):951-63.
19. 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):1443-53.
20.
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. 091231124456033, 2010.
21.
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. 091221135411018, 2010.
22.
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, 2010.
23.
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, 2009.
24.
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,
2009.
25.
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. 6126-6135, 2009.
26.
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. 1138-1143, 2008.
32
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, 2010.
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
proteins
are
not
structured
in
a
unique
averaged structures. We have previously solved
conformation. Rather, they frequently have
the structure of the anti-coccidial peptide PW2 in
regions of conformational diversity.
the presence of SDS, DPC and other mimics of
Our group used a novel view of binding,
membrane environment. Here we applied a
put forward in by several research groups in the
diverse set of strategies of restrained molecular
last 5 to 10 years. In the free state, protein
dynamics simulations (MD) of PW2 in explicit
regions
diversity
DPC micelles. The MD runs enabled the
pre-existing
calculation of discrete conformational states,
displaying
exhibit
conformational
equilibria
among
conformations. In the presence of a ligand, one
dissociating
of these conformations is stabilized, so that the
averaging.
ligand does not need to induce a new
the
effects
Spectroscopic
of
conformational
characterization
of
a
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
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).
structure
determination is strongly influenced by the
Truncated hemoglobin from the nitrogen-
internal dynamics. Proteins and peptides display
fixing bacterium Herbaspirillum seropedicae
flexible regions that undergo motions that are
(Fábio C. L. Almeida)
to
The Herbaspirillum seropedicae genome
conformational diversity. The regions that
sequence encodes a truncated hemoglobin typical
display conformational diversity are frequently
of group II (Hs-trHb1) members of this family.
part of binding epitopes related to biological
We show that Histagged recombinant Hs-trHb1
important events. Nevertheless, these regions are
is monomeric in solution, and its optical
often challenging for the current methods for
spectrum resembles those of previously reported
structural determination, which leads to time-
globins. NMR analysis allowed us to assign
either
thermal
or
slower,
related
34
heme substituents. All data suggest that Hs-
In addition to its systemic activity,
trHb1 undergoes a transition from an aquomet
D(LPR) also inhibits retinal angiogenesis when
form in the ferric state to a hexacoordinate low-
administered in an eye-drop formulation. Finally,
spin form in the ferrous state. The close positions
in preliminary studies, we have showed targeted
of Ser-E7, Lys-E10, Tyr-B10, and His-CD1 in
drug activity in an experimental tumor-bearing
the distal pocket place them as candidates for
mouse model. These results show that drugs
heme
targeting
coordination
and
ligand
regulation.
extracellular
domains
of
VEGF
Peroxide degradation kinetics suggests an easy
receptors are active, affect signal transduction,
access to the heme pocket, as the protein offered
and have potential for clinical application. On a
no protection against peroxide degradation when
larger context, this study illustrates the power of
compared with free heme. The high solvent
ligand-directed selection plus retroinversion for
exposure of the heme may be due to the presence
rapid
of a flexible loop in the access pocket, as
(Giordano et al., 2010, patent)
drug
discovery
and
development.
suggested by a structural model obtained by
using homologous globins as templates. The
Backbone dynamics of the antifungal psd1 pea
truncated hemoglobin described here has unique
defensin and its correlation with membrane
features among truncated hemoglobins and may
interaction by NMR spectroscopy (Fábio C. L.
function in the facilitation of O2 transfer and
Almeida/Ana Paula Valente)
scavenging, playing an important role in the
Plant defensins are cysteine-rich cationic
nitrogen-fixation mechanism. (Razzera et al.,
peptides, components of the innate immune
2009)
system. The antifungal sensitivity of certain
exemplars was correlated to the level of complex
From combinatorial peptide selection to drug
glycosphingolipids in the membrane of fungi
prototype: targeting the vascular endothelial
strains. Psd1 is a 46 amino acid residue defensin
growth factor receptor pathway (Fábio C. L.
isolated from pea seeds which exhibit antifungal
Almeida)
activity. Its structure is characterized by the so-
Inhibition of blood vessel formation is a
called cysteine-stabilized alpha/beta motif linked
viable therapeutic approach in angiogenesis-
by three loops as determined by two-dimensional
dependent diseases. We previously used a
NMR. In the present work we explored the
combinatorial screening on vascular endothelial
measurement
growth factor (VEGF)-activated endothelial cells
Overhauser Effects, R1 and R2 (15)N relaxation
to select the sequence CPQPRPLC and showed
ratios, and chemical shift to probe the backbone
that the motif Arg-Pro-Leu targets VEGF
dynamics of Psd1 and its interaction with
receptor-1 and neuropilin-1. Here, we evaluated
membrane
and validated D(LPR), a derivative molecule
phosphatidylcholine
(PC)
with strong antiangiogenesis attributes. This
dodecylphosphocholine
(DPC)
prototype
glucosylceramide (CMH) isolated from Fusarium
drug
markedly
inhibits
of
heteronuclear
mimetic
systems
Nuclear
with
or
with
neovascularization in three mouse models.
solani. The calculated R2 values predicted a slow
35
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
properties in the
exchange
mus-ms
timescale
supporting the conformation selection as
the binding mechanism. Moreover, the
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.
peptide corresponding to part of Loop1
(pepLoop1: Gly12 to Ser19) is also able to
multiple motions may be related to transient
interact with DPC micelles acquiring a stable
twisting or breathing of the -helix and -sheet.
structure and in the presence of DPC:CMH the
The stages of membrane recognition and
peptide changes to an extended conformation,
disruption by Sd5 over a large time scale range
exhibiting NOE mainly with the carbohydrate
were mapped and demonstrated that Sd5 in
and ceramide parts of CMH.
solution sampled an ensemble of different
conformations, of which a subset is selected
Portrayal of complex dynamic properties of
upon membrane binding. Defensins share similar
Sugarcane defensin 5 by NMR: Multiple
structures, but we demonstrated here that their
motions
dynamics can be extremely diverse.
associated
with
membrane
interaction (Ana Paula Valente)
Defensins
natural
antibiotics
are
essentially
with
potent
ancient
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
Structure of SD5 by solution NMR
36
The structures of several important
Mapping the interactions between a major
pollen allergen and human IgE antibodies.
proteins
(Ana Paula Valente)
characterized as describe below.
The
interaction
of
specific
from
infectious
diseases
were
IgE
antibodies with allergens is a key event in the
Structure and mode of action of microplusin,
induction of allergic symptoms, thus representing
a copper II-chelating antimicrobial peptide
an important target for therapeutic interventions
from the cattle tick Rhipicephalus (Boophilus)
in Type I allergies. We report here the solution
microplus (José Ricardo M. Pires)
NMR structure of Art v 1, the major mugwort
Microplusin,
a
Rhipicephalus
pollen allergen. Art v 1 is the first protein
(Boophilus) microplus antimicrobial peptide
structure with an allergenic defensin fold linked
(AMP) is the first fully characterized member of
to a polyproline domain, which has not been
a new family of cysteine-rich AMPs with
identified in any reported allergen structure in
histidine-rich regions at the N and C termini. In
the PDB. Moreover, the direct interaction of
the tick, microplusin belongs to the arsenal of
polyclonal IgE antibodies from an allergic
innate defense molecules active against bacteria
patient has been mapped on the surface of an
and fungi. We described the NMR solution
allergen for the first time. Our data provide the
structure of microplusin and demonstrate that the
basis for the design of tools for safe and effective
protein binds copper II and iron II. Structured as
vaccination against mugwort pollen allergy.
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
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)
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
demonstrated
that
microplusin
affects
M.
luteus respiration, a copperdependent process. Thus,
we
conclude
antibacterial
that
the
effect
of
microplusin is due to its
ability to bind and sequester
copper II.
Microplusine structure in solution
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
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.
kinoplatids(KSC- Kinetoplastid-specific relatated
do calpains). KSC does not show similarity to
any protein with known function. We determined
Structure and function of Cancer-related
proteins (Prof. Marcius S. Almeida)
Representing an initiative in human
the solution structure of the T. cruzi (Access
code Unitprot: Q4CYU3) protein. This protein
work
contain only the domain KSC.
The
structure
is
cancer structural proteomics, the aim of this
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
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
Structure of domain KSC: 15 lowest energy structure.
of approximately 200 kDa, which would
38
embrace 14 subunits. Surface plasmon resonance
thiol/disulphide oxidoreductases and isomerases,
binding experiments using Biacore X system
and may thus reduce the endoplasmic reticulum
revealed binding and dissociation of BEX3:1-
stress caused by incorrectly folded proteins. We
106/p75-NTR with Kd of 1.4 uM (per BEX3
show for the first time the production of a
subunit), which fits to an one-site binding curve
soluble monomer of CDNF in Escherichia coli.
(R2 = 0.9999). The binding of BEX3 to p75-
This recombinant CDNF, contains four disulfide
NTR has been mapped by NMR.
bounds, and was able to protect the dopaminergic
Programmed cell death 10 (PDCD10).
PDCD10
was
purified
by
neurons isolated from the embryonic day 14
hydrophobic
mouse mesencephalon from the damage caused
interaction chromatography and gel filtration
by 6-OHDA. We are also characterizing the
chromatography. The protein was obtained as a
proteolytic activity that separates the two
dimer. NMR experiments showed that this
domains of CDNF.
protein was structured, and circular dichroism
showed
a
high
content
of
alpha
helix.
Glial cell-line Derived Neurotrophic
Factor (GDNF). Our goal is the study of GDNF
Crystallization trials are being performed, and
mimetic
peptides
interaction
with
their
crystals with 3.6 Å resolution have been
GFRalpha1 co-receptor (GDNF-Family Receptor
obtained.
alpha 1) to identify GDNF regions that activate
Two hybrid associated protein 1 (TWA-
this co-receptor and trigger biological activity,
1). TWA-1 has been purified by ion interaction
for development of small molecules to treatment
chromatography
hydrophobic
of neurodegenerative disorders. DNA sequences
interaction chromatography and gel filtration
encoding a mature GDNF, and its heel region
chromatography. The protein has propensity to
(P9, a sequence of 15 amino acids which shows
form instable aggregates. A condition has been
biological activity and binds to its co-receptor)
found where the protein forms a stable dimer,
and GFRalpha1 domains were subcloned into
and crystallization trials are being performed.
vectors for Escherichia coli. The best expression
followed
by
conditions have been tested to give the highest
Structure and function of
Neurotrophic
factors (Prof. Marcius S. Almeida)
Conserved
for subsequent purification of these various
Neurotrophic
constructions. Recombinante and synthetic P9
This protein showed a
were produced for structural analysis by Nuclear
neurotrophic activity on midbrain dopaminergic
Magnetic Resonance and Circular Dichroism.
neurons using a 6-hydroxydopamine (6-OHDA)
Such as GDNF, P9 can be most biologically
rat experimental model of Parkinson’s Disease.
active
CDNF has two domains; a N-terminal saposin-
Thereby, we are also engaged on synthesis of
like domain, which may bind to membranes; and
tetrameric and dimeric dendrimers of P9.
an presumably intrinsically unstructured C-
Activity assays for P9 using dopaminergic
terminal which contains an internal cysteine
neurons of 14 days-old embryos or SH-SY5Y
bridge in a CXXC motif similar to that of
neuroblastoma
Factor (CDNF).
Dopamine
level of soluble recombinant protein production
as
covalently
cells
linked
have
homodimers.
been
used
for
39
biological trials using 6-hydroxidopamine (6-
phosphatidylglycerol vesicles, the only vesicle
OHDA) as a model for Parkinson’s disease.
that was fused by DV fusion peptide. The threedimensional structure of DV fusion peptide
Structure and Thermodynamics of DNA
bound to DPC micelles was solved by solution
binding proteins and Virus Fusion Proteins
homonuclear NMR with an r.m.s.d. of 0.98 A.
(Prof. Ronaldo M. Borges)
The most striking result obtained from the
In the first goal, we correlated the
solution structure was the hydrophobic triad
oligomeric state of the proteins UmuD e UmuD’,
formed by residues W101, L107, and F108,
which function is mutagenesis and DNA repair
pointing toward the same direction, keeping the
in E. coli. The findings were published in PNAS
segment between G102 and G106 in a loop
(Simon et al., PNAS, 2008) in collaboraton with
conformation. The interaction of DV fusion
Dr. Graham C Walker from MIT (EUA).
peptide
with
phosphatidylcholine-
The second goal we could characterize
phosphatidylglycerol vesicles was also mapped
the process of membrane fusion of the virus
by transfer-nuclear Overhauser enhancement
Dengue II. Dengue virus (DV) infection depends
(NOE) experiments, in which the majority of the
on a step of membrane fusion, which occurs in
NOE cross-peaks were from the hydrophobic
the acidic environment of the endosome. This
triad, corroborating the DPC-bound structure.
process is mediated by virus surface envelope
Substitution of the residue W101 by an alanine
glycoprotein, in which the loop between residues
residue completely abolished membrane binding
D98-G112 is considered to be crucial, acting as a
and, thus, fusion by the peptide and its NOE
fusion
characterized
cross-peaks. In conclusion, the 15-residue DV
functionally and structurally the interaction
fusion peptide has intrinsic ability to promote
between the DV fusion peptide and different
membrane fusion, most likely due to the
model membranes by fluorescence and NMR. Its
hydrophobic interaction among the residues
interaction
W101, L107, and F108, which maintains its loop
peptide.
We
was
have
strongest
in
dodecylphosphocholine (DPC) micelles and
anionic
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
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. 2010 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.. 2010.
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, 105-113
3. 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. 2010. 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, 51125117
4. 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.
2010. Mapping the interactions between a major pollen
allergen and human IgE antibodies. In Structure (London). ,
v.18, 1011-1022
5. PALMIERE, L. C., Lima, L.M.T.R, Freire,
J.B., Bleicher, L., Polikarpov, I., ALMEIDA, F. C. L.,
FOGUEL, Debora. 2010. Novel Zn2+-binding sites in
human transthyretin: implications for amyloidogenesis and
retinol binding protein recognition. In The Journal of
Biological Chemistry (Print). , v.2010, 00-00
6. 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.. 2009. Inhibition of energy-producing
pathways of HepG2 cells by 3-bromopyruvate In
Biochemical Journal (London). , v.417, 717-726
7. 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.. 2009. 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', 736-746
8. Razzera g, GADERMAIER, G., ALMEIDA,
Marcius da Silva, Ferreira, F., ALMEIDA, F. C. L.,
VALENTE, Ana Paula, ABREU, Hector Nicolas Seuanez.
2009. 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,
103-106
9. 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..
2009. 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, 2194-2203
10. Luciano Medeiros, Angeli R, Barreto-Bergter
E., Valente, A.P., KURTENBACH, E., ALMEIDA, F. C.
L.. 2010. Backbone dynamics of the antifungal Psd1 pea
defensin and its correlation with membrane interaction. In
Biochimica et Biophysica Acta. Biomembranes. , v.1798,
105-113
11. Cruz AKM, Andrade GPV, Chavante SF, de
Vasconcelos CL, Garcia RB, Leite EL, Valente, A.P., Sales
MP, Oliveira FW. 2010. Structural elucidation of n galactan
from the eggs of mollusc Pomacea lineata In Carbohydrate
Polymers. , v.79, 975-980
12. L.P. Cinelli, Andrade LR, Valente, A.P.,
MOURÃO, P. A. S.. 2010. Sulfated alpha-L-galactans from
the sea urchin ovary: selective 6-desulfation as eggs are
spawned. In Glycobiology (Oxford). , v.20, 702-709
13. Ano Bom, A. P., Freitas M, Moreira FS,
Ferraz D, Sanches D, GOMES, A. M., Valente, A.P., Y, C.,
SILVA, J. L.. 2010. 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, 03-04
14. 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. 2009. Ferromagnetic Levan Composite: an Affinity
Matrix to Purify Lectin In Journal of Biomedicine and
Biotechnology. , v.01, 03-04
15. 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. 3:187-9.
16. 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. 284:34735-46.
17. 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. 2010 Sep;1804(9):1917-24. Epub 2010 Jun
9.
18. 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,
2010.
19. DIAZ, A., FONTANA, E. C., TODESCHINI,
A. R., SOULE, S., GONZALEZ, H., CASARAVILLA, C.,
PORTELA, M., MOHANA-BORGES, R., MENDONCAPREVIATO, L., PREVIATO, J. O., FERREIRA, F. The
Major Surface Carbohydrates of the Cyst: Mucin-TypeGlycans Decorated by Novel Galactose-Based Structures.
Biochemistry (Easton), v. 48, p. 11678-11691, 2009.
42
AL4
PHARMACOLOGIC PROTEOMIC
.
Coordinator: Russolina Zingali, Instituto de Bioquímica Médica, UFRJ.
Members:
Robson Monteiro
Márcia Soares
Clarissa Damaso
Bianca Cruz Neves
The Proteomic Unity has been serving
many
laboratories
determination
of
from
mass
INBEB
from
for
the
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
Study of Gluconacetobacter diazotrophicus
develepment to optimize the results and to
interaction with plant
identify more peptides. Furthermore we have
Recently as a consequence of previous
results
obtained
with
the
analyses
also looked at the phosphorilation profile of
of
infected cells. The phosphorilation profile of
Gluconacetobacter diazotrophicus we started the
cellular proteins was establisehed for 30 min, 2
study of the interaction between the bacteria and
and 8 hours after the infection. Proteins were
Arabidopis thaliana.
submitted to the Pro-Q Diamond staining that
Arabidopsis thaliana was chosen as
resulted in different profile of phosphorilation
study model, because its genome was fully
probably induced by the infection.
The
sequenced. Arabidopsis plants were inoculated
identification of this proteins by LC-MS/MS and
with Gluconacetobacter in hydroponic medium,
also the purified phosphorilated peptides are
and after 3 days of inoculation the plants had
under analysis.
been separated in leaves and roots. The extracts
of both segments were submitted to 1D SDSPAGE. The extracts of inoculated leaves showed
Snake venom proteomics
The
immunological
recognition
of
to be enhanced by three major proteins bands
B.jararacussu venom by the antisera was
between 50–20 KDa. The trypsinized proteins
determined.
have
jararacussu venom were identified via 2D gel
been
analyzed
using
proteomics
Many of the proteins in B.
technologies. Some of them were identified
