The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.

Foram avaliados 528 isolados clínicos de Pseudomonas aeruginosa de 131 pacientes fibrocísticos atendidos em um centro de referência em fibrose cística (FC) durante o período de 2005 a 2008. 135 isolados clínicos (25,6%) apresentaram subpopulação hipermutante (isolados com altas taxas de mutações) utilizando teste modificado de suscetibilidade aos antimicrobianos. Destes, 9 isolados (6,7%) de 7 pacientes foram classificados como hipermutantes (HPM) segundo estimativa da freqüência de mutação. Após seqüenciamento do gene mutS e análise de mutações relacionadas à inativação do sistema de reparo de erros no pareamento do DNA (Mismatch Repair System - MRS), foi possível observar este tipo de mutação em 5 isolados HPM de 4 pacientes. Avaliamos a formação de biofilme em 45 isolados NHPM, 9 HPM e suas respectivas subpopulações por duas metodologias. Segundo o primeiro método, 24 (53,3%) e 3 (21,4%) isolados NHPM e HPM, respectivamente, foram classificados como não-formadores de biofilme. Pelo segundo método, apenas 4 (8,9%) isolados NHPM e nenhum isolado HPM foram classificados nessa categoria. Os percentuais de resistência aos antibióticos ceftazidima, ciprofloxacino e tobramicina em condições de biofilme foram mais elevados que aqueles obtidos em crescimento plantônico e, em ambas as condições, foi possível evidenciar forte associação entre hipermutabilidade e aumento de resistência antibiótica. A associação dos macrolídeos azitromicina e claritromicina, em concentrações sub-inibidoras, com os antibióticos anti-pseudomonas, demonstrou ação inibitória sobre isolados clínicos de P. aeruginosa em condições de biofilme, o que sugere sua indicação no tratamento de infecções por P. aeruginosa pela sua ação anti-biofilme. / We evaluated 528 clinical isolates of Pseudomonas aeruginosa in 131 CF patients attended at a referral center for cystic fibrosis (CF) during the period of 2005 to 2008. 135 clinical isolates (25.6%) presented hypermutable subpopulation (isolates with high mutation rates) using a modified antimicrobial susceptibility method. Of these, 9 isolates (6.7%) of 7 patients were classified as hypermutable (HPM) according to the estimate of mutation frequency. After gene sequencing and analysis of mutS mutations related to inactivation of the repair system errors in DNA pairing (Mismatch Repair System - MRS), we observed this type of mutation in 5 HPM isolates of 4 patients. We evaluated the biofilm formation in 45 isolates NHPM, 9 HPM and their subpopulations by two methods. According the first method, 24 (53.3%) and 3 (21.4%) isolates NHPM and HPM, respectively, were classified as non-biofilm formers. According the second method, only 4 (8.9%) isolates NHPM and none HPM were classified in this category. The percentages of resistance to antibiotics ceftazidime, ciprofloxacin and tobramycin in conditions of biofilm were higher than those obtained in planktonic growth, and in both conditions, it was observed a strong association between hypermutability and increased antibiotic resistance. The association of macrolides azithromycin and clarithromycin, in sub-inhibitory concentrations, with anti-pseudomonas antibiotics, has shown inhibitory activity on clinical isolates of P. aeruginosa in biofilm conditions, which should be recommended for treatment of CF P. aeruginosa infections due to its anti-biofilm action.

