O papel modulador do gene AIRE - capes

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R.S. Cardoso et al. / Molecular Immunology 43 (2006) 464–472 469Table 2Genes differentially expressed (induced) in gamma-irradiated compared to control FTOCs as displayed by the Cluster and TreeView programCluster Gene name Accession no. Chromosome Function1 Myeloblastosis oncogene NM10848 10 OncogeneKirsten rat sarcoma oncogene 2 NM021284 6 OncogeneCell division cycle 42 NM009861 4 GTPase-signal transductionKu 80, X-ray repair, XRCC5 NM009533 1 DSB repair2 Signal transducer activation of transcription NM011487 1 Transcription factorNeuroprotective protein gene AK129214 2H3 Transcription factorNuclear factor of activated T-cells AK048610 18 Induction of cytokines3 Transcription elongation factor 1 AK048564 1H2 RNA pol II transcriptionTranscription elongation factor 3 AJ223472 4D3 RNA pol II transcriptionRunt-related transcription factor 3 AK053910 4 Enhancer binding proteinThyroid autoantigen Ku70 BC051085 15 DSB repairJanus kinase 3 L40172 8 IL2/IL4 signalingInterleukin receptor IL-11TRA2 X98519 4 Immune response/V(D)J recombinationInterleukin receptor IL-7R NM008372 15 TCR V(D)J recombinationTumor necrosis factor, member7 L24495 6 Survival of activated T-cellsGranzyme A M13226 13 Targeting cell lysis4 Fibroblast growth factor receptor 1 BC010200 8 EmbriogenesisVascular cell adhesion molecule 1 AK089320 3 Cell adhesionRetinoblastome binding protein 7 BC003785 XF4 Negative regulation of transcriptionSRY-box-SOX-4 AK028989 13 Binding to T-cell enhancerArtemis protein AK052369 2A1 TCR V(D)J recombination5 Mitogen activated protein kinase 7 BC070467 8A1.1 Activation of Jun kinasesInsulin-like growth factor binding protein 7 AB012886 5D Regulation of cell growthA-disintegrin and metalloprotease domain 8 BC025584 7F3-F5 Extravasion of leukocytesNuclear transcription factor Y-gamma BC053723 4 Transcription factorTripartite motif protein 28 NM011588 7A1 Transcription factorProtease, serine, 16 (thymus) AK088019 13 Protease, T-cell developmentCalpain 8 NM130890 1H5 Endopeptidase6 X-ray repair, XRCC4 AK038105 13C3 DSB repairHeat shock protein 1-like D85732 17 Cooperation with chaperonesXPC, xeroderma pigmentozum C AK028595 6D DNA excision repair (NER)Interleukin IL-1B NM008361 2 Immune responseInterleukin IL-2 X01772 3 Immune responseInterleukin IL-6 AK089780 5 Immune responseInterleukin Il-7 AK041403 3 Immune response/ V(D)J recombinationLymphotoxin A NM10735 17 Cytotoxic for tumor cellsCyclin D3 AK046638 17 Protein kinaseEGR1, early growth response 1 M22326 18 Transcriptional regulatorRAG-2, recombination activator gene 2 AK040375 2 V(D)J recombinationDNA-activated protein kinase AK088981 16 DSB repair and V(D)J recombinationMMTV endogenous retrovirus B4515481 19 SuperantigenDNA cross-link LR1 BC011094 3F2.2 Nucleotide excision repaira cytokine- and chemokine-mediated signaling pathway.Activity-dependent neuroprotective protein gene (ADNP, accessionno. AK129214, chromosome 2H3) encodes a DNAbindingprotein with transcription factor activity and nuclearfactor of activated T-cells, cytoplasmic 1 gene (NFATC1, accessionno. AK048610, chromosome 18) plays a role in theinducible expression of cytokine genes in T-cells, especially,in the induction of the IL-2 or IL-4 gene transcription.The expression patterns of these genes in non-irradiatedFTOCs suggest a tendency of induction at the early stagesand repression at the late stages.Cluster 3 (correlation = 0.71) also encompasses genes involvedin the regulation of transcription such as TCEA1 andTCEA3 (transcription elongation factor gene 1, accessionno. AK048564, chromosome 1A2 and gene 3, accession no.AJ223472, chromosome 4D3), necessary for efficient RNApolymerase II transcription elongation past template-encodedarresting sites. Cleavage of the nascent transcript by S-II allowsthe resumption of elongation from the new 3 ′ terminus.The Runt-related transcription factor 3 gene (RUNX3 gene,accession no. AK053910, chromosome 4) codes for a proteinthat binds to the enhancers of T-cell receptor genes. Asenhancers of TRs play a role in the control of gene rearrangements(Hempel et al., 1998), RUNX3 could participate in thecontrol of TR V(D)J recombination.G22P1 (Ku70) (thyroid autoantigen gene, accession no.BC051085, chromosome 15) plays a role in DSB repairvia nonhomologous end-joining. Ku70/80 heterodimer is the
470 R.S. Cardoso et al. / Molecular Immunology 43 (2006) 464–472DNA-binding subunit of DNA-PK, an important componentof the repair of DSB via NHEJ (Lees-Miller and Meek, 2003).Absence of Ku70 leads to hypersensitivity to ionizing radiation.Janus kinase 3 gene (JAK3, accession no. L40172,chromosome 8) codes for a tyrosine kinase of the nonreceptortype involved in the IL-2 and IL-4 signaling pathway.This gene belongs to the Tyr family of protein kinases and isimplicated in lymph gland development. The interleukin receptorgenes IL-11RA2 (accession no. X98519, chromosome4) and IL-7R (accession no. NM008372, chromosome 15) arealso present in cluster 3 and may later be associated with IL-7signaling, which plays a role in the V(D)J recombination ofTRs (Muegge et al., 1993; Sollof et al., 1997).In addition, we observed genes associated with apoptosissuch as tumor necrosis