www.lge.ibi.unicamp.br mistérios moleculares da vassoura de bruxa gonçalo guimarães pereira...
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Mistérios Moleculares da
Vassoura de Bruxa
Gonçalo Guimarães PereiraUNICAMP
Salvador - Bahia
23/04/04
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Basis
Organism Gene
BiochemistryDiscover the function of the Gene A
in the Organism X
GenomicsDiscover that the Organism Y
has a gene similar to the Gene A found in the Organism X
A(Y) ~ A(X)
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Genomics Tools
Organism
Raw Sequence
Putative Genes
Cellular Program
Sequencing
Bioinformatics
Expression Analysis
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Genomics Mission
Genomics X
Trial and Error
Hypothesis for Targets Definition
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Genome Xylella fastidiosa
3 Mb
50 kb
1 a 2 kb
50 kb
Sequencing
ATGGGCCTTTGCTTTGCCCCCAAA
TGCCCCCAAAAGGGGGAAAGGGGGAAAAG
ATGGGCCTTTGCCCCCAAAAGGGGGAAAAGCCTGGGGGATGGGCCTTTGC AGGGGGAAAAGTTTGC
Identification of genes
ATG CT
Bioinformatics
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BLAST
Virtual Metabolism
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A.J.G. Simpson1, F.C. Reinach2, P. Arruda3, F.A. Abreu4, M. Acencio5, R. Alvarenga2,
L.M.C. Alves6, J.E. Araya7, G.S. Baia2, C.S. Baptista8, M.H.Barros8, E.D. Bonaccorsi2, S.
Bordin9, J.M. Bové10, M.R.S. Briones7, M.R.P. Bueno11, A.A. Camargo1, L.E.A.
Camargo12, D.M. Carraro12, H. Carrer12, N.B. Colauto13, C. Colombo14, F.F. Costa9, M.C.R.
Costa15, C.M. Costa-Neto16, L.L. Coutinho12, M. Cristofani17, E. Dias-Neto1, C. Docena2,
H. El-Dorry2, A.P. Facincani6, A.J.S. Ferreira2, V.C.A. Ferreira18, J.A. Ferro6, J.S. Fraga4,
S.C. França19, M.C. Franco20, M. Frohme21, L.R. Furlan22, M. Garnier10, G.H. Goldman23,
M.H.S. Goldman24, S.L. Gomes2, A. Gruber4, P.L. Ho25, J. D. Hoheisel21, M.L. Junqueira26,
E.L. Kemper3, J.P. Kitajima27, J.E. Krieger26, E.E. Kuramae28, F. Laigret10, M.R.
Lambais12, L.C.C. Leite25, E.G.M. Lemos6, M.V.F. Lemos29, S.A. Lopes19, C.R. Lopes13,
J.A. Machado30, 36 , M.A. Machado17, A.M.B.N. Madeira4, H.M.F. Madeira12, 37, C.L.
Marino13, M.V. Marques8, E.A.L. Martins25, E.M.F. Martins18, A.Y. Matsukuma2, C.F.M.
Menck8, E.C. Miracca5, C.Y. Miyaki11, C.B. Monteiro-Vitorello12, D.H. Moon20, M.A.
Nagai5, A.L.T.O. Nascimento25, L.E.S. Netto11, A. Nhani Jr.6, F.G. Nobrega8, 38, L.R.
Nunes31, M.A. Oliveira32, M.C. de Oliveira33, R.C. de Oliveira31, D.A. Palmieri13, A.
Paris13, B.R. Peixoto2, G.A.G. Pereira32, H.A. Pereira Jr.6, J.B. Pesquero16, R.B. Quaggio2,
P.G. Roberto19, V. Rodrigues34, A.J. de M. Rosa34, V.E. de Rosa Jr.28, R.G. de Sá34, R.V.