Made available in DSpace on 2015-07-22T20:49:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-10-29. Added 1 bitstream(s) on 2015-08-11T03:25:40Z : No. of bitstreams: 1 Publico-065a.pdf: 467665 bytes, checksum: 5c21c191852c281d4e2eebf6fd7a8d72 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:40Z : No. of bitstreams: 2 Publico-065a.pdf: 467665 bytes, checksum: 5c21c191852c281d4e2eebf6fd7a8d72 (MD5) Publico-065b.pdf: 1808818 bytes, checksum: ea880bd6471f0602f5add685ca9800fe (MD5) / INTRODUÇÃO: A substituição monótona de bases G → A no genoma do HIV-1 é observada desde a década de 1990, entretanto, apenas recentemente esse efeito foi atribuído às APOBECs (apolipoprotein B editing catalytic polypeptide) da imunidade inata. As APOBECs celulares atuam na deaminação de citidina a uridina na fita de DNA viral de polaridade negativa e esse efeito é verificado como excesso de adeninas na fita complementar de DNA viral resultante do processo de transcrição reversa. A presença de hipermutações ocasiona perda da capacidade replicativa da partícula do HIV-1 e pode levar populações virais à extinção. Estudos in vitro demonstraram o efeito antiretroviral das APOBECs3 baseados, principalmente, na verificação de hipermutação. Por outro lado, estudos sistemáticos in vivo são escassos e os dados da literatura são controversos com relação ao efeito da hipermutação no genoma das subpopulações de HIV-1 e na dinâmica da infecção. OBJETIVOS: Este estudo objetivou avaliar os efeitos da hipermutação em pacientes HIVpositivos não tratados correlacionando a freqüência de hipermutação com a carga viral. A carga viral é um importante indicador biológico de replicação do HIV-1 e clínico de progressão para a aids. Assim sendo, foi testado se a presença de hipermutação tem efeito protetor no controle da infecção natural pelo HIV utilizando como parâmetro de apoio a carga viral plasmática do HIV-1 nas 157 amostras de pacientes não tratados testadas para detecção de hipermutação. A integrase constitui um hot spot de hipermutação no genoma viral. MÉTODOS: Foi realizada PCR (Polymerase Chain Reaction) para amplificar um fragmento de 582 pares de bases do gene da integrase e o produto de PCR foi visualizado em gel de agarose com HA-Yellow. O corante HA-Yellow retarda a migração eletroforética de um produto de PCR proporcionalmente ao conteúdo de bases A da sequência. Nosso método de análise foi validado com base em sequências de clones e calibrado em acordo com os resultados gerados pelo programa Hypermut (disponível em http://www.hiv.lanl.gov/content/sequence/HYPERMUT/ hypermut.html). A análise dos dados realizou-se com base na estatística de K-means que permitiu agrupar as amostras clínicas de acordo com sua distância de migração no gel de agarose com HA-Yellow. RESULTADOS: Foi observada hipermutação em 31,2% (n=49/157) destas amostras e houve associação entre presença de hipermutação e maiores níveis de viremia (P=0,02, teste de Mann-Whitney). Adicionalmente, a presença de hipermutação não apresentou associação com menores níveis de linfócitos T CD4 positivos (P=0,06, Teste de Mann-Whitney), nem ao gênero ou à etnia. CONCLUSÕES: Os valores de carga viral detectados em cada indivíduo refletem a quantidade de partículas virais filtradas pelo processo de hipermutação e o substrato da hipermutação é a replicação viral. Assim sendo, nós constatamos que amostras clínicas com altos níveis de replicação viral exibiram o fenômeno de hipermutação mais frequentemente. Em resumo, a detecção de variantes provirais de HIV-1 portando hipermutação correlacionou-se com maiores níveis de carga viral nos pacientes avaliados. Assim sendo, concluímos que a hipermutação, fenômeno bioquímico de substituições G para A no HIV-1, é um processo pervasivo e está associada com níveis mais elevados de replicação viral. Palavras- Chave: HIV-1, APOBEC, imunidade inata, carga viral, hipermutação / TEDE / BV UNIFESP: Teses e dissertações

Foram avaliados 528 isolados clínicos de Pseudomonas aeruginosa de 131 pacientes fibrocísticos atendidos em um centro de referência em fibrose cística (FC) durante o período de 2005 a 2008. 135 isolados clínicos (25,6%) apresentaram subpopulação hipermutante (isolados com altas taxas de mutações) utilizando teste modificado de suscetibilidade aos antimicrobianos. Destes, 9 isolados (6,7%) de 7 pacientes foram classificados como hipermutantes (HPM) segundo estimativa da freqüência de mutação. Após seqüenciamento do gene mutS e análise de mutações relacionadas à inativação do sistema de reparo de erros no pareamento do DNA (Mismatch Repair System - MRS), foi possível observar este tipo de mutação em 5 isolados HPM de 4 pacientes. Avaliamos a formação de biofilme em 45 isolados NHPM, 9 HPM e suas respectivas subpopulações por duas metodologias. Segundo o primeiro método, 24 (53,3%) e 3 (21,4%) isolados NHPM e HPM, respectivamente, foram classificados como não-formadores de biofilme. Pelo segundo método, apenas 4 (8,9%) isolados NHPM e nenhum isolado HPM foram classificados nessa categoria. Os percentuais de resistência aos antibióticos ceftazidima, ciprofloxacino e tobramicina em condições de biofilme foram mais elevados que aqueles obtidos em crescimento plantônico e, em ambas as condições, foi possível evidenciar forte associação entre hipermutabilidade e aumento de resistência antibiótica. A associação dos macrolídeos azitromicina e claritromicina, em concentrações sub-inibidoras, com os antibióticos anti-pseudomonas, demonstrou ação inibitória sobre isolados clínicos de P. aeruginosa em condições de biofilme, o que sugere sua indicação no tratamento de infecções por P. aeruginosa pela sua ação anti-biofilme. / We evaluated 528 clinical isolates of Pseudomonas aeruginosa in 131 CF patients attended at a referral center for cystic fibrosis (CF) during the period of 2005 to 2008. 