electrophoresis
(Figure
being involved in metabolism process as
revealed that anti-jararacussu showed higher
aldolase, GAPDH and photosynthesis as PSBO2,
reactivity to L-aminoxidase (LAOs) and snake
LHCP AB 180 by ESI-Q-TOF. Mass spectra
venom metalloproteinase, (SVMPs) and weaker
analyses resulted in the identification of 149
reactivity towards snake venom serine proteases
proteins most of them induced by the bacteria.
(SVSPs), PLA2, C-type lectin and cysteine-rich
We have demonstrated that the inoculation with
proteins.
G. diazotrophicus changes the pattern of protein
recognized LAOs, SVMPs and SVSPs and Anti-
expression of Arabidopsis leaf and root.
crotalic serum clearly recognized LAOs, C-type
While
1).
anti-jararaca
Western
blots
preferentially
lectin, SVSP, cysteine-rich proteins, SVMP and
Asp49-PLA2.
Dengue virus infection
Our results suggest that the
In order to identify proteins and peptides
contribution of anti-crotalic serum to the
associated with Dengue virus, the secreted
neutralization of B. jararacussu may be due to
proteins of HepG2 cells (infected or not) were
its cross-reactivity with proteins such as C-type
analyzed
lectins, SVSPs, Asp49-PLA2. These results
by
mass
spectrometry.
The
identification of proteins >10 kDa resulted in the
publication by
were published in Toxicon.
Higa et al., 2008. We also
Another venom that was explored by our
analyzed the pool of peptides (<10kDa) by ESI-
group was from the snake genus Micrurus. To
Q-TOF. Up to now, 7 molecules were identified.
gain clues for outlining an alternative antiserum
The method for the analysis of peptide is in
generation strategy based on immunization we
44
have characterized the proteome and the venom
1674.6 KDa, 1009.4 KDa, 1025.4 KDa, 1538.8
gland transcriptome of M. altirostris (Figure 2).
KDa, 1669.6 KDa and 1039.4 KDa for
The venom proteome
contained around 50
bothrojaracin molecules with at least one site of
proteins belonging to eight toxin families.
oxidation (table 2). Mass spectrum of thrombin
Neurotoxins (73%) and PLA2s (14%) comprise
showed eighth different oxidized peptides as
the major toxin families. Other toxin groups
1209.5 KDa, 1332.6 KDa, 1930.8 KDa and
included 3FTxs (7%), Kunitz-type inhibitors
2296.0 KDa, with multiple sites of oxidation.
(2%), PIII-SVMPs (1%), CTLs (<0.1%), CRISP
Oxidation of the complex was performed with
(<0.1%), and unknown molecules (4%). The
the same protocol and the mass spectra clearly
transcriptome (from the pooled glands of an
demonstrate protected sites in the presence of
adult and a neonate specimens) contained 946
bothrojaracin. We also performed deuterium
ESTs grouped into 384 clusters. Venomic and
exchange experiments for thrombin in the
transcriptomic data provided full-length amino
absence and presence of bothrojaracin; a new
acid sequences for the major M. altirostris
interacion site and conformational changes in
toxins. This information allowed homology 3D-
thrombin molecule could be detected.
model-based design of neurotoxin- and PLA2-
We are also using molecular modeling to
derived peptide stretches as targets for antibody
investifate the binding of disintegrin to integrins.
generation in rabbits. The capability of the
Jararacin (JARC) and jarastatin (JAST) are
corresponding antisera to neutralize the toxic
disintegrins present in venom of Bothrops
activities of M. altirostris venom is being
jararaca. They bind to and antagonize the
investigated in our laboratories.
adhesive functions of β3 integrin receptors,
in
which complexes possess two members: αIIbβ3,
collaboration with Dr Inacio Junqueira de
present in platelets, and αvβ3, widely distributed
Azevedo (Instituto Butantan) and Dr Juan
in various cells. In our study comparative
Calvete
de
analyses of the binding of these disintegrins to
in
αIIbβ3 and αvβ3 integrins were performed in
This
results
(Instituto
Valencia,Spain)
The
were
de
obtained
Biomedicina
manuscript
is
preparation.
silico, to observe the degree of affinity between
these complexes. To perform this theoretical
Analysis of protein-protein interaction
analysis, we constructed homology models of
Bothrojaracin is a protein from Bothrops
docking complexes using models of JARC,
jararaca venom that forms complex with
JAST and crystal structures of αIIbβ3, αvβ3, and
thrombin, blocking biological activities. Here we
theoretically evaluated their interaction using a
analyze the extent of bothrojaracin-thrombin
molecular modeling approach. Our results show
interaction using footprinting and ESI Q-TOF
that JARC has more interaction and more
spectrometer. After the induction of oxidation in
affinity with αIIbβ3. On the other hand,
controled medium samples were digested using
JAST/αvβ3
trypsin. MS/MS fragmentation reveal seven
JARC/αvβ3. The results we obtained suggest
different oxidized peptides, with 1067.4 KDa,
that αIIbβ3 has a higher affinity to structures
presents
more
affinity
than
45
similar to JARC. On the other hand αvβ3 have
potent and long-lasting inhibitor of arterial
affinity to structures such as the JAST. The
thrombosis with minor effects on haemostasis.
analysis
of
complex
dynamics
is
under
Another protein studied by our group
evaluation in order to confirm our docking
was the tick anticoagulant Ixolaris that had been
analysis.
previously proven to be an antithrombotic drug.
We now have tested if it interferes with
Exogenous factors as Anti-thrombotic and
glioblastoma progression. We demonstrate that
anti-cancer drugs
Ixolaris effectively blocked the in vitro TF-
Nitrophorin 2 (NP2) isolated from the
dependent procoagulant activity of the U87-MG
blood sucking insect, Rhodnius prolixus is a
human glioblastoma cell line and attenuated
potent inhibitor of the intrinsic pathway of
multimolecular
coagulation upon binding to factor IX (FIX) or
assembly. Notably, Ixolaris inhibited the in vivo
FIXa. The in vivo antithrombotic properties of
tumorigenic potential of U87-MG cells in nude
NP2
mice,
were
investigated.
Surface
plasmon
without
coagulation
observable
complexes
bleeding.
This
resonance assays demonstrated that NP2 binds to
inhibitory effect of Ixolaris on tumor growth was
rat FIX and FIXa with high affinities (KD = 43
associated with downregulation of VEGF and
and 47 nM, respectively), and prolongs the aPTT
reduced tumor vascularization.
without affecting the
PT.
In
order
evaluate
to
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
did not increase the
blood
loss
as
by
tail-
evaluated
transection
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% SDSPAGE. 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
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).
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. 2010 Aug 5;73(9):1758-76.
2. 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. 2010 May 7;73(7):1334-41.
3. 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, DeSimone SG, Soares MR, Foguel D, Zingali RB. Toxicon.
2010 Jun 15;55(7):1222-35.
4. 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. 2010
Jan;50(1):89-96.
5. 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. 2009 Nov;77(7):934-8.
6. Engineering ecotin for identifying proteins
with a trypsin fold. Sathler PC, Craik CS, Takeuchi T,
Zingali RB, Castro HC. Appl Biochem Biotechnol. 2010
Apr;160(8):2355-65.
7. 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. 2009
Nov;20(6):456-64.
8. 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. 2009 Sep;136(11):1339-49.
9. 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-deAzevedo IL, Ho PL, Zingali RB, Perales J, Domont GB. J
Proteomics. 2009 Mar 6;72(2):241-55.
10. 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.
FEMS Immunol Med Microbiol. 2009 Apr;55(3):388-95.
11. 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. 2009 Feb;482(12):25-32.
12. 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. 2010 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. 2010 Sep
13;104(6). [Epub ahead of print]PMID: 20838739
14. 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. 2010
Apr;43(4):403-8.
15. 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. 2010 Jan;277(2):413-27.
16. 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. 2009
Dec;102(6):1183-93.
17. 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. 2009
Nov;7(11):1855-64
18. 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. 2009 Oct;19(5):301-8.
19. 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. 2009
Jun;21(6):1599-604.
20. 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. 2009 Oct 8;283(2):168-75.
21. 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. 2009
Jun;1794(6):873-81.
22.
Lung
adenocarcinoma
and
antiphospholipid antibodies. de Meis E, Monteiro RQ,
Levy RA. Autoimmun Rev. 2009 May;8(6):529-32. Epub
2009 Jan 29.PMID: 19185619 [PubMed – indexed). 48
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
most
effective
intermolecular
chemical defense agents and also as drugs for
interactions with the targets and their effect in
Alzheimer disease. Further reactivation tests
their function. For example, the most common
by NMR also showed that these compounds
targets are enzymes, and the best evaluation of
are good reactivators of the inhibited AChE.
their potential inhibitors are carried out by
Figure 1 shows the inhibition results obtained
enzyme kinetics and inhibition studies, which
by NMR for one new hydrazone.
are
ones
conducted
the
using
100
with
90
80
A c h(5)
emphasis on nuclear magnetic
70
A c O (5)
resonance and ultraviolet. Some
60
A c h(10)
50
A c O (10)
40
A c h(20)
30
A c O (20)
spectroscopy
methods,
tests are also conducted directly
with the cell targets, principally
with
microorganisms,
Plasmodium and
like
Leishmania
20
enzymes that are the targets for
the chemotherapy are obtained
A c h(40)
0
0
cells. Also, in vivo tests are
conducted in some cases. The
A c h(40)
10
20
40
60
80
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).
commercially or by heterologous
The mechanism of reactivation of
expression.
AChE inhibited with war gases was effectively
Antidotes for Intoxication with Pesticides
simulated using QM/MM, using RM1 to
and Warfare Agents
simulate 10 amino acids of the active site (146
We have developed new reactivators
of the enzyme acetylcholinesterase (AChE)
atoms) including the phosphilated Ser203 and
the oxime, as shown in Figure 2.
inhibited with the very toxic organophosphorus
The studies with Yersinia pestis were
compound paraoxon. These compounds are
based on the peptide of interaction of human
neutral oximes, which display a much better
plasminogen (PLG) with the bacteria protein
capacity
the
for invasion of human cells (PLA). This
hematoencephalic barrier and a better effect
peptide was discovered by molecular modeling
than
pralidoxime.
and its structure determined by NMR. The
Simulation of the reactivation process using
future studies are based on the experimental
quantum mechanics procedures indicated that
interaction of the peptide with Y. pestis PLA
other nucleophiles, like hydrazones, have also
by NMR, in order to determine the interaction
potential to function as antidotes. Kinetic
process and to discover forms to avoid the
studies by NMR were able to show that the
invasion of mammal cells by this bacterium.
neutral oximes and hydrazones are effective
The structure of the peptide is shown in Figure
inhibitors of AChE, with important potential as
3.
the
of
permeation
commercial
drug
of
50
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
(NH)
had
its
structure
determined
by
molecular modeling and used to plan new
glected Diseases
The main targets for the development
effective
inhibitors.
The
enzyme
was
of new drugs for the treatment of neglected
expressed linked with maltose binding protein
diseases
dihydrofolate
(MBP), a process that kept the enzyme active
reductase (DHFR) of Plasmodium falciparum
and with great stability and solubility. Enzyme
and Mycobacterium leprae, the nucleoside
kinetic studies with different compounds using
hydrolase
and
ultraviolet spectroscopy lead to the discovery
chagasi. For DHFR in malaria, the structure of
of two new inhibitors with IC50 from 15 to 25
the complete enzyme DHFR-TS and their
M. It was also found that the AIDS drug AZT
chemotherapy resistant mutants were used to
is an effective inhibitor of NH, having
design new type 2 antifolates with IC50 from
potential to be used for the treatment of
80 to 120 pM.
leishmaniosis. Figure 5 shows the NMR basic
are
of
the
enzymes
Leishmania
donovani
New compounds are actually being
synthesized using a novel reaction prepared in
spectra used for the monitoring of the enzyme
kinetics of NH.
our research group, as shown in Figure 4.
The leishmania nucleoside hydrolase
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
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. 179-184, 2010
2.
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, 2010.
3.
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. 113-123, 2010.
4.
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, 2010.
5.
Vieira, A. A.; Gomes, N. M.;
Matheus, M. E.; Fernandes, P. D.; Figueroa-Villar
JD . Synthesis and In Vivo Evaluation of 5-Chloro5-benzobarbiturates as Central Nervous System
Depressants. Journal of the Brazilian Chemical
Society (Impresso), v. 21, p. 1-8, 2010.
6.
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. 407428, 2009.
7.
Delfino, R. T.; Figueroa-Villar
JD. Nucleophilic Reactivation of Sarin-inhibited
Acetylcholinesterase: a Molecular Modeling
Study. Journal of Physical Chemistry. B, v. 113,
p. 8402-8411, 2009.
8.
Figueroa-Villar JD; Tinoco,
Luzineide Wanderley. Spin-lattice relaxation time
in drug discovery. Current Topics in Medicinal
Chemistry (Print), v. 9, p. 811-823, 2009.
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. 531540, 2010.
2.
Batista, Paulo Ricardo; Robert,
Charles Herbert; Maréchal, Jean-Didier; HamidaRebaï, 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.
2850-2859, 2010.
3.
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, 2010
4.
Figueirêdo, P.H.; Moret, M.A. ;
Pascutti, P. G. ; Nogueira Jr., E. ; Coutinho, S. .
Self-affine analysis of protein energy. Physica. A
(Print), p. 2682-2686, 2010
5.
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,
2010.
6.
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, 2010.
7.
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, 2010
8.
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, 2010.
9.
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. 1437-1443, 2009
10.
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. 041908, 2009.
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
and benzimidazole 1,3-dioxide core scaffolds.
MedChemComm, v. 1, p. 216-228, 2010.
2.
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.
1127-1131, 2010.
3.
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. 258-263, 2009
4.
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. 1738-1741, 2009.
5.
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. 811-823,
2009
54
AL6
AND PROTEOMIC HETEROLOGOUS
EXPRESSION
.
Coordinator: Hernán Terenzi, Universidade Federal de Sta Catarina (UFSC)
Major lines of investigation
Lipolytic enzymes applications
Food technology
Biofuels
Chemistry
Lipases
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
2).
Lipases
also
esterification,
On the other hand, a brief toxic
transesterification, acydolysis, alcoholysis and
stimulus allows the cells to acquire tolerance to
aminolysis in addition to the hydrolytic activity
a
on triglycerides (2-4). In the last months, our
preconditioning,it has been reported in a wide
group published two papers (5-6) on purification
variety
and biochemical characterization of lipases and
administration of a NMDA sublethal dose has
one paper leading with esterase activity (7). We
been
also characterized structural features of a
preconditioning against several lethal later
Staphylococcus xylosus lipase and its need of a
injuries. Studies have shown that 24 hours after
metal
administration
cofactor
to
catalyze
thermal
stability
and
more
severe
of
models.
used
as
insult,
In
a
of
this
model
NMDA
of
in
the
chemical
mice,
becomes extremely important for design of the
approximately 50% of animals when exposed to
biocatalysis process. Therefore, the knowledge
quinolinic acid (QA), after this period, the
of
protective effect is reversed, again causing
stability
at
high
temperatures and metal tolerance of lipases
in
convulsions and damage to neuronal tissue.
enables the development and application of
these proteins in industrial processes.
observed
a
neuroprotective
such
is
context,
as
enzymatic activity (unpublished data), which
characteristics
effect
known
Despite the enormous progress in recent
decades, the cellular and molecular mechanisms
Proteomic Analysis of neuroprotection
that involve the preconditioning have yet to be
and pre-condiotioning induce by N-Methyl-D-
elucidated. One of the more recent approaches
Aspartate (NMDA) in mice hippocampi
to extend this knowledge is neuroproteomics
Exposure of neuronal cells to exogenous
or endogenous toxicants can affect many
(Study of brain proteins in a specific condition
or treatment)
biological processes such as the cell signaling.
In this work we aim to identify the
Cells respond to these insults by modulating
proteins
protein
posttranslational
pathways) involved in NMDA pre-conditioning
modifications and the deregulation of protein
and neuroprotection, in mice treated with
expression is associated with many human
NMDA for different period of time using 2D
expression
and
(and
therefore
which
signaling
diseases such as
cancer,
neurodegenerative
disorders
and
acute events such
as
cerebral
ischemia,
traumatic
injury
epilepsy.
brain
and
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
gels analysis and mass spectrometry. The 2DE
spectroscopic methods, recently involving the
gels of total proteins from mice hippocampi
use of circular dichroism spectroscopy.
obtained showed some differences in the
In the last two years, we reported the
proteome profile for each treatment (1, 24 and
complex-DNA interaction of several transition
72 hours). Further analyses to identify proteins
and lanthanides metal complexes in high impact
by mass spectrometry are underway.
scientific journals. Some of these complexes
were developed to mimic the active site of some
Chemical Hydrolases
Interactions
enzymes (bioinspired complexes). We highlight
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
two papers published this year reporting the
DNA cleavage by mononuclear complexes of
copper7 and iron8 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
In addition, we report a new example of
metal
complex
capable
of
hydrolyze
development of DNA cleavage agents in
heterogeneous
catalyst
systems,
i.e.,
phosphodiester bonds in the DNA and peptide
immobilizing the catalyst in specific surfaces
bonds in proteins, i.e., a complex whose activity
like in silica. This approach favors the re-use of
shows catalytic promiscuity. To our knowledge,
the catalyst as already performed by many
this is the third example of metal complex with
biotechnological procedures.
this property. Our efforts also concentrate on the
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. 110-119, 2010.
2.
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, 2010.
3.
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. 242-249, 2010.
4.
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. 1111-1115, 2010.
5.
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. 2580-2582, 2010.
6.
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. 3057-3063,
2010.
7.
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. 1996. Print) , p. 11, 2010.
8.
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. 11-24, 2010.
9.
Kolling, Deise Juliana ; Bertoldo, Jean
Borges ; Brod, Fábio Cristiano Angonesi ; VERNAL,
Javier ; Terenzi, Hernán ; Arisi, Ana Carolina Maisonnave .
Biochemical and Structural Characterization of Two SiteDirected Mutants of Staphylococcus xylosus Lipase.
Molecular Biotechnology , p. 3-8, 2010.
10.
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, 2010.
11.
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,
2010.
12.
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, 2010.