factor receptor superfamily, member7 gene (TNFRSF7, accession no. L24495, chromosome 6)and GZMA (granzyme A gene, accession no. M13226, chromosome13). TNFRSF7 encodes a receptor for tnfsf7/cd27l,possibly playing a role in survival of activated T-cells, whileGZMA codes for a granzyme A precursor, an enzyme necessaryfor targeting cell lysis in cell-mediated immune responses.Therefore, the induction of several genes in cluster 3 suggeststhe occurrence of T-cell differentiation following theirradiation stimulus in irradiated FTOCs.Cluster 4 (correlation = 0.81) contains genes with inducedexpression in irradiated FTOCs similar to cluster 3. Amongthe modulated genes, two may play a role in thymus morphogenesisin FTOC, the fibroblast growth factor receptor 1 gene(FGFR1, accession no. BC010200, chromosome 8) whichmay be essential for generation of mesodermal and endodermallayers and invaginations of various types of cells, and vascularcell adhesion molecule 1 gene (VCAM1, accession no.AK085320, chromosome 3) which is important in cell–cellrecognition and appears to function in leukocyte–endothelialcell adhesion.Two genes implicated in the control of transcription suchas retinoblastoma binding protein 7 gene (RBBP7, accessionno. BC003785 chromosome XF4) which plays a role inthe negative regulation of transcription from the polymeraseII promoter and SRY-box containing gene 4 (SOX4, accessionno. AK028989, chromosome 13) which codes for a transcriptionalactivator that binds with high affinity to the T-cellenhancer 5 ′ -AACAAAG-3 ′ motif, may be associated withthe control of V(D)J recombinations in FTOCs, in concertwith the Artemis gene accession no. AK052369, chromosome2A1, which is directly associated with T-cell receptorgene rearrangements since these genes were included in thesame cluster.Cluster 5 (correlation = 0.78) harbors genes, which wereinduced in irradiated FTOCs. Among these genes, we observedthose associated with cell proliferation such as mitogenactivated protein kinase 7 gene (MAP2K7, accessionno. BC070467, chromosome 8A1.1) which has dual kinaseactivities involving the activation of jun kinases mapk8(jnk1) and mapk9 (jnk2); as well as insulin-like growth factorbinding protein 7 gene (IGFBP7, accession no. AB012886,chromosome 5D) that plays a role in the regulation of cellgrowth.A gene associated with cell organization and biogenesis,A-disintegrin and metalloprotease domain 8 gene(ADAM8, accession no. BC025584, chromosome 7F3-F5),which is possibly involved in extravasation of leukocytes,was induced. As mature T-cells migrate from the thymusto the periphery, this gene could be implicated in suchprocess.Transcription factors such as nuclear transcription factor-Y gamma gene (NFYC, accession no. BC053723, chromosome4), 3FP62 (tripartite motif protein 28 gene) (TRIM 28,accession no. NM011588 chromosome 7A1) form a complexwith a krab-domain transcription factor and increase the efficiencyof krab-mediated repression by recruiting setdb1 tohistone H3 (by similarity).We also observed induction of those genes implicated inprotein metabolism such as PRSS16 [protease, serine, 16(thymus) gene, accession no. AK088019, chromosome 13]which codes for a protease that may play a role in T-cell developmentand is specifically expressed in the developing thymus,and calpain 8 gene (CAP8, accession no. NM130890,chromosome 1H5) that codes for a cysteine-type endopeptidaseand whose enzyme is involved in proteolysis and peptidolysis.Cluster 6 (correlation = 0.77) is formed by genes whosefunctions are directly related to the response to DNA damagestimulus such as X-ray repair complementing defectiverepair in Chinese hamster cells 4 gene (XRCC4, accessionno. AK038105, chromosome 13C3) involved in DSB repairand heat shock protein 1-like gene (HSC70T, accession no.D85732, chromosome 17) whose coded protein in cooperationwith other chaperones, for example HSP70, stabilizespre-existing proteins against aggregation and mediates thefolding of newly translated polypeptides in the cytosol, aswell as within organelles. These chaperones participate inthe recognition of non-native conformations of proteins. Anothergene present in the cluster 6 was xeroderma pigmentosum,complementation group C gene (XPC, accession no.AK028595, chromosome 6D), which encodes a DNA repairprotein, which is involved in DNA excision repair (NER) andmay play a role in DNA damage recognition. XPC seems torecognize DNA helix distortions (Gontijo et al., 2003) and isresponsible by opening the DNA helix in response to DNAdamage (Tapias et al., 2004).Moreover, as the DNA repair XPC protein may alter chromatinstructure, it is plausible that its action could facilitatethe accessibility of the V(D)J machinery to the TR locus.We also found genes directly associated with immuneresponse/activation and with the V(D)J recombinationprocess. There are four interleukin genes, IL-1B, accessionno. NM008361, chromosome 2; IL-2, accession no. X01772,chromosome 3; IL-6, accession no. AK089780, chromosome5 and IL-7, accession no. AK041403, chromosome 3. TheIL-7 is a cofactor associated with modulation of V(D)J
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Universidade de São PauloFaculdade
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Macedo, ClaudiaO papel modulador do