135 clinical isolates (25.6%) presented hypermutable subpopulation (isolates with high mutation rates) using a modified antimicrobial susceptibility method. Of these, 9 isolates (6.7%) of 7 patients were classified as hypermutable (HPM) according to the estimate of mutation frequency. After gene sequencing and analysis of mutS mutations related to inactivation of the repair system errors in DNA pairing (Mismatch Repair System - MRS), we observed this type of mutation in 5 HPM isolates of 4 patients. We evaluated the biofilm formation in 45 isolates NHPM, 9 HPM and their subpopulations by two methods. According the first method, 24 (53.3%) and 3 (21.4%) isolates NHPM and HPM, respectively, were classified as non-biofilm formers. According the second method, only 4 (8.9%) isolates NHPM and none HPM were classified in this category. The percentages of resistance to antibiotics ceftazidime, ciprofloxacin and tobramycin in conditions of biofilm were higher than those obtained in planktonic growth, and in both conditions, it was observed a strong association between hypermutability and increased antibiotic resistance. The association of macrolides azithromycin and clarithromycin, in sub-inhibitory concentrations, with anti-pseudomonas antibiotics, has shown inhibitory activity on clinical isolates of P. aeruginosa in biofilm conditions, which should be recommended for treatment of CF P. aeruginosa infections due to its anti-biofilm action.

Foram avaliados 528 isolados clínicos de Pseudomonas aeruginosa de 131 pacientes fibrocísticos atendidos em um centro de referência em fibrose cística (FC) durante o período de 2005 a 2008. 135 isolados clínicos (25,6%) apresentaram subpopulação hipermutante (isolados com altas taxas de mutações) utilizando teste modificado de suscetibilidade aos antimicrobianos. Destes, 9 isolados (6,7%) de 7 pacientes foram classificados como hipermutantes (HPM) segundo estimativa da freqüência de mutação. Após seqüenciamento do gene mutS e análise de mutações relacionadas à inativação do sistema de reparo de erros no pareamento do DNA (Mismatch Repair System - MRS), foi possível observar este tipo de mutação em 5 isolados HPM de 4 pacientes. Avaliamos a formação de biofilme em 45 isolados NHPM, 9 HPM e suas respectivas subpopulações por duas metodologias. Segundo o primeiro método, 24 (53,3%) e 3 (21,4%) isolados NHPM e HPM, respectivamente, foram classificados como não-formadores de biofilme. Pelo segundo método, apenas 4 (8,9%) isolados NHPM e nenhum isolado HPM foram classificados nessa categoria. Os percentuais de resistência aos antibióticos ceftazidima, ciprofloxacino e tobramicina em condições de biofilme foram mais elevados que aqueles obtidos em crescimento plantônico e, em ambas as condições, foi possível evidenciar forte associação entre hipermutabilidade e aumento de resistência antibiótica. A associação dos macrolídeos azitromicina e claritromicina, em concentrações sub-inibidoras, com os antibióticos anti-pseudomonas, demonstrou ação inibitória sobre isolados clínicos de P. aeruginosa em condições de biofilme, o que sugere sua indicação no tratamento de infecções por P. aeruginosa pela sua ação anti-biofilme. / We evaluated 528 clinical isolates of Pseudomonas aeruginosa in 131 CF patients attended at a referral center for cystic fibrosis (CF) during the period of 2005 to 2008. 135 clinical isolates (25.6%) presented hypermutable subpopulation (isolates with high mutation rates) using a modified antimicrobial susceptibility method. Of these, 9 isolates (6.7%) of 7 patients were classified as hypermutable (HPM) according to the estimate of mutation frequency. After gene sequencing and analysis of mutS mutations related to inactivation of the repair system errors in DNA pairing (Mismatch Repair System - MRS), we observed this type of mutation in 5 HPM isolates of 4 patients. We evaluated the biofilm formation in 45 isolates NHPM, 9 HPM and their subpopulations by two methods. According the first method, 24 (53.3%) and 3 (21.4%) isolates NHPM and HPM, respectively, were classified as non-biofilm formers. According the second method, only 4 (8.9%) isolates NHPM and none HPM were classified in this category. The percentages of resistance to antibiotics ceftazidime, ciprofloxacin and tobramycin in conditions of biofilm were higher than those obtained in planktonic growth, and in both conditions, it was observed a strong association between hypermutability and increased antibiotic resistance. The association of macrolides azithromycin and clarithromycin, in sub-inhibitory concentrations, with anti-pseudomonas antibiotics, has shown inhibitory activity on clinical isolates of P. aeruginosa in biofilm conditions, which should be recommended for treatment of CF P. aeruginosa infections due to its anti-biofilm action.