13.
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
Phenolate-type Ligands. Journal of Physical Organic
Chemistry , v. 23, p. 45-53, 2010.
14.
Ecco G ; VERNAL, Javier ;
RAZZERA, G. ; Martins, Priscila Graziela Alves ;
TERENZI, H . Mycobacterium tuberculosis tyrosine
phosphatase A (PtpA) activity is modulated by Snitrosylation. Chemical Communications (London.
1996. Print) , v. prelo, p. 1, 2010.
15.
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. 165-174, 2009.
16.
Cangahuala-Inocente,
Gabriela
Claudia ; Villarino, Andrea ; Seixas, Daniela ; DumasGaudot, Eliane ; Terenzi, Hernán ; GUERRA, Miguel
Pedro . Differential proteomic analysis of developmental
stages of Acca sellowiana somatic embryos. Acta
Physiologiae Plantarum , v. 31, p. 501-514, 2009.
17.
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.
2711-2713, 2009.
18.
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.
1323-1330, 2009.
19.
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. 7905-7921, 2009.
20.
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. 11-22, 2009.
21.
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, 2009.
22.
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, 2009.
59
60
AL7
BIOCHEMISTRY
.
Coordinator: Carlos H. Inácio Ramos, Instituto de Química (UNICAMP)
Members:
Ljubica Tasic
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 1H NMR and
61
chemometrics
detect
inhibitor developed and it is largely used in
molecular changes in human blood serum
hypertension treatment. On the other hand,
samples by comparing the metabolic profiles of
cyclodextrins (CD) are cyclic oligosaccharides
healthy subjects (control group/ n = 25), patients
whose cone-shaped cavity allows formation of
being treated for bipolar disorder with lithium (n
non-covalent
inclusion
= 15), and patients being treated for bipolar
appropriately
sized
disorder with other medications not including
modifying
lithium (n = 10). 1H NMR data were transported
biological properties. We have reported the
to a data matrix, and chemometrics analyses,
physicochemical characterization and in vivo
based on interval principal component analysis
ACE inhibition evaluation of seven CAP/CD
(iPCA) and partial least-squares discriminant
complexes. The inclusion complexes (IC) were
analysis (PLS-DA), were performed using
prepared by spray drying, freeze drying,
MATLAB 6.5 software. After further processing
kneedling,
the data, the investigated groups (control and
characterized applying the nuclear magnetic
patients with bipolar disorder under different
resonance (NMR), Fourier-transformed infrared
treatments) could be distinguished according to
(FTIR) spectroscopy, X-ray diffraction (DRX),
their metabolic profiles, and the main differential
differential
metabolites found were lipids, lipid-metabolism-
scanning electron microscopy (SEM) techniques.
related molecules (acetate, choline, and myo-
In vivo assays compared the captopril and
inositol), and some key amino acids (glutamate,
CAP/CD complexes (0.5 mg/kg, or 0.09 mg/kg,
glutamine). This strategy showed significant
n=4-7) administration as to evaluate the ACE
potential for exploring pathophysiological and
inhibition
toxicological
bipolar
angiotensin I (Ang I, 30ng/50µL/min) in
disorder and our results suggest that some of the
conscious Wistar rats. The physicochemical
24 identified metabolites may be linked to
analysis demonstrated complete amorphization
lithium
provoked
and complexation of captopril and cyclodextrins,
metabolic changes or may even be directly
indicating the substitution of water molecules
related to the disorder. This work can contribute
inside the CDs cavity by CAP. During the
to find the way for future studies aiming at
infusion of Ang I, the administration of all
identifying potential biomarkers for bipolar
CAP/CD complexes induced a reduction in
disorder.
mean arterial pressure (MAP) similar to that
and
was
performed
features
other
involved
medication
to
in
quests
or
host
with
molecules
physical,
thus
chemical,
lyophilization methods,
thermal
by
complexes
analysis
continuously
(DSC),
infusion
and
and
and
of
observed upon captopril administration. The
Supramolecular Interactions in Captopril and
nanoparticles obtained by kneading method
Cyclodextrins complexes: Physico-chemical
(CAP/-CD:KM) showed a potent and long-
and
lasting inhibitory activity (~22h) upon the Ang I
Angiotensin-I-converting
enzyme
pressor
Inhibition Evaluations
Captopril
(CAP)
angiotensin-I-converting
was
enzyme
the
first
(ACE)
effect. Therefore,
obtained
results
suggest that the inclusion complex of captopril
and
-CD
can
function
as
a
novel
62
antihypertensive formulation that may improve
Hsp70s assist in the process of protein
captopril therapeutic use by reducing its oral
folding through nucleotide-controlled cycles of
dose administration in one per day thus
substrate binding and release by alternating from
providing better life conditions for almost 15%
an ATP-bound state in which the affinity for
of
substrate is low to an ADP-bound state in which
word
population
that
suffers
from
hypertension.
the affinity for substrate is high. It has been long
(A)
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
(B)
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
experiments,
with
our
previous
results
biochemical
suggest
that
conformational changes caused by nucleotide
binding are a consequence of the movement in
position of both nucleotide- and substrateFigure 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 (H4) and red (H-8) on CAP molecule are the most altered
by complexation as measured in ROESY NMR and seen
by CICS.
binding 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, 230-233.
Main publications:
Heat shock protein 40 (Hsp40) cochaperones assist in cellular protein folding and
Borges,
J.C.,
(2009).
degradation through the binding and delivery of
nucleotide-induced
non-native proteins to heat shock protein 70
changes on the structure of human 70 kDa
(Hsp70). The mechanism for substrate transfer
heat shock protein Hsp70.1 by analytical
from Hsp40s to Hsp70 is unknown. Two recent
ultracentrifugation. BMB Reports., 42, 166-
studies provide new details that shed light on
171.
novel mechanisms for substrate recognition by
Characterization
Ramos,
of
C.H.I.
63
prevent
protein
aggregation by acting as
thermosensors
enhance
and
cell
to
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
substrate
have
distinct
specificities.
Therefore, they are useful
models to study how class
I SHsps work. Here we
present data on the effects
of
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.
heat
on
oligomerization
chaperone
activity
the
and
of
SsHsp17.2 and SsHsp17.9.
Using several biophysical
and biochemical probes,
Hsp40s
and
a
common
mechanism
for
polypeptide transfer to Hsp70.
we show that the effects of heat are completely
reversible, an important property for proteins
that act at heat shock temperatures. SsHsp17.2
Tiroli-Cepeda, A.O., Ramos, C.H.I. (2010).
and SsHsp17.9 dodecamers dissociated to
Heat causes oligomeric disassembly and
dimers at temperatures ranging from 40 to 45 _C
increases the chaperone activity of small heat
and this dissociation was followed by enhanced
shock proteins from sugarcane. Plant Physiol.
chaperone activity. We conclude that high
Biochem. 48, 108-116.
temperature affects the oligomeric state of these
Small
heat
shock
proteins
(sHsp)
constitute an important chaperone family linked
chaperones, resulting in enhanced chaperone
activity.
to conformational diseases. In plants, sHsps
64
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, 164-173.
2.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, 727-743.
3.Gava, L., Ramos, C.H.I. (2009) Human 90 kDa
heat shock protein Hsp90 as a target for cancer
therapeutics. Curr. Chem. Biol. 3, 330-341.
4.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,
166-171.
5.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, 119-125.
6.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, 230-233.
7.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,
968-980.
8.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, 3078-3088.
9.Correa, D.H.A., Ramos C.H.I. (2009). Insigths
on the structure of amyloid fibrils from site-directed
mutagenesis. Protein Pept. Lett.. 16, 1519-1525.
10. Durán, N.; Durán, M.; Tasic, L.; Marcato, P.
D. Nanocrystal technology in pharmaceuticals. Química
Nova 2010, 33(1), 151-158.
11.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), 9755-9763.
12.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, 492-498.
13.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, 108-116.
14.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, 553-563.
15.Fattori, J.; Prando, A.; Martini, A.M.;
Rodrigues, F.H.S.; Tasic, L. (2010) Bacterial Secretion
Chaperones. Protein & Peptide Letters, 2010. (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 A2 Inhibitor from Bothrops
jararacussu Snake
SnakePlasma.
Plasma.Current
CurrentTopics
Topics
in Medicinal
jararacussu
in Medicinal
Chemistry (CTMC),.
(CTMC),. In
In press.
press.
Chemistry
65
66
AL8
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 14alfa 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
fluconazol is decreased affinity of lanosterol 14-
mutation and explain why such modifications
alfa demethylase (14-DM) towards the drugs,
might alter the drug affinity towards C.
as
neoformans 14-DM (Figure 1).
a
consequence
of
mutated
residues.
Knowledge about the interaction profile of drugs
into
the
binding
site
provides
In order to further investigate this
valuable
matter, crystallographic studies with wild-type
information to circumvent this kind of problem,
and mutant 14-DM are being undertaken.
however there is no crystallographic structure of
Meanwhile, we decided to explore ligand-based
C. neoformans 14-DM available. In such cases,
in silico tools that might be helpful to design
it is possible to gain some structural insight from
more effective antifungals. Aiming to contribute
homology based models that are built from the
to this goal, 2D QSAR studies were carried out
aminoacid sequence of the the target structure
for a series of 33 azoles derivates that had been
and the 3D coordinates from a related protein,
tested
whose sequence identity is greater than 30%.
neoformans strain. The best PLS model shows
Using the coordinates from 14-DM from
good fit and internal consistence (r2= 0.89 and
against
a
fluconazole-resistant
C.
q2(LOO)=0.82, with 3 PCs) and
average predictive power (r2pred=
0.73) (Figure 2).
Analysis
vector
of
suggests
regression
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
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
(i.e
modeling was also carried out. The
best model (A/B/C/H/Ch) shows
excellent
consistence
and
(r2=
0.97
internal
and
2
q (LOO)= 0.79, with 6 PCs), but
very
poor
predictive
power
(r2pred=0.28). This result suggests
that fragment based QSAR models
molecular
predictive
fit
power,
holograms)
whereas
have
limited
descriptor-based
QSAR models can be useful not only to shed
some light on the structure-activity relationship
68
of azole derivates that were evaluated against
diverse dataset of 122 hydroxy-ethylamine
fluconazole-resistant C. neoformans strains, but
(HEA) derivatives, whose inhibitory potency
also to guide the design of novel and more
(IC50) against BACE-1 ranges from 4208 to 8.70
potent antifungals.
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
2
2
2
R = 0.89, Q (LOO)=0.82 AND R
(3 PCS)
PRED=
shows
good
internal
consistency (r2= 0.80 e q2= 0.80) and
0.73
predictive
power,
r2pred=
0.75)
(Figure 3-left panel). With the
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-
intention to facilitate QSAR model
analysis, fragment based QSAR
models
(HQSAR)
developed
were
using
also
molecular
holograms. The best HQSAR (A/B/C ) model
presented q² = 0,77 (Leave-One-Out), r² = 0,89
and r2pred =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.
1 inhibition. In order to do so, we selected a
69
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. 73-79, 2010.
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. 1421-1427,
2010.
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. 510-519, 2010.
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. 1693-1705, 2010.
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. 124-129, 2010.
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. 134-139, 2010.
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. 355-360, 2010.
Note: The results described herein will be
submitted to publication within 3 months
70
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
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., 2008. JBC. 283, 31541-31550).
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., 2010. Mol. Microbiol. 76, 1358-1375).
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,
Electron tomography of a T. gondii tachyzoite
form showing the PLV (green), selected as the
cover of Molecular Microbiology
using antibodies against TcPDEC2 showed that
this phosphodiesterase is mainly localized in the
porin features and the secondary structure
contractile vacuole complex (CVC) region,
prediction indicates a β-barrel pattern with 18
preferentially in the spongiome. Functional
transmembrane domains. Antiserum produced
analysis and expression of truncated sequences
against the recombinant porin revealed that this
showed that a FYVE domain is required for the
protein is mainly located in the symbiont
correct docking of the TcPDEC2 in the CVC and
envelope, forming porin channels with slight
that it down regulates the regulatory volume
cation-selectivity when reconstituted in a lipid
decrease (Schoijet et al., Mol. Microbiol. 2010,
bilayer. Taken together, our data show that the
in press).
C. deanei endosymbiont has a porin channel that
is phylogenetically and structurally similar to
classical porins of Gram-negative bacteria.
Endosymbiosis in Trypanosomatids.
Biochemical, molecular, ultrastructural
and computational methods were used to to
Endocytosis in T. Cruzi
deanei
The endocytic pathway of of T. cruzi
endosymbiont porin. A search of the annotated
epimastigotes is comprised by two entry sites,
symbiont genome database identified a sequence
the cytostome and the flagellar pocket, an early
that presents identity to porins of the Bordetella
endosomal network and reservosomes, the final
genus. The protein’s primary sequence presents
compartment, which stores exogenous proteins
identify
and
characterize
the
C.
72
and lipids besides enzymes produced by the
intervention. Our group used atomic force
parasite. We have published the proteome of
microscopy (AFM) to examine the ultrastructure
isolated reservosomes in 2009.
LC-MS/MS
of Trypanosoma cruzi, obtaining valuable
analysis identified a total 442 T. cruzi-specific
information on the organisation of the flagellar
proteins; of these, 286 had predicted function
sub-structure. AFM images revealed novel
and 156 were classified as hypothetical proteins.
flagellar components such as the presence of
We could confirm the presence of several
periodically-spaced protrusions organised along
proteins validated by previous work and identify
a flagellar furrow and oriented through the major
new proteins from different classes such as
flagellar axis between the axoneme and the
enzymes, proton pumps, transport proteins and
paraflagellar rod. The nature and functional role
others.
of this structure are still unknown, although the
We
have
protein
hypothesis that the furrow might physically
immunologically similar to BILBO-1, that have
separate the two distinct domains of the flagellar
been recently described as involved in endo-
membrane that comprise the surface of the
exocytosis and essential for flagellar pocket
axoneme and the paraflagellar rod (PFR) has
biogenesis
cruzi
been raised. To test whether the furrow was
epimastigotes, BILBO-1 was found at the
present or not only in PFR-bearing flagella,
flagellar pocket and flagellum.
different protists containing or lacking the PFR,
in
localized
T.
brucei.
the
In
T.
Electron tomography and dual beam
were analysed by AFM. Analysis of T. cruzi,
scanning electron microscopy are been used to
Trypanosoma
study
endosomal
megaseliae, which present distinct PFRs, showed
compartments within the parasite and have
similar and equivalent furrows along the main
already revealed that several reservosomes can
axis of their flagella, whereas Crithidia deanei,
fuse
network
Giardia lamblia and Tritrichomonas foetus (in
simultaneously. Reservosomes also appear to
which the PFR is reduced or absent) lacked a
fuse directly with the endoplasmic reticulum,
furrow. Our results strongly suggest that the
defining a new degradative pathway for internal
flagellar furrow is a characteristic feature of
proteins. We have isolated the crystalloid lipid
PFR-containing
inclusions of reservosomes and demonstrated,
perspectives for its functional role in the
using GC-MS, that they are formed by high
definition of sub-domains on the flagellar
amounts of cholesterol. Different from mammal
membrane.(Rocha GM et al.,Micron 41:939-44)
the
with
distribution
the
early
of
the
endosomal
brucei
flagella
and
and
Herpetomonas
opens
new
cells, T. cruzi epimastigotes are able to mobilize
cholesterol from crystals.
Interaction of protozoa with host cells
Trypanosoma cruzi- Dynasore acts as a
Paraflagellar Rod (PFR) Structure
potent inhibitor of endocytic pathways. We
Understanding the structural aspects of
observed that, in macrophages and LLC-MK2
the flagellum may be important for the
the parasite internalization was drastically
identification of novel targets for therapeutic
diminished when we used 100 mM dynasore. T.
73
cruzi adhesion index was unaffected. By
microdomains in adhesion and internalization of
scanning electron microscopy and comparing
T. gondii. The participation of cholesterol
peritoneal macrophages to LLC-MK2 cells
enriched microdomains in invasion of T. gondii
revealed differences. In LLC-MK2 cells, the
into LLC-MK2 and murine macrophages was
parasites were only associated with cellular
evaluated through transient depletion of host
microvilli, whereas in peritoneal macrophages,
cells
trypomastigotes were not completely engulfed
cyclodextrin (MβCD); Filipin. Cholera Toxin B
by a host cell plasma membrane. We explored
subunit (GM1 marker) and Lidocaine were also
cholesterol
with
either
methyl-beta-
whether membrane rafts participate
in the entry of Trypanosoma cruzi's
trypomastigotes
macrophages
cyclodextrin
into
using
and
murine
methyl-beta-
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
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.
the diverse signaling events which
take place during egress, kinases seem to play a
tested.
Adhesion to macrophages was not
crucial role. Although parasite egress induction
affected by any of the drugs, but was
was only slightly affected by wortmanin and
significantly diminished in LLC-MK2 cells.
staurosporin, the specific inhibitor of tyrosine
This effect was not reversible. On the other
kinase, genistein, blocked the exit of parasites to
hand,
more than 50%. The actin polymerization
instances for both cell types.
invasion
was
diminished
in
all
inhibitor cytochalasin D also blocked the
Ultrastructural organization of parasitic
induced egress of T. gondii. Dynasore that
protozoa- The conoid, in Toxoplasma gondii is a
blocks dynamin had little or no effect on egress
small cone-shaped structure composed of a
of
spiral
T.
gondii.
Participation
of
host
cell
of
microtubules,
two
intraconoidal
74
microtubules, and two polar rings. It shows a
tachyzoites. Micronemes were seen in a radial
mechanical activity in invasion of host cells. We
distribution around the conoid. Dense granules
combined
Field
were scattered in the cytoplasm. Rhoptries were
Electron
grouped in one side of the cell and the necks
Microscopy techniques. For TEM fixation was
made a sinuous route up to the conoid and
in 2.5% glutaraldehyde and 1% tannic acid in
micronemes were also concentrated around the
0.1M cacodylate buffer, Post fixation was
base of the conoid. Stereological measurements
carried out in 1% OsO4 in the same buffer. For
showed that rhptries occupied 2-3% of the total