Santelli2, H.E. Sawasaki14, A.C.R. da Silva2, A.M. da Silva2, F.R. da Silva3,27, W.A. Silva
Jr.15, J.F. da Silveira7, M.L.Z. Silvestri2, W.J. Siqueira14, A.A. de Souza17, A.P. de Souza3,
M.F. Terenzi23, D. Truffi12, S.M. Tsai20, M.H. Tsuhako18, H. Vallada35, M.A. Van Sluys33,
S. Verjovski-Almeida2, A.L. Vettore3, M.A. Zago15, M. Zatz11, J. Meidanis*27 & J.C.
Setubal*27.
Publication
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More
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Xylella fastidiosa - Pierce's Disease Strain: Grape phytopathogen
Leifsonia xyli subsp. xyli (Sugarcane phytopathogen)
Leptospira
Eucalyptus
Coffee
Caw
and more....
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Regional Genomes
Leishmania chagasi
Schistosoma mansoni
Trypanossoma cruzi
Paracoccidioides brasiliensis
Human diseases
Agriculture improvement
Herbaspirillum seropedicae
Gluconacetobacter diazotrophicus
Eucalyptus
Phytopathogen
Crinipellis perniciosa
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Samba, football and .....
Genomics
Brazilian science
Fruits of co-operationJul 20th 2000 | SAO PAULOFrom The Economist print editionSAMBA, football and...genomics. The list of things for which Brazil is renownedhas suddenly got longer. Only a few days after publishing, on July 13th, the first-ever sequence of the genome of a plant pathogen, scientists at Sao Paulo’s stateresearch agency, Fapesp, were due to announce, on July 21st, anothersuccess—the composition of 279,000 human expressed-sequence tags, smallpieces of DNA that allow genes to be located along chromosomes. Only inAmerica and Britain have more than that number of human ESTs beenidentified.
Though they are of global significance, both of these advances are also of particularinterest to Brazilians. A number of the ESTs in question are derived from geneslinked to cancer of the head and neck, which for some reason is unusually common inBrazil. And the plant pathogen sequenced, Xylella fastidiosa, is an insect-bornebacterium that has been ravaging Brazil’s orange groves, causing their trees toproduce shrivelled fruit and costing growers an estimated $100m a year. ............
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Practical Results
????????????
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Cacau Production in Brazil
0
50
100
150
200
250
300
350
400
450
1981
1984
1987
1990
1993
1996
1999
Production
Th
ousa
nd T
ons
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International Price
0
10
20
30
40
50
60
1981
1983
1985
1987
1989
1991
1993
1995
1997
1999
U$/@
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Cacau Income
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1981
1983
1985
1987
1989
1991
1993
1995
1997
1999
Income
U$
Bil
lion
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Witche’s Broom Loses
-250
-200
-150
-100
-50
0
1981
1983
1985
1987
1989
1991
1993
1995
1997
1999
Loses
U$
Mil
lion
U$ 1.380.000.000,00300.000 jobs
100.000 ha Forest
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Goals of the Witch's Broom Genome Project
Pragmatic
2. Create the basis to understand the Witch's Broom disease, indicating possible solutions
1. Build a Genomics Network at the State of Bahia to face biological problems
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Symptoms - 1
Fotos - João de Cássia - CEPEC-CEPLAC