Malt lymphoma is a malignant disease that can arise in a variety of extra nodal sites. Previous studies indicate that tumour arise from more mature B-cells. Our purpose was to examine the presence of clonality and somatic hypermutation of immunoglobulin (IgVн) of MALT lymphomas. Paraffin-embedded tumour samples from13 MALT lymphoma were subjected to rearrangement analysis, by using PCR, heteroduplex gels and sequence analysis. Successful amplification was seen in 10/13 cases and sequences of IgVн genes were obtained in 6/13, all of them were mutated. The percentage of mutation compared to germline sequences was 1,1% to 8,6% monoclonal rearrangemang. It was demonstrated that 5 of 7 clones were derived from the Vн3 family, 2 from Vн1 and 1 from the Vн 4 family.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19228号 / 医博第4027号 / 新制||医||1011(附属図書館) / 32227 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 竹内 理, 教授 髙田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The pathogenesis of lupus and other autoimmune diseases driven by antibody-antigen complexes involves interactions between genetic and environmental factors. The genetic factors can be separated into factors that dysregulate adaptive immunity, innate immunity or cell death. One genetic risk factor that can affect both innate and adaptive immunity is the inhibitory Fcγ receptor, FcγRIIB. Reduced or loss of function mutations in FcγRIIB lead to an increased risk of autoimmunity. Using the murine IgG2a specific B cell receptor (BCR) transgenic (Tg) mouse, AM14, our lab discovered that delivery of nucleic acid ligands via the BCR activates B cells by dual engagement of the BCR and endosomal toll like receptors (TLR) 7 and/or 9. Mechanistic studies interrogating the role of downstream signaling effectors and intracellular trafficking in the attenuation of BCR-TLR responses by FcγRIIB were limited by our inability to deliver immune complexes (IC) to non-Tg B cells or form brightly fluorescent IC. To deliver IC to non-Tg B cells, I developed a BCR adapter (BCRAM) that delivers IC to IgM-positive B cells. To track the uptake and trafficking of IC, I developed a panel of antibodies specific for streptavidin (SA). Complexes formed with biotinylated molecules and fluorescent streptavidin could be delivered to AM14 B cells or macrophages and tracked via flow cytometry and/or confocal microscopy. BCRAM and fluorescent IC were used to understand how FcγRIIB attenuated BCR-TLR responses. I found that both DNA IC and RNA IC responses were enhanced by FcγRIIB ablation. Interestingly, a naturally-occurring somatic mutation in the Fc domain of the nucleic acid-binding antibody PL2-3 prevented regulation by FcγRIIB and reduced binding to activating FcγR. Paradoxically, I found that SHIP-1, a negative regulator activated downstream of FcγRIIB engagement, promoted BCR-TLR9 responses independent of FcγRIIB. I hypothesized that FcγRIIB attenuates BCR-TLR9 responses by interfering with sensing by the endosomal TLRs. Using a pH sensing IC, I found that engagement of FcγRIIB leads to residence of the IC in a higher pH compartment. These findings demonstrate that FcγRIIB regulates the activation of autoreactive B cells by modulating the trafficking of nucleic acid containing IC to TLR7 and TLR9 associated intracellular compartments in B cells.