FESEM the membrane was extracted with non-
cell volume, while micronemes filled less than
ionic detergents. We can already show that the
0.5% of it. Dense granules filled the higher
conoid is more cylindrical than conoidal and in
volume: 4-5%.
Transmission
Emission
Scanning
(TEM)
and
(FESEM)
its retracted position remains under the posterior
In
trypanosomatids,
cell
division
polar ring and surrounded by the inner pellicle.
involves morphological changes and requires
Tiny bridges connecting the conoid to the lateral
coordinated replication and segregation of the
portion of the posterior polar ring were also
nucleus,
observed.
endosymbiont-containing trypanosomatids, like
3-D
reconstruction
tachyzoites
of
T.
kinetoplast
and
flagellum.
In
of
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,
replicas,
and
freeze
fracture
partial
3-D
reconstruction from serial sections or
electron-tomography. Using the slice
and
view
tool
DualBeam™
of
FEI's
(FIB/SEM)
Helios
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
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
Crithidia deanei, this process is more complex,
T.cruzi expressing luciferase was used to
as each daughter cell contains only a single
evaluate the progression of Chagas’ Disease in
symbiotic
the
the murine model. The luciferase gene (firefly,
prokaryote must replicate synchronically with
Promega) was amplified by PCR and cloned in a
the host protozoan. In this study, we used light
vector Topo II blunt. The luciferase gene was
and
with
removed from TOPO vector and cloned in the
threedimensional reconstruction approaches to
integrative expression vector of T.cruzi, pTREX
observe the endosymbiont shape and division
(Vásquez & Levin, 1999). Next, the linearized
during C. deanei cell cycle. We found that the
plasmid was inserted in the epimastigote forms
bacterium replicates before the basal body and
by eletroporation. The metacyclogenesis of
kinetoplast segregations and that the nucleus is
genetic modified epimastigotes (GMO) was
the last organelle to divide, before cytokinesis.
carried out in the LLC-MK2 cells. The
In addition, the endosymbiont is usually found
trypomastigote forms (GMO) were
bacterium,
electron
indicating
microscopy
that
combined
used to
5
close to the host cell nucleus, presenting
infect mice(10 /animal) and the infection could
different shapes during the protozoan cell cycle.
be
(Motta et al. PLos One, 5(8): e12415, 2010)
bioluminescence produced by the infected tissue
monitorized
by the the detection of
and organs, utilizing the IVIS Lumina image
Experimental
Chemotherapy
in
system
(Xenogen).
New
tests
are
being
performed to evaluate the sensibility of this
Trypanosomatids
Trypanosoma cruzi -
Epimastigotes
methodology.
treated with 20, 40 and 50 µM of posaconazole
The effects of different topoisomerase
and observed by FESEM showed extensive loss
inhibitors and DNA binding drugs were tested
of integrity of the plasma membrane with
on the cellular proliferation and ultrastructure of
parasite
the
body deformation.
Moreover, the
Trypanosoma
cruzi
Blastocrithidia
Observations by TEM of epimastigote treated
comparative model, with a more relaxed kDNA
with posaconazole in same concentrations
organization. Our results showed that the
showed
eukaryotic
and
vacuolization
in
was
topoisomerase
used
form.
flagellar pocket also demonstrated deformation.
swollen
culicis
epimatigote
I
as
a
inhibitors,
mitochondria, intense changes of the Golgi
camptothecin and rebeccamycin, were the most
complex,
and
effective compounds in T. cruzi proliferation
alterations in the flagellum. In intracellular
arrest. The eukaryotic topoisomerase II inhibitor,
amastigote form treated with posaconazole,
mitoxantrone, but not merbarone, was effective
cytoplasm vacuolization and plasma membrane
against
shedding were observed. Amastigotes treated
topoisomerase II inhibitors, norfloxacin and
with posaconazole plus amiodarone showed
enoxacin, targeted the kinetoplast specifically,
similar vacuolization in the cytoplasm, changes
thus
in Golgi complex and intense plasma membrane
rearrangement in B. culicis. Among DNA
shedding.
binding drugs, berenil caused remarkable kDNA
cytoplasm
vacuolization
cell
proliferation. The
promoting
prokaryotic
ultrastructural
kDNA
76
disorganization.
With
the
of
Golgi complex at concentrations and times of
camptothecin, there have been no previous
treatment lower than those of dinitroanilines.
evaluations of the compounds tested here on
The in vivo assay miltefosine was effective when
trypanosomatid ultrastructure. We conclude that
the mice were treated orally with doses of 40 and
inhibitors of the same class have different effects
50 mg/kg/day for 21 days. Treated-mice
on
and
presented a significant reduction in the size of
ultrastructure. The results obtained in this work
the lesions, which disappeared and did not grow
may help us to reveal the mechanism of action of
again after a hundred days of the end of
different
treatment. However, the treatment performed
trypanosomatid
exception
proliferation
topoisomerase
inhibitors
in
trypanosomatids.
with trifluralin was ineffective, since it was not
Leishmania amazonensis- Our group has
possible to observe reduction in size of the
evaluated the effects of 13 phospholipids
lesions in the infected-mice. Thus, these studies
analogues and dinitroanilines (trifluralin and
suggest that TC 95, a phospholipid analogue, is a
oryzalin) against promastigotes and intracellular
compound with an interesting leishmanicidal
amastigotes of Leishmania amazonensis. The
activity against L. amazonensis in vitro and that
miltefosine and trifluralin have also evaluated in
miltefosine is effective for the treatment of
vivo against murine models of cutaneous
experimental cutaneous leishmaniasis caused by
leishmaniasis by L. amazonensis infection. From
L. amazonensis.
assays in vitro with phospholipids analogues,
only one compound, the TC 95, was chosen,
Experimental
because the IC50 values were lower than the
Toxoplasmosis
Chemotherapy
in
values obtained to the other compounds. The
8 thiolactomycin (TLM) analogues were
effect of dinitroaniline was also studied, because
tested against tachyzoite-infected LLC-MK2
the TC 95 is a hybrid molecule between
cells. The TLM analogues demonstrated anti-T.
miltefosine and trifluralin. The results of
gondii activity, arresting tachyzoite proliferation
antiproliferative effects for promastigotes and
with IC50 values in the micromolar level after
intracellular amastigotes showed that the TC 95
24 h and 48 h of treatment. Metabolic labelling
has a higher leishmanicidal activity than the
of extracellular parasites treated with TLM
dinitroanilines and miltefosine. Treatment with
analogues using [3H]acetate demonstrated that
TC 95 caused changes in the structure of plasma
these drugs affected acylglycerol synthesis. The
membrane
electron
rapid reduction of parasite load suggests that
microscope and also in its integrity showed by
these compounds have selective cytotoxic effects
fluorometry assays with Sytox Blue. Using light
against
and electron microscopy, we could observe
microscopy demonstrated that TLM analogues
changes in the morphology, cell division at the
interfered with membrane-bounded organelles
stage of cytokinesis, accumulation of lipid
and parasite division and this in turn affected
bodies and important lesions in organelles such
parasite development and survival (Martins-
as mitochondrion, endoplasmic reticulum and
Duarte et al., Parasitol Int. 58:411-15, 2009).
observed
by
scanning
T.
gondii.
Transmission
electron
77
The activity of the antifungals fluconazole (FLZ)
significant survival difference compared to
and itraconazole (ITZ) was also tested against T.
untreated
gondii in mice infected with the Me49 strain. As
20mg/kg/day ITZ significantly reduced the brain
previously
also
cyst burden compared to untreated mice but did
demonstrated a selective effect against T. gondii
not exert significant protection against death.
in vitro; the IC(50) values obtained for FLZ
The results obtained in this work are rather
were 8.9 microM and 3.1 microM after 24h and
promising as ITZ and FLZ are safe and low-cost
48 h of treatment, respectively. A 10-day
drugs available on the market (Martins-Duarte et
treatment of mice with orally or intraperitoneally
al.,
reported
for
ITZ,
FLZ
Exp
mice.
The
Parasitol
administration
124:466-9,
of
2010)
administered 20mg/kg/day FLZ showed a
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, 2010.
2.
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):485-95.
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, 421-434,
2010.
4.
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, 1358-1375, 2010.
5.
Dynasore, a dynamin inhibitor, inhibits
Trypanosoma cruzi entry into peritoneal macrophages.
Barrias, E S; Reignault, LC ; De Souza, W; Carvalho, T
MU. PLoS One. 2010 20;5(1):e7764
6.
Encystation process of Giardia
lamblia:morphological and regulatory aspects. Silvestre
JB; Lemgruber L ; De Souza, W. Archives of
Microbiology, 554, 1-9, 2010.
7.
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, 1432-1455, 2010.
9.
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):217-25.
10.
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, 269-278, 2010.
11.
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, 2010.
12.
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, 2010.
13.
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,
319-324, 2010.
14.
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; MartinsDuarte, ES; Lemgruber L; Attias, M; Vommaro RC.
Scientia Medica (PUCRS), 20: 40, 2010.
16.
Review on Trypanosoma cruzi: Host
Cell Interaction. De Souza W, Carvalho TMU, Barrias ES.
Int J Cell Biol. 2010; pii: 295394
17.
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, 2010.
18.
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):e12415
20.
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, 170-176, 2010.
21.
Toxoplasma gondii: Fluconazole and
itraconazole activity against toxoplasmosis in a murine
model. Martins-Duarte, ES; Lemgruber, L; De Souza, W;
78
Vommaro, RC. Experimental Parasitology, 124, 466-469,
2010.
22.
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):939-44
23.
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, 1665-1676, 2009
24.
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, 149-154,
2009.
25.
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):154-60.
26.
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):11477-90.
27.
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. 2009 46(12):956-63.
28.
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, 487-499, 2009.
29.
Dynamin
inhibitor impairs Toxoplasma gondii invasion.Caldas LA,
Attias M, de Souza W. FEMS Microbiology Letters, 301,
103-108, 2009.
30.
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, 67-124, 2009.
31.
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), 9,120-129, 2009.
32.
Giardia lamblia:
Characterization of ecto-phosphatase activities.
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.
34.
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, 4918-4924, 2009.
35.
Molecular
characterization and intracellular distribution of the alpha 5
subunit of Trypanosoma cruzi 20S proteasome. Gutierrez,
B; Osorio, L; Motta, MCM; Huima- Byron, T; ErdjumentBromage, H; Muñoz, C; Sagua, H; Mortara, R; Echeverria,
A; Arayaa, JE ; González, J. Parasitology International, 58,
367-374, 2009.
36.
Particularities of
mitochondrial structure in parasitic protists (Apicomplexa
and Kinetoplastida). De Souza W, Attias M, Rodrigues JC.
International Journal of Biochemistry & Cell Biology, 41,
2069-2080, 2009.
37.
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):594-602.
38.
Prodigiosin is
not a determinant factor in lysis of Leishmania (Viannia)
braziliensis after interaction with Serratia marcescens Dmannose 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):84-90.
39.
Sterol
Biosynthesis Pathway as Target for Anti-trypanosomatid
Drugs.
De Souza W,
Rodrigues JC. Interdiscip
Perspect Infect Dis. 2009; 642502.
40.
Structural
organization of Trypanosoma cruzi. De Souza W. Mem
Inst Oswaldo Cruz. 2009; 104 Suppl 1:89-100.
41.
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, 1782-1794,
2009
42.
Thiolactomycin
analogues as potential anti-Toxoplasma gondii agents.
Martins-Duarte ES, Jones SM, Gilbert IH, Atella GC, De
Souza W, Vommaro RC. Parasitol Int. 2009 ;58(4):411-5.
43.
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, 665-673, 2009.
44.
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, 2009.
Amazonas JN, Cosentino-Gomes D, Werneck-Lacerda A, Pinheiro AAS, Lanfredi-R
Molecular and Biochemical Parasitology 168, 102-108, 2009.
79
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
10.1007/978-3-642-12863-9_5, Springer-Verlag, Berlin –
Heidelberg, 2010, p. 115-130.
2. 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, 2010
4. 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, 2010
5. 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. 151-161.
6. 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. 393-432.
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.
40-62.
80
AL10
ASSOCIATE LABORATORY OF GENOMIC,
PROTEOMIC, MODELING AND NANOSCOPY
OF BIOLOGICAL SYSTEMS
Coordinator: Paulo Mascarello Bisch, IBCCF/UFRJ
Members:
Gilberto Weissmuller
Geraldo Antônio Cidade
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,
2850 – 2859.
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
millisecond scale, involved, for instance, in the
atomic force spectroscopy, a method now
catalytic activity of enzymes, are still difficult
currently used by our group, was used to study
to be attained and analysed. The proposed
the interaction of a HIV fusion inhibitor with
method combines a short dynamics, typically
lipid films, in a collaborative work with the
50 ns, with a normal modes analysis, given a
group of Dr. Castanho from University of
robust picture of important motions involved
Lisbon. The paper reveals important structural
in catalytic activity, as exemplified in the
details of this interaction and the mechanism of
article for the HIV-1 protease.
inhibition, which should be used as a guide to
design new and more efficient inhibitors.
Structural
Changes
of
the
Paraflagellar Rod during Flagellar Beating
Molecular analysis of VCA1008: a
in Trypanosoma cruzi. Gustavo Miranda
putative phosphoporin of Vibrio cholerae.
Rocha, Dirceu Esdras Teixeira, Kildare
Goulart, Carolina L. ; Lery, Letícia M.S. ;
Miranda,
Paulo
Diniz, Michelle M.P. ; Vianez-Junior, João L. ;
Mascarello Bisch, Wanderley de Souza.,
Neves-Ferreira, Ana Gisele C. ; Perales, Jonas
PLoS ONE 5(6): e11407. (2010).
; Bisch, Paulo M ; von Krüger, Wanda M.A.
Gilberto
Weissmüller,
The unusual combination of Electron
Microscopy with Atomic Force Microscopy
was used in a fruitfull collaboration with the
FEMS Microbiology Letters, 298 (2009) 241–
248
As
a
consequence
of
previous
group of Prof. Wanderley de Souza by
proteomic analysis, we have identified a new
showing within molecular details the structural
putative phosphoporin involved in the response
changes during flagellar beating in T. cruzi.
of phosphate starvation. We have made a
Our work is pioneer showing the powerful
detailed molecular analysis of the expression
combination of these techniques.
and modeling the structure of this new porin,
which should have an important role on the
Unravelling the molecular basis of
adaptation during human colonization by V.
the selectivity of the HIV-1 fusion inhibitor
cholerae. The studies of the expression
sifuvirtide
conditions,
towards
bioinformatics
and
computer
rich rigid membranes. Henri G. Franquelim
modeling have helped us to confirm the
A. Salomé Veiga, G. Weissmüller, Nuno C.
function of the new protein and have shown
Santos
the effectiveness of such approach.
and
Miguel
A.R.B.
Castanho,
Biochimica et Biophysica Acta (BBA) –
Biomembranes, Volume 1798, Issue 6, June
2010, Pages 1234-1243.
One of the crucial steps on the virus
Effects of light intensity and light
quality on growth and circadian rhythm of
saxitoxins
production
in
invasion of cells is the virus fusion with the
Cylindrospermopsis
cell lipid membrane. The combination of
(Cyanobacteria). Ronaldo Leal Carneiro,
several spectroscopic techniques, including
Maria Elisângela Venâncio dos Santos, Ana
raciborskii
82
Beatriz Furlanetto Pacheco and Sandra Maria
Cyanobacteria, a very important contaminant
Feliciano de Oliveira e Azevedo. Journal of
of waters, especially in our region. The initial
Plankton Research, Volume 31, Number 5,
work concerns the conditions of toxin
Pages 481–488, 2009.
production as a function of light radiation of
Covering a new subject, we have
the cyanobacteria culture. Our groups will
started a new collaboration with the group of
investigate proteome and protein expression
Dr. Sandra Azevedo about toxin production in
regulation of thir bacteria.
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, 2850 – 2859.
2- 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): e11407.
(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 1234-1243.
4. 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) 241–
248
5- 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 481–
488,
2009.
83
84
AL11
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
Experimental
chemotherapy
in
been conducted by Roberta Ferreira Cura das
trypanosomatids- We have analyzed the effects
Neves, now a PhD student that presented her
of natural compounds such as snake and bee
master theses, also related to shedding process,
venons on the proliferation and ultrastructure
in
searching to define the kind of cell death process
undergraduated student, Grazielle Lima Cruz in
involved in parasite death. This topic has been
the same topic. During the last 12 months,
developed by 2 students: Camila Marques Adade
Roberta has been analyzing the presence of
that has recently concluded her PhD theses (July
metalloproteases in the shedding vesicles from
2010) and continues in the laboratory as a Pos-
trypomastigotes of different strains of T. cruzi
Doc student; The second student is Gabriela
and the effect of cell surface ligands, such as
Santos Ferreira das Chagas, an undergraduated
cationized ferritin and concanavalin A, in
student co-oriented by Camila Adade that works
stimulating shedding process. Nowadays the
with bee venons. Recently, Camila published a
students are accumulating shedding vesicles to
paper and a review about chemotherapy in T.
analyze
cruzi. Figure 2
replicas.
The analysis of shedding process in T.
cruzi-
The
their
2009.
Roberta
ultrastructure
Ultrastructural
co-orients
in
an
criofracture
analysis
of
and
trypanosomatids isolated from fishes and toads-
the
This topic is developed by a PhD student, Moara
components of the shedding vesicles from
Lemos that analyses at the scanning and
differente strains of the parasite. In this topic we
transmission
have analyzed the ultrastructure, composition
ultrastructure of trypanosomes obtained from the
(presence of proteases and antigens), and
blood of Brazilian fishes and toads. The
signaling pathways involved in the process of
development of a tridimensional model using
shedding of different vesicles by trypomastigote
tomography is in the planes for the next year.
immunocytochemical
ultrastructure
August
detection
of
electron
microscopes
the
and amastigote forms of T. cruzi. This topic has
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
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. 2010
2 - Laboratório de Ultraestrutura e Biologia
Biomineralization of magnetosomes-
Celular de Procariotos - coordinated by Dr.
We have studied the biomineralzaition of
Ulysses Lins focus its attention to the study of
magnetosomes in magnetotactic bacteria for
the biology, diversity and biomineralization in
several years. We have studied the purity of the
magnetotactic bacteria.
magnetosome particles and have shown that they
can incorporate small amounts of magnese in
The main subjects of our interest are:
their crystalline structure (Figure 4).
Cultivation of magnetotactic bacteria-
Biology and diversity of magnetotactic
Magnetotactic
bacteria