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Fotos - João de Cássia - CEPEC-CEPLAC
Symptoms - 2
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Fotos - João de Cássia - CEPEC-CEPLAC
Symptoms - 3
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Crinipellis perniciosa
Fotos - João de Cássia - CEPEC-CEPLAC
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Life Cycle
Differentiation
Meiose
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Complexity X Strategy
Prokaryotes
Eukaryotes
Fungi - Saccharomyces cerevisiae
Human
ATG CT
ATGCT
ATG CT
ATGCT
~ 3 Mb1G/1Kb
~ 20 Mb1G/2Kb
~3.000 Mb1G/75 Kb
ATG (?) CT
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0,0%
20,0%
40,0%
60,0%
80,0%
100,0%
0,0% 100,0% 200,0% 300,0% 400,0% 500,0% 600,0%
Sequenced
Effe
ctiv
e c
ove
rag
e
Normal % singlets % contigs % assembled
Sequencing X Coverage
Singlets
Contigs
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60539
29778
12161
6032
2962
1201
597
303
129
356110
175474
93509
42908
2091413739
6110
2982
1322
1
10
100
1000
10000
100000
1000000
1% 10% 100% 1000%
C over age ( r eads)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Average Maximum > 1000 > 1
1X 2X
303
>1000bp
Gaps X Coverage
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Sequencing Strategy
20 Kb
40 reads: 500 bp/each
3 reads
Genomic Shotgun
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Bahia Genomics Network
UESCCEPLACCENARGENUNICAMP
DNA CoordinationUNICAMP
UFBA-FarmáciaUFBA- IBUEFSUCSAL
Databank
BioinformáticaUNICAMP/UESC
Interface
Cacau Research Community
Libraries Libraries
Sequences Sequences
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Data Treatment: “on going” annotation
Shotgun
Blast
Pre-Annotated Read
Local clustering
ORFs
Assembling
BACs
Complete Genome
Representative Read
Chips Molecular Mapping
InactivationStructural Analysis
Proteomics
Metabolic Maps
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Bioinformatics Services
Serviços
Labs productivity Labs productivity graphs Labs productivity graphs (cumulative) Quality
Administrative services
Access control Access control (graph)
Bug reports
Sequence submission Nomenclature Nomenclature edition Submission test
Keyword search in Blast files Anotation (test) Annotated seeds Contigs progress Sequence pattern search Blast against C. perniciosa Blast results - All reads Sequence analysis tools (Emboss)
Library Control Strain
Geração Controle Análise
Gene Project
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Gene Project*
Data bank: Blasted reads
Selected reads
Assembled ReadsConsensus
Annotated Gene
*Carazzolle, MF, Digiampietri, L, Araújo, MRS, Formighieri, EF, Tsukumo, F & Pereira, GAG
Selection: File, Keyword, Blast, Seq. pattern, all
Assembling: PPC, SaturationBlast of consensus
Analysis: ORF finder, Comparison, Prediction
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Chitin Metabolism
UDP-N-AcetilglicosaminaUDP-N-Acetilglicosamina
CHITINCHITIN
N-Acetylglicosamine-1- phosphateN-Acetylglicosamine-1- phosphate
Glucosamine-6- phosphateGlucosamine-6- phosphate
GlutamineGlutamine
GlutamateGlutamate
AcetylCoAAcetylCoA
CoACoA
UTPUTP
N-AcetylglucosamineN-Acetylglucosamine
UDPUDP
2. 7. 7. 232. 7. 7. 23
ChitobioseChitobiose
N-Acetylglucosamine-6-hosphateN-Acetylglucosamine-6-hosphate
2. 3. 1. 42. 3. 1. 4
5. 4. 2. 35. 4. 2. 3
2. 7. 1. 592. 7. 1. 59pyrophosphatepyrophosphate
2. 6. 1. 162. 6. 1. 16
ChitosanChitosan 3. 5. 1. 413. 5. 1. 41