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Dedico Especialmente este TrabalhoM
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AgradecimentosAgradeço do fundo do
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ÍndiceRESUMO .....................
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INDICE DE FIGURASFigura 1. Desenvol
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Promiscuous gene expression in medu
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INTRODUÇÃO1. INTRODUÇÃO1.1. A m
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INTRODUÇÃORecentemente surgiram e
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INTRODUÇÃOmigram para a região s
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INTRODUÇÃOCD4 + CD8 + , e Ccl21 (
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INTRODUÇÃOos timócitos duplo-pos
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INTRODUÇÃOgrande diversidade nas
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INTRODUÇÃOAs células mTECs são
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INTRODUÇÃODerbinski (2004). Os ti
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INTRODUÇÃOinvertebrados multicelu
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INTRODUÇÃOCoraçãoLínguaEstôma
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INTRODUÇÃO1.3. O papel do gene Au
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INTRODUÇÃOdefinido pela atividade
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INTRODUÇÃOpara abordarmos nossa p
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INTRODUÇÃOesses modelos levam em
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Hipóteses
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Objetivos
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Material e Métodos
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MATERIAL E MÉTODOSFigura 6. Padrã
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MATERIAL E MÉTODOScom estes RNAs f
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MATERIAL E MÉTODOSdos complexos de
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MATERIAL E MÉTODOSAo RNA total rec
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4.5. PCRs semi-quantitativas para o
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MATERIAL E MÉTODOSminutos a 4 °C.
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MATERIAL E MÉTODOS(Merck), soluç
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MATERIAL E MÉTODOSEnsaios de co-hi
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MATERIAL E MÉTODOSRNA TotalANELAME
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MATERIAL E MÉTODOSa) Preparação
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MATERIAL E MÉTODOScentrifugação
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MATERIAL E MÉTODOScanais (Cy3 e Cy
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MATERIAL E MÉTODOSSpotfinderQuanti
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MATERIAL E MÉTODOSOnde Xp(i) e Xc(
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MATERIAL E MÉTODOSO programa SAM p
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MATERIAL E MÉTODOSextrair automati
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DELINEAMENTO EXPERIMENTAL5. DELINEA
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RESULTADOS6. RESULTADOS6.1. Inibiç
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RESULTADOSPCR semiquantitativa gene
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RESULTADOS6.3. RT-PCRs semi-quantit
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RESULTADOS11.00%6,50%AdequadaNão A
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RESULTADOS6.6. Quantificação e no
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RESULTADOSPara um melhor entendimen
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RESULTADOSFigura 25. Agrupamento hi
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RESULTADOSFigura 26. Gráfico ilust
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RESULTADOS6.8. Influência do silen
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Discussão
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DISCUSSÃOA tolerância do repertó
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DISCUSSÃOCom relação à técnica
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DISCUSSÃOgene AIRE (do inglês aut
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DISCUSSÃOdesse modo a tolerância
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DISCUSSÃOPML (leucemia promielocit
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DISCUSSÃOBoehm et al. (2003) afirm
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DISCUSSÃOprograma Genenetwork (Wu
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Conclusões
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Referências Bibliográficas
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REFERÊNCIAS BIBLIOGRÁFICASBarthlo
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REFERÊNCIAS BIBLIOGRÁFICASDissert
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REFERÊNCIAS BIBLIOGRÁFICASGotter
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REFERÊNCIAS BIBLIOGRÁFICASMatsumo