bacteria- During the period, we advanced in the
microorganisms.
But,
description of magnetotactic bacteria in extreme
understanding of the cell biology it is mandatory
environments. To achieve that goal we analyzed
to grow these cells in pure cultures. For that we
samples collected from sediments with extreme
have established a collaboration effort with
conditions: high temperature, low temperature,
Professor Dennis Bazylinski from University of
high salinity and high sulfur content. At least
Nevada, LV, EUA. Dr. Bazylinski is one of the
four new types of magnetotactic bacteria were
world
discovered
microorganisms. So far, we have been able to
(ultrastructure,
and
their
in
in
the
magnetotactic
magnetosomes) and phylogenetic (16S rDNA
freshwater magnetotactic bacteria. Also, we are
sequencing)
now trying the isolate and grow one strain from
were
of
advance
cultivate at least three strains of marine and
characteristics
analysis
experts
fastidious
the
(Figure 3).
mineral
morphological
leading
to
are
described
sediments collected in Brazil.
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.
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): 563-570,
2009.
2) 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): 6883-6899,
2009.
3) 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, 2009.
4) 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 AntiParasitic 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 SoutoPadrón. A Review on the Ultrastructure of
Trypanosoma cruzi. in EMERGING CHAGAS
DISEASE, CHAPTER FIVE Pp. 40-62 - eISBN:
978-1-60805-041-3, 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: 250–253, 2009.
2) 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: 3331-3343, 2009.
3) 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: 353–365, 2009.
4) 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:
193-201, 2009.
5) 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: 1450-1459, 2009
6) 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: 110-116, 2010.
7) J. P.Albuquerque, C. N. Keim, U. Lins.
Comparative analysis of Beggiatoa from
hypersaline and marine environments. Micron 41:
507-517, 2010.
8) 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, 2010.
9) 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: 3220–3227, 2010.
10) C.T. Lefevre, F. Abreu,; M.L.
Schmidt, U Lins, R.P. Frankel, B.P. Hedlund, D.
Bazylinski,
Moderately
thermophilic
89
magnetotactic bacteria from hot springs in Nevada
USA. Applied and Environmental Microbiology
76: 3740-3743, 2010.
11) 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,
2011.
12) 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, 2011.
13) 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, 2011.
CHAPTER BOOK
1) U. Lins , D. Bazylinski, D. (2009).
Magnetotaxis. In: Moselio Schaechter. (Org.).
Encyclopedia of Microbiology. 3 ed.EUA: Elsevier
Inc., pp. 229-241.
90
AL12
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,
fluorescence
such
as
microscopy,
videomicroscopy,
scanning
and
91
transmission electron microscopy. The analyses
5)
We have published an important
revealed that approximately 55% of the parasites
article concerning the effects of jasmonates in
were in pseudocyst form at each preputial
trichomonas. Jasmonates are a group of small
sample, whereas approximately 25% of T. foetus
lipids that are produced in plants and function as
displayed pear-shaped body.
stress hormones. We tested this drug against
Trichomonas vaginalis and we demonstrated
3) We have isolated organelles using cell
using flow cytometry, JC-1 and scanning and
fractionation form trichomonas. The main
transmission electron microscopy that MJ
organelles were: hydrogenosomes, costa and
induced the cell death of T. vaginalis parasites.
Golgi. One article is in preparation, showing two
specific proteins found in trichomonas Golgi.
6)
Concerning Giardia lamblia, we
have obtained gerbils to in vivo infection.
4)
The
antiproliferative
and
Processes of encystation and excystation were
ultrastructural effects of sterol biosynthesis
performed in vitro, successfully. We have used
inhibitors against T. vaginalis were investigated.
immunocytochemistry to compare the behavior
It was found that 22,26-azasterol and 24(R,S),25-
of the encystation specific vesicles (ESV) and
epiminolanosterol, known inhibitors of 24(25)-
try to find the granules responsible for the
sterol
exhibited
carbohydrates portion of the cyst wall. Using
vaginalis
immunofluorescence we detected two granules
trophozoites cultured in vitro. Morphological
populations, which is an important result. In
analyses showed that azasterols induced changes
addition, cytochemistry for acid phosphatase
in the ultrastructure of T. vaginalis. The most
demonstrated a positive reaction in a distinct
significant alterations
sub-population of vesicles localized in Giardia
methyltransferase,
antiproliferative
effects
on
T.
were (1)
membrane
blebbing and disruption, (2) cell wrinkling and
when they are under excystment.
(3) the formation of cell clusters. In addition,
autophagic vacuoles, Golgi duplication arrest, an
7) In another report we showed the
abnormal Golgi enlargement and damaged
cytopathic effect of Trichomonas vaginalis,
hydrogenosomes
using oviduct cells. It occurs due to mechanical
were
also
cytotoxicity
assays
observed.
the
stress and subsequent phagocytosis of the
cultured mammalian cell lines MDCK showed
necrotic cells. The investigation was done using
no effect of the azasterols on the viability and
a primary culture of bovine oviduct epithelial
proliferation of these cells at a concentration that
cells (BOECs), grown either in monolayers or as
significantly inhibited the proliferation of T.
floating cells. Trophozoites displaying different
vaginalis, indicating a selective antiparasitic
virulence levels were co-incubated with BOECs
action. Taken together, these results suggest that
for times varying between 1 min and 48 h.
azasterols could be important compounds in the
Analyses
development
videomicroscopy, scanning and transmission
Nonspecific
of
novel
using
chemotherapeutic
approaches against T. vaginalis.
were
performed
using
electron microscopy, colourimetric assays and
92
cytochemistry. Injury was observed as early as 1
Phagocytosis occurred by trichomonads avidly
h after incubation, while after 12 h the host cells
eating
were
fresh
containing the nucleus and other organelles, but
trichomonad isolate was used. Trichomonads
living or intact cells were not ingested. Necrotic
attack the host cells by clustering around them.
fragments were rapidly digested in lysosomes, as
Mechanical stress on the microvilli of the host
shown by acid phosphatase and ruthenium red
cells was observed and appeared to induce
assays where only the BOECs were labelled.
plasma membrane damage and cell death. After
The lytic capacity of the trichomonads was more
membrane injury and lysis, fragments of the
pronounced in host cell suspensions.
severely
damaged
when
a
large
portions
of
epithelial
cells
necrotic cells were ingested by trichomonads.
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.
1.Giardia lamblia behavior during
encystment: how morphological changes in shape
occur. Parasitology International. Parasitol Int.
2009 Mar;58(1):72-80.
2. Hydrogenosomes under microscopy- a
review- Marlene Benchimol-Tissue and Cell.
Tissue Cell. 2009 Jun;41(3):151-168. Epub 2009
Mar 17.
3. Cytoskeleton in Trichomonads. Trends
in Cell & Molecular Biology. Vol.4, pp 25-39
(2009).
4. Tritrichomonas foetus: budding from
multinucleated
pseudocysts.
Pereira-Neves,
Antonio and Benchimol, Marlene-Protist, 2009,
160, 536-551.
5. Cytopathic effects of Tritrichomonas
foetus on bovine oviduct cells. V. Midlej, R. Vilela,
A. B. Dias, M. Benchimol. Veterinary ParasitologyVeterinary Parasitology 165 (2009) 216–230
6. 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 2009. 10, 330-338.
7. 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.
2009 Dec;58(4):424-37.
8. 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. 2010 Mar
2;5(3):e9439.
9. 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) 387-393
10.
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. 2010.
11. Trichomonas vaginalis kills and eats –
evidence for phagocytic activity as a cytopathic
effect. Midlej, V., M. Benchimol. Parasitology.
2010 Jan;137(1):65-76.
93
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. 2010. Aceito em julho de
2010.
13. Identification of Tritrichomonas foetus
pseudocysts in fresh preputial secretion samples
from bulls. Antonio Pereira-Nevesa, b, Carlos
Manuel Camperoc, Alfredo Martínezd and Marlene
Benchimola, 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. 1-26.
2. 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. 266-.
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.1.
3. Siano Miguel, D. Esdras Teixeira,
BENCHIMOL, M.-Dinâmica da Terra. Rio de
Janeiro : CEDERJ, 2009, v.1.
4. 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
AL13
STRUCTURAL BIOTHECNOLOGY
Coordinator: Celso B. Sant'Anna Filho, INMETRO
Members:
Wanderley de Souza
Kildare Miranda
THE Gustavo Conde Menezes
Daniela Lourenço
Danielle Cavalcanti
Isabel Port-Carreiro
The newly established Biotechnology
Laboratory, at National Institute of Metrology,
Standardization
and
INMETRO, has
a
Quality
–
composed by
6
Industrial
group
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
composed of polysaccharides (cellulose and
hemicelluloses)
biomass,
and
including
lignin.
Lignocellulosic
sugarcane,
has
been
The feedstocks usually adopted for
biodiesel
production
are
vegetable
oils.
Recently, much attention has been paid to the
considered as potential source to second
exploration
generation biofuel production. The technology
productivity of microorganisms greatly exceeds
used to conversion of fermentable sugar in
the vegetable productivity and non-arable land is
bioethanol involves pretreatment, which aim to
used
improve digestibility of biomass, such as acidic
oleaginous yeasts and microalgaes and analyze
and thermal degradation. The PCW molecular
the oil content using Nile Red staining and
architecture remains unclear and it has been
Confocal Laser Scanning Microscopy (Figure 2).
to recalcitrance of biomass to
A
deconstruction.
We have applied high resolution
for
of
microbial
production.
oils.
We
The
isolated
oil
some
related
B
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
5
µ
Figure 2. Confocal laser scanning micrographs of microalgae
M
mass spectrometry, we have also analyzed the
Ankistrodesmus (a) and a yeast (b). Nile red staining shows the
lipid bodies (yellow ) inside these microrganisms.
protein of PCW sugarcane, to get some
information for improving the process of ethanol
Other focus of our work is the study of
saccharification (Figure 1d).
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
transformation
of
structure
amastigotes
during
the
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;
INBEBof2009-2010
INBEBMicroscopy.
2009-2010 ANNUAL
REPORTof proteins of
(c) Error signal
Atomic Force
(d) 1D-PAGE
sugarcane cell wall.
96
isolated kDNA networks of trypanosomatids
etched and rotary-replicated cells (Figure 4c) to
using AFM. The results allow an examination of
obtain detailed information of the PFR structures
kDNA at high resolution (Figure 3). We were
in regions of the flagellum in straight and in bent
able to identify regions of overlapping kDNA
state. The images obtained show that the PFR is
molecules and sites where several molecules
not a fixed and static structure. Measurements of
cross.
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
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.
using
graphic
computation, we proposed
an animated model for the
PFR
structure
during
The function played by the PFR is not
flagellar beating, providing a new way to
well established. To this, we have started to
observe the PFR filaments during flagellar
investigate by atomic force microscopy (Figure
beating. A stationary frame of the flagellum in
4a-b) and transmission electron microscopy of
straight state can observed in Figure 4d.
replicas of quick-frozen, freeze-fractured, deep-
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
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. 1782-1794, 2009.
2.
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. 67-124, 2009.
3.
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. 1462-1472, 2009.
4.
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.
115-130.
5.
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, 2010.
6.
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, 2009.
7.
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): 629-33.2010.
8.
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. 939-944,
2010.
9.
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, 2010.
10.
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, 2010.
11.
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, 11751182, 2009.
12.
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, 11477-11490, 2009.
13.
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
AL14
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
compounds called physalins, which showed
spreading ability, followed by cytoskeleton
antileishmanial activity in vitro and in vivo for
rearrangement, a high number of cytoplasmatic
cutaneous leishmaniasis. Thus, we consider
projections and enhancement of the phagocytosis
interesting to analyze the effects of the aqueous
process, besides a greater superoxide production.
extract obtained from roots of P. angulata
Moreover, it acted decreasing the growth of
against promastigotes of L. amazonensis in vitro
promastigotes and intracellular amastigotes. In
and its effects on host cell.
vivo topical treatment with HMP-ointment
We have recently demonstrated that P.
promotes healing process and suppressing ulcer
angulata aqueous extract effectively inhibits
dissemination. In addition, many collagen fibers
growth of promastigotes forms and promotes
were found in infection sites of HMP-treated
ultrastructural
animals.
bioproduct
parasite
analyzed
hydroxymethyl-γ-pyrone
alterations.
is
the
Other
5-hydroxy-2is
effectively inhibits the growth of parasites in
produced by some species of Aspergillus fungi,
vitro and in vivo, mainly by the activation of
has bacteriostatic activity and inhibition effect of
host cell microbicidal response and does not
the tyrosinase enzyme in the process of melanin
have cytotoxic effects on the host cells. Thus,
biosynthesis.
antileishmanial
both HMP and the extract from Physalis
activity and the effects on host immune cells are
angulata could be useful as alternative source
not well known. Macrophages treated with 50
for a new antileishmanial agent.
However
(HMP),
its
which
These results demonstrated that HMP
µg/mL of HMP showed increased cytoplasm and
FIG. 1. Effects of Physalis angulata extract on growth curve of Leishmania amazonensis promastigotes.
CTL: control; GLU: glucantime.
100
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
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
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. 1075-1081, 2010.
2. 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. 154-160, 2009.
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), 2010.
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-2hydroxymethyl-4-Pyrona as a macrophage activation agent
to combat Cutaneous Leishmaniasis. 2009
International: RODRIGUES, Ana Paula
Drummond ; Silva, E.O. ; Santo, A.S. ; Carvalho, ASC ;
ALVES, C. N. ; NASCIMENTO, J. L. M. . Use of 5hydroxy-2-hydroxymethyl-4-Pyrona as a macrophage
activation agent to combat Cutaneous Leishmaniasis. No.
Reg PCT/BR2009/000254. 2010.
102
AL15
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 1947. 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
DEC effectively suppressed the effects
mice. Forty-eight male C57BL/6 mice were
of monocrotaline challenge on lung cells: a)
separated in groups: control group (C, DEC25 e
optical
some
DEC50) and alcoholic group (CEtOH, EtOH25 e
alterations after MCT treatment, such as
EtOH50). After the induction of alcoholism,
emphysema, and accumulation of inflammatory
mice were submitted 12 days of treatment with
cells, associated with edema fluid. DEC reversed
DEC solutions in concentrations of 25 and
these effects, showing preserved epithelium; b)
50mg/kg orally. Biochemical analyses were
Transmission Electron Microscopy revealed
performed and liver fragments were processed
some alterations after MCT treatment, such as
for light microscopy and transmission electron
fluid accumulation in the lung, characteristic of
microscopy. The level of AST increased
edema,
dilated
significantly in the control group subjected to
mitochondria with degenerated cristae. DEC turn
alcohol (CEtOH) compared with the control
the lung morphology close to the control group;
group (C). We observed a significant reduction
c)
and
of serum AST and alcoholic groups DEC-treated
decreased
EtOH25 and EtOH50 compared with the group
labeling to the inflammatory mediators tested
that received no treatment CEtOH. The serum
(IL-6,
treatment.
ALT,
pulmonary
avaliations showed no significant differences
microscopy
and
demonstrated
pneumocytes
with
Immunohistochemistry
Immunocytochemistry
VCAM-1)
Conclusions:
revealed
after
DEC
MCT-induced
alkaline
phosphatase
and
bilirubin
dysfunction was attenuated by DEC treatment,
among the groups.
probably via inhibition of a number of steps in
CEtOH group showed evident hepatocellular
arachidonic acid pathway, thereby preventing
damage. However, in EtOH25 and EtOH50
the
groups, we observed a reduction of damage
production
of
eicosanoids,
important
inflammatory intermediates.
Histological analysis of
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
INFLAMMATORY
study
entitled
ANTI-
Ultrastructural analysis of hepatocytes EtOH25
EFFECT
OF
and EtOH50 group presented well-preserved
DIETHYLCARBAMAZINE
IN
HEPATIC
organelles.
Immunohistochemistry
CEtOH
CELLS OF ALCOHOLIC C57BL/6J MICE
group revealed expression of inflammatory
analyzed
markers IL-6, eNOS, CCR2, VCAM and ICAM,
the
protective
effect
of
diethylcarbamazine in hepatic cells of alcoholic
however
the
EtOH50
group
showed
no
104
immunoreactivity for any of these markers.
twenty animals received 25mg/kg body weight
According to the present results, DEC is a
of Sildenafil for 4 weeks administered in the
potential drug for the treatment of chronic
drinking water, and the dose was monitored by
inflammation induced by alcoholism
daily weighing of the water bottle. The control
LI
LI
LI
LI
V
V
LI
**
A
LI
V
B
C
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
is Sildenafil that is an important inhibitor of the
phosphodiesterase-5. In fact, Sildenafil is a
group, also composed by twenty animals,
received only pure water.
Leydig cells in control group showed
novel, orally treatment approach for pulmonary
morphological
hypertension. Its pharmacological action is the
cytoplasm
inhibition of cGMP-specific phosphodiesterase
endoplasmic reticulum, lipid droplets, scattered
type 5 (PDE5) enzyme, which is abundant in the
lysosomes, Golgi, mitochondria with tubular
pulmonary vasculature. The therapeutic scheme
cristae, and rough endoplasmic reticulum.
with high daily doses improves the pulmonary
Sildenafil-treated Leydig cells showed some
vascular haemodynamics and therefore the
morphological alterations as a vesicular smooth
exercise
the
endoplasmic reticulum, large vacuoles in the
expression of PDE5 was detected in Leydig and
peripheral cytoplasm, presumably opening into
myoid cells of rat testis, it is necessary to
the extracellular space. The mitochondria were
investigate whether compounds that interfere on
enlarged with disarranged or discontinued
PDE5 activity could affect the steroidogenesis.
cristaes, and several vesicles were observed on
In
entitled
whole peripheric membranes, probably involved
PHOSPHODIESTERASE TYPE 5 INHIBITOR
in hormonal secretion. All of these alterations
STIMULATES
CELL
described above are typical of an activated cell.
some
Immunocytochemistry revealed an augmented
ultrastructural alterations in mice Leydig cells
expression of StAR protein, P450scc and