3. 5. 1. 25
2. 4. 1. 16
3. 2. 1. 143. 2. 1. 143. 2. 1. 523. 2. 1. 52
Fructose-6- phosphateFructose-6- phosphateGlucose-Glucose-6-phosphate6-phosphate
5. 3. 1. 95. 3. 1. 9
Glucosaminide
3. 2. 1. 1323. 2. 1. 132
GlucosamineGlucosamine3. 2. 1. -3. 2. 1. - 2. 7. 1. 12. 7. 1. 1
ATPATP
ADPADP
ATPATP ADPADPNc, Sc, Sp, CpNc, Sc, Sp, Cp Nc, Sc, CpNc, Sc, Cp
Only Only NcNc NoneNone
Goes-Neto, Priminho, et. AlUEFS
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Control Infected
1 3
2 7
3 14
4 21
DAIPhases
5 35
6 61
7 130
Biochemistry of Infection - 1
Ph.D. Work: Leandra Scarpari; Supervisor: Gonçalo Pereira; Co-supervisors: Paulo Mazzafera e Alan PomellaAcknowledge: Almirante Cacau
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Biochemistry of Infection - 2
Component > Control > Inoculated No Diference
Açucares- Solúveis totais XXX- Sacarose XXX- Amido XXX- Hemicelulose XXX- Açúcares redutores XXX- Celulose XXX- Pectinas XXX
AminoácidosQuantitativo XXXQualitativo *** ASP, GLU ASN
Fenóis totais XXXTaninos XXXNitrato XXXFlavonóides totais XXXClorofilas e carotenóides XXX
HormôniosEtileno XXXAuxinaCitocinina
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
1 2 3 4 5 6
Controle
Inoculado
Açúcares Solúveis Totais (ug/mg)
Açucar Solúvel
0,0
20,0
40,0
60,0
80,0
100,0
120,0
1 2 3 4 5 6
Sadio
Inoculado
Hemicelulose
Hemicelulose
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
CONTROLE1 CONTROLE2 CONTROLE3 CONTROLE4 CONTROLE5 CONTROLE6
ASP
GLU
ASN
AA - Controle
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
INOCULADO1 INOCULADO2 INOCULADO3 INOCULADO4 INOCULADO5 INOCULADO6
ASP
GLU
ASN
AA - Inoculado
0,0
500,0
1000,0
1500,0
2000,0
2500,0
3000,0
3500,0
4000,0
4500,0
1 2 3 4 5 6
Controle
Inoculado
Aminoácidos (nmoles/mg)
AA - Total
0,0
5,0
10,0
15,0
20,0
25,0
1 2 3 4 5 6
Controle
Inoculado
Amido (ug/mg)
Amido
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
1 2 3 4 5 6
Controle
Inoculado
Flavonóides Totais
0
0,5
1
1,5
2
2,5
3
1 2 3 4 5 6
Clorofila Total (ug/mg)
Clorofila Total
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Biochemistry of Infection - 3
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
ASP GLU ASN SER GLN ALA GABA GLY +THR
CONTROLE1
CONTROLE2
CONTROLE3
CONTROLE4
CONTROLE5
CONTROLE6
C
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
ASP GLU ASN SER GLN ALA GABA GLY +THR
INOCULADO1
INOCULADO2
INOCULADO3
INOCULADO4
INOCULADO5
INOCULADO6
I
Acido Glutâmico
Asparagina
The infection triggers the plant Programmed Cell Death - PCD
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1 2 3 4 5 6 7 8 9 101. S. cerevisiae + H. wingei (Bio Rad)
2. CP02 (Biótipo C)
3. CP09 (Biótipo C)
4. FA42 (Biótipo C)
5. Ilhéus (Biótipo C)
6. FA104 (Biótipo S)
7. SCFT (Biótipo L)
8. FA322 (Biótipo L)
9. S. pombe
10. S. cerevisiae
C S L
Molecular Features
Rincones, J, Meinhardt, L, Vidal, B & Pereira, GAG
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CP02 (25-06)
3.12.8(2)
4.65.23.5(3)
29 Mb
Genome Size
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Genome Variability
Bahia I II IIAmazônicos
Probably only two clones of C. perniciosa have been introduced in Bahia
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Telomeric PCR
I II
BahiaA1-R
1018 bp
1636 bp2036 bp3054 bp
517 bp
I II
BahiaA1
Meinhardt, L, Rincones, J. & Pereira, GAG
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Amazônicos
Rondônia????