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REFERÊNCIAS BIBLIOGRÁFICASby mito
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REFERÊNCIAS BIBLIOGRÁFICASRossi S
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REFERÊNCIAS BIBLIOGRÁFICASof thym
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Anexo I123
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ANEXO IEST IMAGE:582370 Expressed s
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ANEXO IDhps IMAGE:583332 Deoxyhypus
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ANEXO IMapkapk2 IMAGE:572979 MAP ki
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ANEXO IGm608 IMAGE:583350 Gene mode
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ANEXO ICcdc72 IMAGE:640628 Coiled-c
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ANEXO ITranscribed locusTranscribed
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ANEXO ITranscribed locusTranscribed
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ANEXO Irepair deficiency, complemen
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ANEXO Imember 1EST IMAGE:583824 Exp
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ANEXO ITranscribed locus IMAGE:5830
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ANEXO ITfdp1 IMAGE:640119 Transcrib
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ANEXO I2410022M11Rik IMAGE:640727 R
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ANEXO Icontaining 1Kif14 IMAGE:5836
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ANEXO Imember 3In multiple clusters
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ANEXO IAnkrd10 IMAGE:583740 Ankyrin
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ANEXO IVcam1 IMAGE:576563 Vascular
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ANEXO ITegt IMAGE:639877 Testis enh
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ANEXO I3Ehmt2Euchromatic histone ly
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ANEXO I20 homolog (yeast)Ube1c IMAG
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ANEXO IRasa3 IMAGE:582174 RAS p21 p
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ANEXO I4930566A11Rik IMAGE:583433 R
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Anexo II
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ANEXO II1500002B03RikIMAGE:1330385
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ANEXO IIEST IMAGE:640173 Expressed
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ANEXO IICnot10 IMAGE:576406 CCR4-NO
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ANEXO IIEST IMAGE:576142 Expressed
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Anexo III
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ANEXO III197Ifit1RelaIMAGE:575632Gr
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ANEXO III199Nde1Nol5aIMAGE:640341Rp
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Manuscrito da Tese
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MANUSCRITOAbstractThe expression of
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MANUSCRITOdominated the scenario in
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MANUSCRITOserum autoantibodies, all
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MANUSCRITOwere transfected with 10
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MANUSCRITOwere analyzed using the M
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MANUSCRITOpurified from ex vivo mou
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MANUSCRITOinformation theory as ARA
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MANUSCRITOKyewski B, Derbinski J, G
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MANUSCRITOBruno R, Sabater L, Tolos
Page 236 and 237: MANUSCRITOFigure 3. The gene expres
Page 238 and 239: MANUSCRITOABFigure 5. Genetics netw
Page 240 and 241: SÚMULACURRICULUM VITAE (Janeiro 20
Page 242 and 243: ESTÁGIO NO EXTERIORSÚMULA• Labo
Page 244 and 245: SÚMULAtranscript finishing initiat
Page 246 and 247: SÚMULAlinhagens isogênicas de cam
Page 248 and 249: SÚMULABOLSAS DE ESTUDO• Bolsa de
Page 250 and 251: T cell signaling gene networks duri
Page 252 and 253: IntroductionThe thymus is an import
Page 254 and 255: ontogeny using such technology has
Page 256 and 257: laboratory using a Generation III A
Page 258 and 259: cDNA microarray data analysisStatis
Page 260 and 261: Gene networksAs previously mentione
Page 262 and 263: cells, including human herpesvirus-
Page 264 and 265: gene was mostly indirectly positive
Page 266 and 267: lymphocytes and finally associates
Page 268 and 269: 4) Finally, Prrg2 was also identifi
Page 270 and 271: 14. Kishimoto H, Sprent J (2000) Th
Page 272 and 273: proliferation of a rat astrocyte ce
Page 274 and 275: IMMUNOLOGYORIGINAL ARTICLEOnset of
Page 276 and 277: Promiscuous gene expression in muri
Page 282 and 283: R.S. Cardoso et al. / Molecular Imm
Page 290 and 291: Molecular Immunology 42 (2005) 1043
Page 292 and 293: D.A.R. Magalhães et al. / Molecula
Page 294 and 295: D.A.R. Magalhães et al. / Molecula
Page 296 and 297: Molecular and Cellular Biochemistry
Page 298 and 299: 225Table 1. Target cDNA sequences a
Page 300 and 301: 227coincided (CBA/J) with expressio
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