and increased testosterone levels after chronic
testosterone in isolated Leydig cells, when
treatment with the phosphodiesterase type 5
compared to the controls. The serum testosterone
inhibitor. One experimental group composed by
levels were significantly higher in 25mg/kg
capacity
a
of
patients.
recent
STEROIDOGENESIS,
Since
study
LEYDIG
we
reported
rich
characteristics
in
as
anastomosing
areas
of
tubular
105
Sildenafil administrated mice when compared to
cellular
control
steroidogenesis
animals
(Mann-Whitney,
Z=3.26,
morphology
in
suggests
abnormal
sildenafil-treated
group;
p<0.01). The results obtained in the present
however, it is necessary to perform a hormonal
study are consistent with the hypothesis that the
assay to confirm these results.
accumulation of cGMP, by PDE5 inhibition, and
The therapeutic use of Sildenafil
activation of its dependent pathways could be
pulmonary
involved
pulmonary
in
the
androgen
biosynthesis
hypertension
vascular
in
improves
the
haemodynamics.
The
stimulation. Important clinical implications of
treatment involves the administration of high
hormonal disorders should be taken into account
doses daily, and until now, little is known about
for patients with pulmonary hypertension.
the action of this scheme on neuronal cells. In
In relation to the effects of chronic
the CNS, the cerebellum present constitutively
treatment of Sildenafil on mice ovary, the work
PDE5, and it has been showed that cGMP-
ULTRASTRUCTURAL
OF
pathways protect oligodendrocytes [1]. Besides,
SILDENAFIL TREATMENT IN LUTEAL
mice lacking iNOS exhibit more demyelination
CELLS showed that Luteal cells from control
in demyelinating model [2]. This study entitled
group showed lipid droplets and mitochondria
SILDENAFIL
PDE5
with tubular cristae. Ribosomes and enlarged
THERAPEUTIC
OPPORTUNITY
smooth endoplasmic reticulum were visible.
MYELIN REPAIR was conducted to investigate
Luteal
group
the iNOS role in myelination and astrocytes
showed numerous large mitochondria, with
physiology, and if Sildenafil oral treatment acts
tubular cristae, and lipid droplets were visible.
in iNOS-/- mice nervous tissue. Ten B6.129 P2-
Several ribosomes were present in cytoplasm,
Nos2 (knockout iNOS) mice, 21-day-old, were
scattered or attached to the endoplasmic
used for each group. One group received
reticulum. However, the smooth endoplasmic
25mg/kg of Sildenafil/8 weeks, administered in
reticulum was compacted compared to control
the drinking water. The control group received
group. Nevertheless, morphology from control
pure water. The genetic background C57BL/6
and
normal
wild type without treatment was used for
steroidogenesis. Biochemical analyses showed
comparison. The animals were sacrificed by
that serum levels of HDL, LDL, VLDL,
perfusion and pieces of cerebellum were
cholesterol
no
processed for electron microscopy. The GFAP
significant differences between groups. Previous
(glial fibrillary acidic protein) levels were
studies
detected
cells
from
treated
and
(Donato
EFFECTS
Sildenafil-treated
group
suggests
triglyceride
et
al,
presented
2009)
using
by
INHIBITOR:
FOR
immunohistochemistry.
phosphodiesterase-5 inhibitor Vardenafil showed
Morphometrical analysis revealed that myelin
different results, as a diminished serum level of
was thicker in wild type (p < 0.01) and iNOS-/-
HDL after treatment. This can be explained by
Sildenafil (p < 0.001), comparing to iNOS-/-
the fact that although Sildenafil and Vardenafil
control; the mean of g-ratio (the ratio of the
inhibit phophodiesterase-5, they may interact
inner axonal diameter to the total outer
with different cellular mechanisms. In our study,
diameter), was less in wild type (p < 0.05) and
106
iNOS-/-Sildenafil (p < 0.01), than in iNOS-/-
increase in this protein labeling was seen, which
control, indicating thinner myelin in this group.
returned to physiological levels after Sildenafil
The
presented
treatment. The results obtained in the present
noteworthy ultrastructural alterations in myelin,
study are consistent with the hypothesis that the
graded as grade II (diffused local disarrangement
inducible
of myelin sheath resembling as a ―collar‖ but
downregulate the cGMP signaling, damaging the
preserving structural arrangement), and wild
myelin structure, and causing a clear reactive
type and iNOS-/-Sildenafil were graded how
gliosis,
grade I (well-preserved myelin with few local
accumulation of cGMP by PDE5 inhibition
disarrangement), in a myelin disarrangement
could improve the myelination in cerebellum,
scale arbitrarily defined (I-III). The GFAP, an
and extinguish the reactive gliosis. These results
astrocytes activation (reactive gliosis) marker,
point to important potential clinical applications
showed physiological expression in the wild
potential of Sildenafil in demyelinating disease,
type, however, in iNOS-/-control, an intense
such as multiple sclerosis
axons
of
iNOS-/-control
generated
in
NO
cerebellum.
absence
However,
can
the
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
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.429 - 437, 2010.
2.
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,
2010.
3.
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.454 - 462, 2009.
4.
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.845 - 850, 2009.
5. 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-09-0073.R1. Reproductive Biology and
Endocrinology. , v.1, p.8 - 13, 2010.
6. 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.4782 - 4789, 2009.
108
AL16
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
containing the Yamanaka factors. In fact we
cardiogenesis.
have been able to produce iPSC-like cells using
preparation of lentiviral vectors containing each
only three factors (Klf4, Sox2, Oct3/4), without
of these factors and will transduce fibrobasts
the need for c-Myc, with great efficiency and in
containing a cardiac myosin heavy chain
shorter times (first colonies detected at 4 days
promoter linked to a GFP reporter gene to screen
after
this
for the relevant factors. During the course of this
improvement to the endogenous expression of
project, Deepak Srivastava, at the Gladstone
some of the pluripotency factors in the MSC
Institute in California, described the direct
from menstrual blood and neonatal tissues.
differentiation of mouse dermal and cardiac
Differentiation of the iPSC-like cells into cell
fibrobasts into cardiomyocytes, demonstrating
types of ecto, meso and endodermal origin has
the feasibility of this project.
transduction).
We
attribute
We are now starting the
been shown, and we are currently working on
All our in vitro experiments are geared
developing teratomas from these cells to prove
towards generating cells that can be used for
that they are bona-fide iPSC.
cardiac repair. In our in vivo experiments we are
We
are
also
working
on
direct
using
four
animal
disease
cardiovascular
cardiomyocytes. This project is at a preliminary
infarction
stage, where we have cloned 15 transcription
cardiomyopathy
factors
hypertension model, and an induced diabetes
play
prominent
roles
during
model,
a
of
differentiation of human cardiac fibrobasts into
that
alterations:
models
a
chronic
model,
a
myocardial
chagasic
pulmonary
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
INBEB
2009-2010
INBEB 2009-2010 ANNUAL REPORT
gradient
method.
110
hearts and vasculature of these animals, using
Chagasic and dilated cardiopathies and in stroke
the multi-imaging facility that was created by
patients,
our Institute.
mononuclear cells.
using
bone
marrow
derived
We have also actively participated in
clinical trials performed in patients with
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
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
1. Ribeiro, V. P. ; Maia, A. C. V. ; Werneck-DeCastro, 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.
290-296, 2010.
2. Louzada, Ruy A. N. ; Oliveira, Patricia F. ;
Cavalcanti-De-Albuquerque, Joao Paulo A. ; CunhaCarvalho, Leandro ; Baldanza, Marcelo R. ; KasaiBrunswick, 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, 2010.
3. Nihei, Oscar K ; Fonseca, Paula C ; Rubim,
Nara M ; Bonavita, Andre G ; Lyra, Jurandy Spo ; NevesDos-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,
2010.
4. 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. 377-389, 2010.
5. 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, 2010.
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,
2010.
7. 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. 20051298, 2010.
8. 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. 20546573, 2010.
9. Da Fonseca, L. M. B.; Gutfilen, Bianca;
Batistela, V.; Castro, P. H. R.; Goldenberg, Rcs; KasaiBrunswick, 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 IntraArterial Injection. Experimental Neurology, V.
221, P. 122-128, 2010.
10. 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, 2010.
11. Carvalho, Adriana Bastos ; Campos De
Carvalho, Antonio Carlos . Heart Regeneration: Past,
Present And Future. World Journal Of Cardiology, V. 2, P.
107-111, 2010.
12. 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. 18-24, 2010.
13. 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. 989-995, 2010.
14. Azevedo-Pereira, R.L ; Medei, E. ; MendezOtero R ; Marcondes J ; Alves-Leon S.V. "Isolation
Ofneurosphere-Like Bodies From An Adult Patient With
Refractory Temporal Lobeepilepsy..". Arquivos De NeuroPsiquiatria Epub, 2010.
15. 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 2010.
16. 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. 431437, 2010.
17. 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.
244-251, 2010.
18. 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. 524-539, 2009.
19. 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): 403-412, 2009.
20. 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. 75-81, 2009.
113
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. 1140-1149, 2009.
22. 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,
2009.
23. 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, 2009.
24. Campos De Carvalho A. C. . Physiology In
Brazil: Past And Present. Journal Of The Physiological
Society Of Japan, V. 71, P. 148-150, 2009.
25. 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, 2009.
26. 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. 539-541,
2009.
27. 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 LType Calcium Current Blockade And Reduced Intracellular
Calcium Transients. British Journal Of Pharmacology, V.
158, P. 580-587, 2009.
28. 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 NeuroPsiquiatria (Impresso), V. 67, P. 633-638, 2009.
29. 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. 719-724, 2009.
30. 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,
2009.
31. 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. 1307-1311, 2009.
32. 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, 2009.
33. 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. 416-426, 2010.
34. 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.
377-389, 2010.
114
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
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 < 0.001 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 < 0.001 vs SHAM and SHAM + BMMC. #P < 0.001 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
Ca2+-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
Importantly,
or
recovery
BMMC
(Fig.
infusion
2;
shifted
[2]).
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 Ca2+-ATPase in
basolateral membranes of proximal tubule
cells in ipsilateral and contralateral kidneys,
but BMMC infusion maintains control levels.
(A) Representative immunoblotting for Ca2+ATPase (5F10 antibody). (B) Densitometric
representation
of
the
Ca2+-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
CTR
ATPDP
ATPDP/MSC
A
B
*
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%
CO2 and 95% air at 37 0C) 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
Clinical, epidemiological and
in renal function due to undernutrition.
experimental data confirm the critical role of the
o
Laboratory N 17 has a branch
kidneys in the long-term regulation of systemic
at Federal University of Pernambuco, where
blood pressure and in essential hypertension,
pioneer studies showing that programming by
which is strictly correlated to the ability of the
perinatal
chronic
kidneys to fine-tune the level of sodium
undernutrition leads to severe cardiovascular and
excretion. Generation of hypertension appears to
renal dysfunctions in adult life have been
be correlated, at least in part, with high levels of
developed over the last decades. In the period
Ang II in the renal cortex. In this context two
covered by this report we published together 4
main findings emerged from the studies carried
studies where we demonstrated for the first time
out in this period. First, we found that high
that renal lesions occur at the level of sodium-
levels of Ang II in the kidney are associated with
transporting ATPases and the signaling cascades
the generation of Ang-(3-4), a peptide that
involved in their regulation. The main finding
antagonizes the influence of Ang II on cellular
was to demonstrate that undernutrition leads to a
calcium via stimulation of the plasma membrane
constitutive up- or down-regulation of key
Ca2+-ATPase. We described the interconnected
protein kinases that participate in the regulation
enzymatic pathways of Ang-(3-4) formation in
of these ATPases by angiotensin II (Ang II) and
renal
derived
homeostasis (Figs. 4,5; [4,5]).
undernutrition
peptides.
Most
or
importantly,
we
cortex and its effects
on calcium
demonstrated that prevention of deleterious
Second, we demonstrated that Ang II
effects of early undernutrition can be obtained at
type 1 receptor is involved in the regulation of
the level of gene transcription and protein
renal Na+-ATPase in spontaneously hypertensive
expression [3].
rats (SHR), and long-term treatment with an
antagonist of this receptor (losartan) blocks the
3) Cellular and molecular basis for the action
onset of hypertension in adult rats (Fig. 6; [6]).
of hormones and autacoids in renal tissue.
The results indicate a clear correlation between
AT1 receptor activation in SHR and increased
117
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].
Figure 5. Femtomolar Ang-(3-4) reactivates the
Ang II-inhibited Ca2+-ATPase after binding to
AT2 receptors. (A) Basolateral membranes
preincubated or not with 10-10 M Ang II, as
shown, were assayed for Ca2+-ATPase activity as
described under Materials and Methods in [5], in
the presence or absence of Ang-(3-4) and the AT2
receptor
antagonist
PD123319
in
the
combinations and concentrations shown on the
abscissa. (B) Ca2+-ATPase activity was measured
with the combination of 10-10 M Ang II and
increasing concentrations of Ang-(3-4) as shown.
pA1/2 ~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];
physiological
(ii)
we
activation
demonstrated
of
cyclic
that
AMP-
dependent protein kinase (PKA) modulates
4) Alterations of copper transporters in
ATP7B (Fig. 6; [7]); (iii) we described the
neglected diseases.
serines of highest scores for phosphorylation by
Progress in understanding the
PKA in eukaryotes (Ccc2 in Saccharomyces
basis for altered copper homeostasis in the
cerevisiae; ATP7A and ATP7B in humans) that
neglected Menkes and Wilson diseases can be
appear to be involved in the regulation of
summarized as follows: (i) we succeeded for the
copper-transporting ATPases (Fig. 7).
first time in precise measuring of copper affinity
in the femtomolar range (2.6  10-17 M) for
118
Figure 6. The AT1 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 (524) 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 (Ser258 in Ccc2, Ser653 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.
the mechanisms associated with opportunistic
colonization by Candida sp. in HIV and in other
In this field we have been able
immunocompromised individuals. We showed
to shed light on the role of cell surface ATPases
that C. albicans from HIV+ individuals has an
and phosphatases (ecto-ATPases and ecto-
ecto-phosphatase
phosphatases) in the oral manifestations of
isolates from HIV- subjects (Fig. 8A; [8]) and,
Human Immunodeficiency Virus (HIV) and in
most important, yeast expressing higher levels of
significantly
higher
than
119
surface phosphatase activity showed greater
Finally, it is important to mention that a
adhesion to epithelial cells, thus favoring the
clear relationship also exists between these
early steps for host cell invasion (Fig. 8B; [8]).
surface activities and physiological adaptative
In this regard we also demonstrated that
responses of the microorganisms during their life
2+
expression of a Mg -stimulated ecto-ATPase is
cycle. Nutrients acquisition, which is vital for
increased in C. parapsilosis, which is the second
parasite life (growth and development and, in
most common species found in the bloodstream
many cases, host invasion), evidence a central
of seriously infected nosocomial patients (Fig. 9;
strategy for acquire essential substances via
[9]). Again, a direct relationship between ecto-
surface ATPases and phosphatases [9,10].
ATPase activity and adhesion was observed.
More important, we provided evidence showing
that inhibitors of these ecto-ATPases and ecto-
6) Integrated conclusion.
The association established
in the
phosphatases can be envisioned as a new
Laboratory 17 of the National Institute of
potential class of specific antimycotic agents.
Structural
Inhibition of enzyme activity resulted in
succeeded during these 2 years in allowing the
decreased levels of yeast adhesion (Fig. 8B; [8]).
comprehension – at least in part – of the
A
Biology
and
Bioimaging
has
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 610.27 
166.36 and 241.25  78.96 picomoles of 4MU/h/107 cells for HIV+ and HIV- group,
respectively (Mann-Whitney test (P < 0.05).
Note: o2, o10, *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
Figure 9. Expression of Mg2+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].
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. 2010. 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 NPRA/cGMP/PKG pathway. Peptides (New York, N.Y. 1980).
, v.31, p.910 - 918, 2010.
3.
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.1220 - 1227,
2010.
4.
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.360 - 366, 2010.
5.
LARA, L. S., Diogo Vives, Juliana S.
Correa, Flavia P. Cardozo, Maria Fernanda MarquesFernades, 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.117 - 122,
2010.
6.
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.354 - 359,
2010.
7.
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: 431-437.
10.
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: 436-441.
11.
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: 386393.
12.
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. 2010 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. 2010 10:735-746.
14.
Vieira FS, Corrêa G, Einicker-Lamas
M, Coutinho-Silva R. Host cell lipid rafts: a safe door to
microorganisms?. Biol Cell.2010 102: 391-407.
15.
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. 2010 26:481-498.
16.
Axelband F., Dias J., Ferrão F.,
Einicker-Lamas M. Nongenomic signaling pathways
triggered by thyroid hormones and their metabolite 3iodothyronamine on the cardiovascular system. J. Cell
Physiol. DOI: 10.1002/jcp.22325 (versão eletrônica). 2010.
17.
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. 2010 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.
2010 498:57-61.
121
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. 2009 24:585-94.
20.
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. 2009 158:47-56.
21.
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. 2009 24:1959-1965.
22.
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. 2009 36:1157-1163.
23.
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. 2009 48:437-445.
24.
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. 2009 155:81-90.
25.
Lindoso RS, Verdoorn KS, EinickerLamas M. Renal recovery after injury: the role of Pax-2.
Nephrol Dial Transplant. 2009 24: 2628-2633.
26.
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: 15-21.