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Genetic Solution
Creation of strict sanitary barriers between Amazonia and Bahia
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Transposons
S C C L
Class ISuper-family Gypsy/Ty3
Marisa Queiroz - UFV
M L S U 1 2 3 4 5 6 P B
394
ReverseTrancriptase
S C C L
Impala-like
S C C L
Restless-like
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Variability Dynamics
Homobasidiomycete
Sexual Phase
???
Meioses
1. Cromossomal variations can be immediately fixed2. Variability may raise by mutation andtransposons 3. Why we do not see higher variability in Bahia????
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Mitochondria
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Senescence
Kalilo - like
109 kb
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Senescence Waves
Hugo
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Alternative Oxidase
C. perniciosa Alternative Oxidase
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Recommendations
1. Strobirulin based fungicides are ineffective at the field
2. Inhibition of mitochondria may be effective to control the disease and could be achieved by combining strobirulin and Sham analogues
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Acidocalcisome ???
Nuclei
???
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Homeostase
Ca
Ca
Ca
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Necrosis Factor
Phytophthora
Crinipellis
C. perniciosa
VV VS G M
RT-PCR do gene indutorde etileno e necrose
~ 4 paralogsNo intronsSecreted
UESC & UNICAMPExpressão, Estrutura e
Regulação
Etileno/Massa
20
60
100
140
180
220
260
300
1 2 3 4 5 6 7
coleta
controle
inoculado
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Fátima Alvim - UESC
Necrosis Induction
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Dr. Lyndel Mainhardt
Plant X Pathogen
BT
NC
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Biotrophyc Hyfas
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First Global Expression
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Ethylene Formation
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Chemical Genomics
ATOM 1 N PHE 13 41.568 48.879 96.268 1.00 23.86 N ATOM 2 CA PHE 13 42.820 48.397 96.955 1.00 23.90 C ATOM 3 C PHE 13 43.984 48.568 95.986 1.00 23.35 C ATOM 4 O PHE 13 43.926 49.391 95.071 1.00 21.30 O ATOM 5 CB PHE 13 42.687 46.912 97.362 1.00 22.95 C ATOM 6 N SER 14 45.042 47.799 96.191 1.00 23.62 N ATOM 7 CA SER 14 46.177 47.890 95.297 1.00 24.80 C ATOM 8 C SER 14 45.721 47.249 94.008 1.00 26.03 C ATOM 9 O SER 14 44.772 46.466 94.005 1.00 27.80 O ATOM 10 CB SER 14 47.368 47.133 95.860 1.00 22.46 C ATOM 11 N ASP 15 46.348 47.619 92.900 1.00 27.00 N ATOM 12 CA ASP 15 46.006 47.011 91.632 1.00 25.59 C ATOM 13 C ASP 15 46.328 45.539 91.796 1.00 24.64 C ATOM 14 O ASP 15 45.507 44.694 91.497 1.00 26.72 O ATOM 15 CB ASP 15 46.816 47.642 90.503 1.00 24.84 C
Target Structure
http://www.rcsb.org/pdb/20098 Structures (18/02/03)
Chemical Libraries
Molecular Docking
Molecular Design
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“Omics” Era
Genomics
Metabolomics
TranscriptomicsProteomics Economics
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Malaria
Nature 419, 498 - 511 (2002)
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The Regress
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Price Reaction
Preço interno na Bahia - Diferencial s/Bolsa de NY(em $/t)
-200,00
-100,00
0,00
100,00
200,00
300,00
04/0
1/99
13/0
2/99
25/0
3/99
04/0
5/99
13/0
6/99
23/0
7/99
01/0
9/99
11/1
0/99
20/1
1/99
30/1
2/99
08/0
2/00
19/0
3/00
28/0
4/00
07/0
6/00
17/0
7/00
26/0
8/00
05/1
0/00
14/1
1/00
24/1
2/00
02/0
2/01
14/0
3/01
23/0
4/01
02/0
6/01
12/0
7/01
21/0
8/01
30/0
9/01
09/1
1/01
19/1
2/01
28/0
1/02
09/0
3/02
18/0
4/02
28/0
5/02
500
750
1.000
1.250
1.500
1.750Bolsa NIPrêmio/Desconto
Difer. Bolsa
Fonte: Thomas Hartman
Início
.