27.
Mariano AC, Santos R, Gonzalez MS,
Feder D, Machado EA, Pascarelli B, Gondim KC, MeyerFernandes JR. Synthesis and mobilization of glycogen and
trehalose in adult male Rhodnius prolixus. Arch Insect
Biochem Physiol. 2009 72: 1-15.
28.
Dutra PM, Vieira DP, MeyerFernandes JR, Silva-Neto MA, Lopes AH. Stimulation of
Leishmania tropica protein kinase CK2 activities by
platelet-activating factor (PAF). Acta Trop. 2009, 111:
247-254.
29.
Cosentino-Gomes D, Russo-Abrahão
T, Fonseca-de-Souza AL, Ferreira CR, Galina A, MeyerFernandes JR.Modulation of Trypanosoma rangeli ectophosphatase activity by hydrogen peroxide.Free Radic Biol
Med. 2009 47: 152-158.
30.
Sodré CL, Moreira BL, MeyerFernandes 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: 657-663.
31.
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: 242246.
32.
Moreira OC, Rios PF, Esteves FF,
Meyer-Fernandes JR, Barrabin H. CrATP as a new
inhibitor
of
ecto-ATPases
of
trypanosomatids.
Parasitology. 2009,136: 35-44.
33.
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: 137-143.
34.
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, 2009.
35.
Assaife-Lopes, Natália, Wengert, Mira,
Ana Acacia S. Pinheiro, LEAO-FERREIRA, L. R., CarusoNeves, 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, 2009.
36.
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, 2009
37.
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.693
699,
2009.
122
AL18
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
Bosentan-treated
TCT Dm28c
activation of TLR2. In the settings
of natural chagasic infection, it is
PBS
PBS
well accepted that trypomastigotes
cardiovascular
TCT
T=0
are occasionally released from
infected
TCT
cells.
Viewed from the perspective of
pathogenesis, a major challenge in
this project is to determine if
trypomastigotes also activate the
T= 30min
cardiac microcirculation via the
same mechanism, perhaps evoking
interstitial edema in the chagasic
heart. As a first step towards
addressing
this
question,
we
T= 60min
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
T= 90min
far
performed in infected paw tissues,
our preliminary studies (Fig.1)
revealed
induce
that
trypomastigotes
potent
T= 120min
microvascular
responses, which peak between 3090
min.
Consistent
with
previous
findings,
microvascular
reactogenicity
trypomastigotes
reduced,
either
was
our
the
T= 150min
of
drastically
by
BK2R
antagonist (HOE-140) or ETR
antagonists (bosentan) (Fig.1). A
T= 180min
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
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
needle
puncture
slightly
activates
the
accidental tissue injury (sterile trauma), it is
endothelin/kinin pathway, but the reaction is
reasonable to predict that the innate immune
rapidly resolved.
system has evolved safeguards to prevent
excessive exposure of tolerogenic DCs to
Immunological
studies
on
ACE
proinflammatory peptides produced under such
inhibitors, kinins and innovative vaccination
trivial
strategies. In previous studies, we have shown
however, the
that lymphoid-resident dendritic cells sense the
surpassed, or not, depending on the phenotypic
endogenous peptides (such as kinins) liberated
profile and intensity of innate response ignited
in extravascular tissues. Acting as a prototypic
by any given pathogen. The first precedent that
endogenous
(BK)
transient blockade of ACE leads to a robust
activates their cognate G-protein coupled
upregulation of Th1 responses via the BK2R
receptors
lymphoid-resident
pathway was obtained in a subcutaneous model
dendritic cells, converting these specialized
of T. cruzi infection (Monteiro et al., 2006).
antigen-presenting cells into inducers of T cell
However, In the case of mucosal infection by
effector development. By the time that we
P.gingivalis, the synergism between TLR2
submitted our workplan to INBEB, we were
ligands and kinin-releasing proteases was
unaware that of recently published peptidome
sufficiently strong to induce Th1 and/or Th17
data showing that BK is present in the prenodal
responses via the BK2R pathway, irrespective
(human) lymph. This interesting finding
of ACE blockade (Monteiro et al., 2009).
implies
precursor
Preliminary studies conducted in our lab
molecules circulating in the plasma, but also
(supported by the GCE program of Gates
produced
foundation/Phase I) suggest that it may be
adjuvant,
expressed
that
in
by
kininogens
various
bradykinin
(kinin
tissues)
undergo
circumstances.
processing in interstitial spaces, even in
possible
healthy
protective
individuals
(steady
state).
Once
to
During
infection,
peptidase barrier
may be
harness
CD8+
T-dependent
immunity
against
intracellular
collected by efferent lymphatics, kinins-along
pathogens such as T. cruzi through the
with C5a and a wide range of self-peptides
administration of a single dose of captopril,
originally produced in extravascular tissues
prior to vaccination and booster.
(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
125
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):3700-11.
2. 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):1005-14.
3. 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):e479.
4. 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):43546.
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):1251-60. 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 1:187-98. 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. 2010 Oct 21. doi: 10.1111/j.13652249.2010.04270.x. [Epub ahead of print]
126
AL19
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 2009- 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
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
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
99m
labeled
with
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, 99mTc
BMMC brain SPECT
revealing accumulation of
the BMMCs in the left
hemisphere 2 hours after
cell transplantation.
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),
hipoxia-
ischemic 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; RibeiroResende 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:
In the clinical studies, we have used
stem cells labeled with
99m
Technetium in order
to analyze the migration and homing of the
128
transplanted cells to the lesioned region in the
Evaluate the safety and effectiveness of
patients (Fig 1). However, the half-life of this
different types of labeled cells in different
radioactive compound is of approximately 6 hs
animal models of disease (nervous system,
which gives us only a maximum of 24 hs to
heart and kidney) with respect to toxicity and
visualize migration and homing of the injected
limit of detection;
cells. To solve this problem we have
Verify whether the transplanted cells migrate
investigated the possibility of labeling different
to the lesion sites through the same reactions
stem cells (pluri and multipotent) with
as well as monitor their destination by MRI at
commercial available SPIONs and also with
different times after the transplant;
SPIONs specially generated by our group. We
Evaluate the possible role of labeled cells in
were able to establish protocols for each cell
animal models of cell therapy:
type and for the different SPIONs (Fig 2).
These are ongoing projects and we are
Using these protocols we have also tested the
still in the process of doing the experiments
proliferation,
differentiation
and analyzing the results. We have however
capacity of the labeled cells and concluded that
preliminary results with respect to toxicity and
the incorporation of SPIONs does not affect
limit of detection. We have found that we can
any
detect
of
viability
and
these
cellular
functions.
500.000
mesenchymal
stem
cells
labeled with SPIONs using a 1.5 T RMI (Fig
The
3). The cells were injected stereotaxically into
results of these
the striatum of an adult rat and the signal was
studies
were
still present 60 days after the injection. We
for
suggest that with the 7 T equipment (that has
submitted
publication
just become available) it will be possible to
(Jasmin et al.,
detect even a smaller number of labeled cells.
2010
,
submitted).
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 (5x105) 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
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. 2010
Aug 18. [Epub ahead of print] PubMed PMID: 20719093.
2: 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):403-12. PubMed PMID:
20163207.
3: Pimentel-Coelho PM, Mendez-Otero R. Cell
therapy for neonatal hypoxic-ischemic encephalopathy.
Stem Cells Dev. 2010 Mar;19(3):299-310. Review.
PubMed PMID:19916801.
4: Barbosa da Fonseca LM, Gutfilen B, Rosado
de Castro PH, Battistella V, Goldenberg RC, KasaiBrunswick 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. 2010 Jan;221(1):122-8. Epub 2009 Oct 22.
PubMed PMID:19853605.
5: 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. 2010 Jan 8;1306:149-58. Epub 2009 Sep 30.
6: Barbosa da Fonseca LM, Battistella V, de
Freitas GR, Gutfilen B, Dos Santos Goldenberg RC,
Maiolino A, Wajnberg E, Rosado de Castro PH, MendezOtero R, Andre C. Early tissue distribution of bone marrow
mononuclear cells afterintra-arterial delivery in a patient
with chronic stroke. Circulation. 2009 Aug11;120(6):53941. PubMed PMID: 19667245.
7: 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:
19370156; PubMed Central PMCID: PMC2667250.
8: 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: 19368806.
9: 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.
2010 Mar;19(3):351-8. PubMed PMID: 19296724.
10: 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. 2009
Feb;42(2):189-96. PubMed PMID: 19274347.
11: Ribeiro-Resende VT, Pimentel-Coelho PM,
Mesentier-Louro LA, Mendez RM,Mello-Silva JP, Cabral-
da-Silva MC, de Mello FG, de Melo Reis RA, MendezOtero R.Trophic activity derived from bone marrow
mononuclear cells increases peripheral nerve regeneration
by acting on both neuronal and glial cell populations.
Neuroscience. 2009 Mar 17;159(2):540-9. Epub 2009 Jan
7. PubMed PMID: 19174184.12: Gubert F, Zaverucha-doValle C, Pimentel-Coelho PM, Mendez-Otero R, Santiago
MF. Radial glia-like cells persist in the adult rat brain.
Brain Res. 2009 Mar3;1258:43-52. Epub 2008 Dec 24.
13: 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.
2009
Apr;29(2):140-7. Epub 2008 Aug 14. PubMed PMID:
18713310.
14: 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. 2010 Aug 25.[Epub ahead of
print] PubMed PMID: 20739294.
15: 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.99 - 103, 2009.
16: 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.51:71-75.
17: 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):311-321.
18: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):e1000870.
20: 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):1183611845.
21: 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):198-206.
AL20
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
supportive therapy, their mortality remains
Another important research field of the
unacceptably elevated. The diagnosis of ALI and
Laboratory of Inflammation and Metabolism is
ARDS is based on clinical, radiological and gas
the application on biomedical images in the
exchange parameters, but those are late events
study of neuroinflammation, aging and dementia.
occurring after molecular signaling and fluid
For decades, magnetic resonance imaging (MRI)
accumulation in the lung. Traditional methods of
has provided non-invasive assessment of many
imaging
neurological
(chest
tomography)
x-rays
have
small
and
computed
sensitivity
and
disorders,
neuroinflammatory
as
well
diseases.
specificity in the early diagnosis. Positron
macrostructural,
cellular
emission
with
measurements.
T1-weighted
been
images
tomography
[18]fluorodeoxyglucose
(PET)
(18FDG)
has
show
gray
as
MRI
and
and
acute
allows
metabolic
gradient
white
echo
matter
considered a noninvasive and highly sensitive
abnormalities. T2-weighted spin echo images
imaging technique that can be used to quantify
highlight
pulmonary inflammation. In the Laboratory of
Advanced diffusion-weighted imaging (DWI)
Inflammation and Metabolism we are interested
shows
in describing the pattern of 18F-FDG uptake in
remyelination. Phosphorous MR spectroscopy
the lung parenchyma of experimental models of
(31P-MRS) provides simultaneous in vivo
ALI in rats and mice (Figure 1). Additionally, we
bioenergetic assessments, such as ATP, PCr
are interested in the early uptake pattern and in
(phosphocreatine) and Pi (inorganic phosphate).
the kinetics of glucose incorporation by the lung
Intracellular pH can be assessed by the chemical
parenchyma of rodents with ALI, as well as in
shift of Pi relative to PCr.
the molecular mechanisms responsible for such
assessments may reflect changes in energy
event.
metabolism and mitochondrial function due to
fluid
from
demyelination,
MicroPET/CT imaging
Lung uptake of FDG-1 8F after LPS in rats
neuroinflammation.
axonal
damage
and
Longitudinal
pathological processes.
More sophisticated MRI
techniques may have applicability
to
Control
HU = -325.9
HU = -274.3
2 hours
severe
neuroinflammatory
conditions, such as sepsis, and
measurements of reactive oxygen
Bq/ml = 43.1
Bq/ml = 83.4
species
(ROS).
Perivascular
edema,
and
spectroscopic
abnormalities
can
be
demonstrated by MRI and 1H6 hours
HU = -344.0 (+/- 59.0 )
HU = -290.7 (+/- 53.8)
24 hours
MRS in a mouse sepsis model
(Bozza et al., JCBFM 2010). The
Bq/ml = 138.1
Bq/ml = 97.6
Imaging and Neuroinflammation
detection of oxidative damage
and reactive oxygen species (ROS) by MR
techniques is now possible through the use of
132
Gd-based spin trapping contrast agents that track
patients with mild cognitive impairment (MCI)
the formation of protein radicals. It is unknow
show
whether characteristic MR abnormalities define
hypometabolism.
subjects with age-associated chronic low-level
speculation
neuroinflammation, or whether disease activity
diagnosed by FDG PET, but this has not been
may be assessed by either traditional or novel
supported by longitudinal studies. MRI and FDG
MR techniques.
PET may provide useful indicators that MCI has
similar,
that
but
less-severe,
regional
This
might
to
the
can
be
pre-clinical
lead
AD
Positron emission tomography (PET) is
progressed into frank AD, particularly when
used for evaluation of dementia, seizures, and for
longitudinal studies can be performed. However,
assessment of recurrent tumor versus radiation
the utility of MRI or FDG PET for characterizing
necrosis,
MCI and chronic neuroinflammation has not
and
may
neuroinflammatory
be
applicable
disorders.
to
Activated
been studied.
microglia, monocytes and macrophages show an
increase
in
expression
peripheral
amyloid plaques and neurofibrillary tangles,
benzodiazepine receptors (PBR). PBR binding
which contain beta-amyloid peptides (Abeta) and
ligands, such as [(11)C]PK11195 are currently
highly phosphorylated
under development and investigation and may
deposition leads to an increase in beta-amyloid
play a future role in assessing neurodegenerative
plaques, the initial neuropathological change in
where
18F
AD. Radiotracers for in vivo imaging beta-
fluorodeoxyglucose (FDG) PET shows increased
amyloid in brain is an important focus of
uptake in acute cerebral inflammation, and
research development. The most widely used and
decreased uptake in the late stages of the
studied of these agents is N-methyl-[(11)C]2-(4'-
diseases. Increased uptake of glucose analogs is a
methylaminophenyl)-6-hydroxybenzothiazole
very early event (< 6h) in experimental sepsis,
([(11)C]PIB).
possibly from excitotoxity or activation of
tomography (PET) has been validated as
microglial orastrocytes (Figure 2). Decreased
showing increased binding in subjects with AD,
FDG uptake in neocortical regions of the brain
compared to normals. (11)C]PIB binding also
24h after endotoxin, and could be due to
occur in subjects with mild cognitive impairment
neuronal injury or dysfunction. FDG PET may
(MCI) and also some elderly normal patients
provide assessment of both the acute and chronic
without neurocognitive effects. Abeta deposition,
phases of neuroinflammation.
the primary pathological feature of AD, has also
inflammation
plays
of
The brains of AD patients reveal beta-
a
role.
In AD, decreased uptake of FDG occurs in
been
shown
tau
(11C)PIB
occur
proteins.
positron
in
Abeta
emission
response
to
the parieto-temporal, cingulate, and medial
lipopolysaccharide-induced
temporal cortices. MR findings of AD show
in animal models. (11C)PIB uptake may, in some
early hippocampal and medial temporal volume
cases, indicate a population of individuals with
loss. The discrepancy between FDG PET and
ongoing neuroinflammation due to chronic or
MRI findings may be due to technical limitations
recurrent low level systemic inflammation.
of PET in measuring small structures. Many
However, this has not been studied.
neuroinflammation
133
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.
1.
Bozza, Fernando A ; Salluh, Jorge I.F. .
An urban perspective on sepsis in developing countries.
Lancet Infectious Diseases, v. 10, p. 290-291, 2010.
2.
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, 2010.
3.
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. 40-43, 2010
4.
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, 2010.
5.
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. 348-353, 2010.
6.
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. 00-00, 2010.
7.
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. e1000963,
2010.
8.
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. 00-00, 2010.
9.
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, 2010.
10.
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. 284-292, 2010.
11.
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, 2010.
12.
Caiaffa, C.D. ; Stiebler, R; Oliveira,
M.F. ; Lara, F.A. ; Paiva-Silva, G.O. ; Oliveira, P.L. . Sn-
protoporphyrin inhibits both heme degradation and
hemozoin formation in Rhodnius prolixus midgut. Insect
Biochemistry and Molecular Biology, p. 1-8, 2010.
13.
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, 2010.
14.
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, 2010- 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. 993-1006, 2010.
16.
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, 2010.
17.
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, 2010.
18.
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. 2010 Mar 17;2(23):23ra19.
19.
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. 2010 Jan;38(1):347-8
20.
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. 2009 Dec;8(12):5431-41.
21.
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. 2010 Feb;30(2):440-8.
22.
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. 2010
Jan;38(1):9-15.
23.
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. 2010 Jan;24(1):218-28.
24.
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. 2009
Jul;104(4):531-48. 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. 2009
Aug;50(8):1205-13.
26.
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. 2009 Dec;24(4):556-62.
27.
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. 2009 Feb;53(2):210-7.
28.
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. 2009 Feb;40(2):123-34.
29.
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. 856-865, 2009
30.
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, 2010.
31.
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, , 2009.
32.
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, 2009.
33.
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. 993-1006, 2010.
34.
Carvalho, N C ; Beda, A ; de Abreu, M
G ; Spieth, P M ; Granja-Filho, P ; Jandre, F C ; GiannellaNeto, 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. 1151-1162, 2009.
35.
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. 1109-1120,
2009.
135
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.
644-653, 2009.
37.
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,
2009.
38.
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, 2009.
136
3. COOPERATIVE
AND
EDUCATIONAL
ACTIVITIES
137
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
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.