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www.lge.ibi.unicamp.br/vassoura
Homepage
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Team
COORDENAÇÃOGonçalo Amarante Guimarães Pereira -Júlio Cascardo
COORDENADORES DE CENTROS CEPLAC Raúl René Meléndez Valle e Karina GramachoEMBRAPA - Dário Gratapaglia UESC -Júlio Cascardo e Dário Ahnert UNICAMP - Gonçalo Guimarães PereiraUFBA - FC - Marilda GonçalvesUFBA - IB – Luciana Veiga UEFS - Aristóteles Goes Neto e Carlos Priminho PirovaniUCSAL - Luzimar Gonzaga
GERÊNCIA E ASSESSORIA JURÍDICAEliane Laranja Dias e Leonardo Grecco
COORDENADORAS DNA Carolina Cotomacci - Dirce Maria Carraro
CONSULTORES DNA Anderson Ferreira da Cunha e Helaine Carrer
PLANTA X PATÓGENOLeandra Scarpari, Allan Pomella e Lyndel Meinhardt
BIOINFORMÁTICA Marcos Renato Araujo, Luciano Digiampetri,Marcelo F. Carazzolle e Eduardo Formigheri
SEQÜENCIAMENTOCarolina Cotomacci, Raquel, Anrdéa de Assis, Camilla RamosVitor Almeida, Robson, Monica, Luana, ReimarMaíza Alves Lopes e Bruno Motta Oliveira
MAPEAMENTO MOLECULARJohana Rincones, Lyndel Maynhardt
CICLO SEXUAL Carolina Cotomacci
ECOLOGIA MOLECULAR / MITOCÔNDRIA Naiara Sardinha Pinto, Eduardo Formigheri
EXPRESSÃO GLOBALMaricene Sabha e Leandra
TRANSFORMAÇÃO DO FUNGO Marisa Queiroz
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GENÉTICA VEGETAL Acássia Benjamim Leal Pires Alana Maria Cerqueira de OliveiraFábio Gelape Faleiro Fátima Cerquira Alvim José Antonio Peters José Bonadie MarquesJosé Luis Pires Júlio Cézar de Mattos Cascardo Leandro Lopes Loguércio Luana Mahé Costa Gomes Milton Macoto Yamada Mônica Menezes Passinho Mônica Rosa Bertão Reinaldo Figueiredo dos Santos Rita de Cássia Siqueira Bahia de Assis Uilson Vanderlei Lopes Wilson Reis Monteiro
BIOQUÍMICA Paulo Mazzafera
SISTEMÁTICA DE FUNGOS Aristóteles Goes Neto
SISTEMÁTICA VEGETAL Cíntia Schultz Coimbra FISIOLOGIA VEGETAL Marco Antonio Galeaas Aguilar Raúl René Meléndez Valle
FITOPATOGIA Carlos Priminho PirovaniEdna Dora M. N Luz Givaldo Rocha Niella Jadergudson Pereira João de Cássia do Bomfim Costa João Louis Pereira José Lauereano José Luis Bezerra Karina Peres Gramacho Marival Lopes de OliveiraStela Dalva M. N. Silva
MICROSCOPIA ELETRÔNICAStela Silva e Paulo Joazeiro
Team - 2
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Funding
Fapesp
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Economical Perspectives
Amex Biotech x Nasdaq x Dow Jones
-
200
400
600
800
1.000
1.200
1.400
1.600
jan/90 jan/91 jan/92 jan/93 jan/94 jan/95 jan/96 jan/97 jan/98 jan/99 jan/00 jan/01
AMEX Biotech Nasdaq Dow Jones
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Biotech Process
Alcohol Fermentation
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