AL7, PI Prof. Carlos Ramos: At the laboratory at UFRJ as
part of a collaborative effort.

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
 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 810, 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
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
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
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, 2010. 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
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 (2009 - 2010):
AL 1:
Master
Guilherme Augusto Piedade de Oliveira. Aspectos
Clínicos e Termodinâmicos da Leucemia Mielóide Crônica
(LMC). 2009. 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. 2010. 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.
2010. 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. 2009. 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.. 2010. 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. 2009.
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. 2009. 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
Peptídeos de Fusão com Membranas Biomiméticas e
Implicações para o Desenvolvimento de uma Vacina
Inativada por Alta Pressão Hidrostática. 2005-2009. 2009.
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. 2009. 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. 2010. Tese (Doutorado
em Química Biológica) - Universidade Federal do Rio de
Nível Superior.
AL 2:
Master
Ricardo Sant’Anna de Oliveira. Pequenas
moléculas como inibidores de agregação da proteína
amiloidogênica transtirretina (TTR). 2009. 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 Snitrosomiosina sob condições fisiológicas.. 2009.
Dissertação (Mestrado em Quimica Biologica) - Instituto de
Bioquimica Medica - UFRJ, Coordenação de
Martha Meriwether Sorenson.
Luciana Elena de Souza Fraga Machado. Efeito
do fenol na interação acto-S1.. 2009. 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.. 2009. 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.. 2009. Dissertação
(Mestrado em Química Biológica) - Universidade Federal
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. 2009.
Debora Foguel.
Leonardo de Castro Palmieri. Complexo
transtirretina humana e zinco: caracterização estrutural,
funcional e termodinâmica. 2009. 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.. 2009. Tese
(Doutorado em Quimica Biologica) - Instituto de
Bioquimica Medica - UFRJ, Coordenação de
Martha Meriwether Sorenson.
AL 3:
Master
José Eduardo da Silva Rabelo. Caracterização
funcional e termodinâmica de proteínas de alta mobilidade
de Aedes aegypti (AeHMGB1). 2010. Dissertação
(Mestrado em Ciências Biológicas (Biofísica)) - Instituto de
biofísica Carlos Chagas Filho, Coordenação de
Ronaldo da Silva Mohana Borges.
Marcela da Silva Rosa. Identificação de novos
alvos moleculares para diagnóstico e prognóstico da dengue
através de técnicas proteômicas. 2009. 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. 2009. Tese
Pessoal de Nível Superior. Advisor: Fabio Ceneviva
Lacerda Almeida.
Guilherme Razzera Maciel. Estudos Estruturais
dos enovelamentos proteicos de defensinas e globinas
atraves de RMN. 2009. Tese (Doutorado em Química
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. 2010. Dissertação (Mestrado em Química
Tecnológico. Advidor: Russolina Benedeta Zingali
Saulo Martins Vieira. Análise da Atividade da
Trombina com Substratos Fluorogênicos Baseados no
Receptor PAR 1. 2009. 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). 2009. 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. 2009.
Dissertação (Mestrado em Quimica Biologica) - Instituto de
Bioquímica Médica /CCS / UFRJ, Coordenação de
Russolina Benedeta Zingali.
Morgana
Guimarães
Soares.
Efeito
do
anticoagulante Ixolaris no crescimento tumoral de gliomas
humanos implantados em encéfalo de roedores. 2009.
Dissertação (Mestrado em Química Biológica) Universidade Federal do Rio de Janeiro, Conselho Nacional
146
Robson de Queiroz Monteiro.
Aline da Costa Cruz. Diagnóstico sorológico da
infecção pulmonar por Pseudomonas aeruginosa em
crianças com Fibrose Cística. 2009. 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. 2009. 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. 2010. 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. 2010. 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. 2010. Tese
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. 2009. 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. 2010. 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 2,4diaminopirimidina-5-carboxialdeído para Preparação de
Novos Compostos Heterocíclicos como Potenciais
Antimalariais. 2010. 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. 2010. Dissertação (Mestrado em
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. 2009. Dissertação
(Mestrado em Ciências Biológicas (Biofísica)) Universidade Federal do Rio de Janeiro, Coordenação de
Pedro Geraldo Pascutti.
Reinaldo de Oliveira Júnior. Dissociação
molecular sob alta pressão hidrostática por simulação
computacional. 2009. Dissertação (Mestrado em Ciências
Biológicas (Biofísica)) - Universidade Federal do Rio de
Nível Superior. Advisor: Pedro Geraldo Pascutti.
Tácio Vinício Amorim Fernandes. Estudo do
Enovelamento de Proteínas por Dinâmica Molecular e
Generalized Simulated Annealing. 2009. Dissertação
(Mestrado em Ciências Biológicas (Biofísica)) Universidade Federal do Rio de Janeiro, Conselho Nacional
Doctoral
Tatiana Santana Ribeiro. Síntese e Avaliação de
Oximas como Antídotos para Intoxicação com
Organofosforados Neurotóxicos. 2009. Tese (Doutorado em
Química) - Instituto Militar de Engenharia, Coordenação de
Jose Daniel Figueroa Villar.
Magdalena Nascimento Rennó. Avaliação de
Inibidores da Nucleosídeo Hidrolase de Leishmania
donovani. 2009. 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. 2009. Tese (Doutorado em
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.. 2009.
Tese (Doutorado em Ciências Biológicas (Biofísica)) Universidade Federal do Rio de Janeiro, Conselho Nacional
Gabriel Limaverde Soares Costa Sousa. Estudo
Estrutural das Proteínas Antiangiogênicas Endostatina e
Anastelina e Potenciais Implicações na Terapia contra o
Câncer.. 2009. Tese (Doutorado em Ciências Biológicas
(Biofísica)) - Universidade Federal do Rio de Janeiro,
Tecnológico. Advisor: Pedro Geraldo Pascutti
Diego Enry Barreto Gomes. Modelos de
Confinamento para Organofósforo Hidrolase em
Nanoestruturas. 2010. Tese (Doutorado em Ciências
e Tecnológico. Orientador: Pedro Geraldo Pascutti
147
Rafael de Cássio Bernardi. Estrutura e Dinâmica
de Anestésicos Locais em Biomembranas. 2010. 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.
2010.
Dissertação
(Mestrado
em
Biotecnologia) - Universidade Federal de Santa Catarina,
Superior. Advisor: Hernan Francisco Terenzi.
Carolina Tavares. Proteômica de micoplasmas.
2010. Dissertação (Mestrado em Pós Graduação
Bioquímica) - Universidade Federal de Santa Catarina,
Tecnológico. Advisor: Hernan Francisco Terenzi.
Franciele Luanne Fischer. Nucleases Químicas.
2010. 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. 2010.
Dissertação (Mestrado em Biotecnologia) - Universidade
Federal de Santa Catarina, . Advisor: Hernan Francisco
Terenzi.
Doctoral
Claus Troger Pich. Interação de complexos metálicos com
ácidos nucléicos. 2009. 0 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. 2010.
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. 2009. 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. 2010.
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. 2009.
Dissertação (Mestrado em Ciências Biológicas (Biofísica)) Universidade Federal do Rio de Janeiro, Coordenação de
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. 2009. Dissertação (Mestrado em Ciências
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. 2009. 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. 2009. 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. 2009. 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. 2009.
Dissertação (Mestrado em Ciências (Microbiologia)) Universidade Federal do Rio de Janeiro, Coordenação de
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. 2010. Tese
(Doutorado em Ciências (Microbiologia)) - Universidade
148
Ulysses Garcia Casado Lins.
Júlia Peixoto de Albuquerque. Caracterização
morfológica da população de bactéias oxidantes de enxofre
dos gêneros Beggiatoa. 2009. Tese (Doutorado em Ciências
(Microbiologia)) - Universidade Federal do Rio de Janeiro,
Tecnológico. Advisor: Ulysses Garcia Casado Lins.
Destino: Programa de Pós-Doutorado FIOCRUZ
camundongos. 2010. Tese (Ciências Biológicas)
Universidade Federal de Pernambuco
Karina Lidianne Alcântara Saraiva. tratamento
camundongos pré-púberes e adultos com inibidor
fosfodiesterase-5 e avaliação de seus efeitos sobre a
espermatogênese. 2010. Tese (Saúde Pública) - Centro
Pesquisas Aggeu Magalhães
AL 12:
Master
Luiza de Lima e Silva Bagno. Estudo da função
cardíaca no transplante de células progenitoras de tecido
adiposo em ratos com infarto cicatrizado.. 2009. 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. 2009. 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. 2009. Dissertação (Mestrado
em Ciências Biológicas (Fisiologia)) - Universidade Federal
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. 2009. 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. 2009. 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.
Ivone Rosa de Andrade. Caracterização de
proteínas do Complexo de Golgi de Tritrichomonas foetus.
2009. Dissertação (Mestrado em Ciências Morfológicas) Universidade Federal do Rio de Janeiro, Coordenação de
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.. 2009. 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. 2010. 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. 2010.
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. 2010.
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. 2009. Dissertação (Ciências
Biológicas) - Universidade Federal de Pernambuco
Bruna Santos da Silva. Caracterização dos efeitos
da Dietilcarbamazina (DEC) sobre a Ovogênese de
Camundongos. 2009. Dissertação (Ciências Biológicas) Universidade Federal de Pernambuco
Tiago Bento de Oliveira. Novas tiazolidinadionas:
síntese, caracterização estrutural, modelagem molecular e
avaliação da atividade antiinflamatória. 2010. 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
de
da
a
de
AL 16:
Master
Doctoral
Ricardo Luiz de Azevedo Pereira. Avaliação do
inibidor de cisteína proteases E64 na diferenciação de
células-tronco embrionárias em células neurais. 2009. 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. 2009. 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. 2009. 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. 2009. Dissertação
149
(Mestrado em Ciências Biológicas (Biofísica)) Universidade Federal do Rio de Janeiro, Coordenação de
Adalberto Ramon Vieyra.
Sharon
Landgraf
Schlup.
Mecanismos
moleculares envolvidos no desenvolvimento da Hipertensão
primária. 2009. Dissertação (Mestrado em Ciências
Biológicas (Fisiologia)) - Universidade Federal do Rio 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. 2009. Dissertação (Mestrado em
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.. 2009. Dissertação (Mestrado em
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 ectoATPdifosfohidrolase
em
Candida
parapsilosis:
caracterização bioquímica e possível envolvimento na
virulência e aquisição de adenosina.. 2009. Tese (Doutorado
em Química Biológica) - Instituto de Bioquímica Médica,
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. 2009.
Dissertação (Mestrado em Ciencia Biológicas) - Instituto de
biofísica Carlos Chagas Filho, Coordenação de
Julio Scharfstein.
Larissa Nogueira de Almeida.
Doctoral
Ilka Maria Bakker Coelho de Abreu. Mecanismo
de Invasão Tissular no Câncer de prostata. 2010. 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. 2009. 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. 2009.
Dissertação (Ciências Biológicas (Biofísica)) - Universidade
Federal do Rio de JaneiroValeria Battistella Amado dos Santos. Seguranca
do transplante autologo de celulas mononucleares da
medula ossea em pacientes com acidente vascular cerebral
isquemico subagudo. 2009. 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. 2009.
Dissertação (Mestrado em Medicina (Radiologia)) Universidade Federal do Rio de Janeiro, Coordenação de
Léa Mirian Barbosa da Fonseca.
Maria Verônica Fonseca Torres de Oliveira.
Desenvolvimento da marcação da doxorrubicina com Tc99m - Estudos " in vitro' e " in vivo". 2009. Dissertação
(Mestrado em Medicina (Radiologia)) - Universidade
Federal do Rio de Janeiro, Coordenação de
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. 2009. Dissertação (Mestrado em Ciências
e Tecnológico. Co-Advisor: Arthur Giraldi Guimarães.
Doctoral
Camila Zaverucha do Valle. Potencial Terapeutico das
celulas de medula ossea na regeneracao do nervo optico.
2010. Tese (Doutorado em Ciências Biológicas (Fisiologia))
- Universidade Federal do Rio de Janeiro, Coordenação de
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. 2010. 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. 2010. 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. 2009. 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. 2009. 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. 2009.
Tese (Doutorado em Medicina (Radiologia)) - Universidade
Federal do Rio de Janeiro, Coordenação de
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: 2010. 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
CAMUNDONGOS
INTOXICADOS
POR
MICROCISTINA-LR. Início: 2010. Dissertação (Mestrado
em Ciências Biológicas (Fisiologia)) - Universidade Federal
Científico e Tecnológico. (Advisor Fernando Bozza).
Patrícia Duque Estrada Jacintho. Efeito da pressão
positiva expiratória nas vias aéreas na arritmia sinusal
respiratória. 2009. Dissertação (Mestrado em Engenharia
Biomédica) - Universidade Federal do Rio de Janeiro,
Tecnológico. Advisor: Alysson Roncally Silva Carvalho.
Ana Caroline de Paiva Gandara. Caracterização da
produção de espécies reativas no trato digestivo do barbeiro
Rhodnius prolixus. 2010. Dissertação (Mestrado em
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. 2009. Dissertação
(Mestrado em Engenharia Biomédica) - Universidade
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.
2009. Dissertação (Mestrado em Engenharia Biomédica) Universidade Federal do Rio de Janeiro, Coordenação de
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.
2009. 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.
2009. 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. 2009. Tese (Doutorado em Medicina
(Radiologia)) - Universidade Federal do Rio de Janeiro,
Superior. Advisor: Fernando Augusto Bozza.
Christine Cruz Oliveira. Degradação de heme e
inflamação: Estudos sobre o papel inibitório da biliverdina
na migração de leucócitos. 2009. Tese (Doutorado em
Nível Superior. Advisor: Aurélio Vicente Graça de Souza.
Diego S Menezes. Modulação do metabolismo
redox em tripanossomatideos como novas ferramentas
quimioterápicas. Início: 2009. 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. 2009. Tese
do Rio de Janeiro. Advisor: Marcus Fernandes de Oliveira.
151
152
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
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
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
INBEB
from
developed
three activities with
the students of the
State
School
Martins
da
José
Costa.
State school José Martins da Costa
155
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
activity
first
day
occurred
of
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
Professor Emiliano Medei at the school
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
156
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%
addition,
).
In
71%
said the activity
at
the
school
made them feel
better
about
science.
157
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
4. PERSPECTIVES
AND FUTURE
DEVELOPMENTS
159
Organization of the individual research groups into a National
Institute for Science and Technology has made it possible to create
synergistic and increasingly productive interactions. For INBEB, this first 18
months has involved constructing a multidisciplinary approach to a number
of the scientific questions in the original proposal. The catalytic effect of
having formal collaborations among the different Associated Laboratories
has been mutually beneficial, leading to synergistic actions that combine
structural, dynamics, molecular biology, and micro- and macro-imaging
techniques. It is especially gratifying to see that the younger members of the
groups (graduate students and post-docs) demonstrate enormous enthusiasm
and creativity, which holds great promise for a new generation of
imaginative leaders in these areas. One of the principle goals of INBEB is to
support these young professors as they build up their own research groups.
We expect that now, in the nextyear of INBEB activities, having
installed a magnetic resonance imaging facility for small animals and
transferred the microscopy equipment to the new CENABIO-II building, we
will be able to report rapid progress in applying these frontier technologies
to the projects of the Associated Laboratories, as well as in training students
to use them well. We believe that the results will be reflected in our ability to
publish top-notch research in high-impact publications.
Strengthening our ties with IDOR (Instituto D´Or for Research and
Teaching), thereby closing the gap between basic and clinical research
(translational research), is another of our primary goals. In addition, over the
next 12 months we will give high priority to initiating the construction of a
new building, CENABIO III, to house the microscopy equipment, with the
aim of creating the largest and most advanced collection of equipment in
Latin America for NMR of macromolecules, small animals imaging and
microscopy.
160
161

Activities Reports - INCT

Transcrição

Documentos relacionados

Vicente del Rio is an architect with a graduate diploma in Urban and

ICMT 2_First Announcement - University of Southampton Blogs

BRASIL TELECOM S

university of porto, portugal 13 14 june 2011 jocelyn benoist

Race Guide - The Color Run

Title Authors Affiliation Materials and the making of

1 AMAZÔNICA Journal of Anthropology AMAZÔNICA is an

www.cmrocha.com.br

Mentoring - Observatório Juventude, Ciência e Tecnologia

Brazil uses geospatial technology