Dans le but de reconnaitre et répondre de manière efficace à une très grande variétés d’agents pathogènes, les cellules B ont développé au cours des mécanismes de diversifications des immunoglobulines contrôlés par des processus génétiques complexes comme la recombinaison V(D)J, l’hypermutation somatiques (SHM), et le changement de classe par recombinaison (CSR). L’ensemble de ces processus est contrôlé par différentes voies de réparations de l’ADN. L’anémie de Fanconi est une maladie génétique rare caractérisée par une défaillance progressive de la moelle osseuse, des anomalies de développement et un risque accru de développer des leucémies et des cancers oesopharyngés. La voie FANC est impliquée dans la réparation des pontages de l’ADN et dans le maintien de la fourche de réplication en cas de stress génotoxique. Il est également bien décrit que la voie FANC joue un rôle important dans la coordination des voies de réponses aux dommages à l’ADN. Dans ce travail de thèse, nous nous sommes intéressés au rôle de la voie FANC dans les processus de diversifications des immunoglobulines.En utilisant des souris déficientes pour le gène Fanca, nous montrons que la voie FANC (ou FANCA) participe à la recombinaison V(D)J en contrôlant, dans la moelle osseuse, la transition des cellules B, du stade pre-B au stade de cellules B immatures. Ceci se ferait probablement par le contrôle de la transcription des gènes codant les chaines légères κ des immunoglobulines. Nous montrons également que Fanca pourrait avoir un rôle dans l’addition de nucleotides aux extrémités codantes, en régulant d’une manière indéterminée l’activité et/ou l’activation de l’enzyme TdT ou de la polymérase Polµ. Par ailleurs, nous avons montré que Fanca est nécessaire pour l’induction des mutations de type transitions A/T pendant le processus de SHM en régulant l’expression ou la stabilisation de Polη. Enfin, Fanca (ou la voie FANC) participe à l’inhibition de la recombinaison non homologue (NHEJ) et est requis durant le CSR pour stabiliser les duplexes entre 2 régions de microhomologies qui facilitent le recrutement d’endonucléases et réguler l’accès des DNA polymérases aux cassures de l’ADN. / To recognize and respond dynamically to an enormous variety of different pathogens, B lymphocytes of the immune system have evolved controlled genetic processes at their immunoglobulin (Ig) loci that are known as Ig diversification including V(D)J recombination, somatic hypermutation (SHM), and class switch recombination (CSR). These complex and vulnerable processes are orchestrated by multiple DNA repair pathways. Fanconi anemia (FA) is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and an increased risk of leukemia and cancer. FANC pathway has been implicated in DNA interstrands crosslinks (ICL) repair and in the rescue of stalled replication forks. The FANC pathway also plays a fundamental role in coordinating the DNA repair pathways. Several lines of evidence suggest a possible involvement of the FANC pathway in Ig diversification processes, thus we are particularly interested in revealing function of FANC pathway during these processes. By using Fanca-/- mice, our results first show that during V(D)J recombination, Fanca (or FANC pathway) participates to the control of the transition from pre-B to immature B cells in bone marrow (BM), probably through transcriptional activation of post-rearranged κ light chain. In addition, Fanca might play a role in nucleotide addition at coding end, possibly by regulating either TdT or Polµ activity/activation. Secondly, we found that Fanca is required for the induction of transition mutations at A/T during SHM via regulation of Polη expression/stabilization. Finally, Fanca (or FANC pathway) inhibits short-range recombination and is required during CSR to stabilize duplexes between 2 short microhomology regions that facilitate the recruitment of endonucleases to trim overhangs and avoid unscheduled access of polymerases to DNA ends.

L’enzyme Activation-induced cytidine deaminase (AID) initie la diversification des immunoglobulines (Ig) par l’induction de dommages à l’ADN. Alors que les lésions induites aux gènes des Ig sont cruciales pour l’établissement de réponses immunes hautement spécifiques et adaptées, ce même type de lésions provoquées ailleurs dans le génome contribue à la transformation cellulaire et à l’apparition de cancer. Les mécanismes impliqués dans la protection de l’intégrité génomique des cellules B restent à définir. D’une part, nous avons développé une approche de protéomique locus-unique en couplant une technique d’identification de protéine par biotinylation de proximité avec l’outil d’édition du génome CRISPR/Cas9. Cette technique innovante, dont nous avons fait la preuve de principe pour des loci abondants, pourra être utilisée pour identifier le protéome des différentes cibles génomiques d’AID. D’autre part, nous avons caractérisé le rôle de Parp3, Parp9 et Med1, identifiées comme partenaires d’AID, éclairant ainsi les mécanismes qui contrôlent l’activité d’AID et la réparation des lésions induites par AID lors de la diversification des Ig. / Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) diversification by inducing DNA damage. While on-target lesions are crucial for mounting highly specific and adaptive immune responses, off-target lesions contribute to malignant cell transformation. Despite its implications, the events following AID recruitment that enforce genome integrity in B cells remain poorly defined. It is not understood why multiple non-Ig loci bound by AID are not mutated or why AID-induced DNA lesions may lead to mutations or DNA breaks. To address this question, we developed a single-locus proteomic approach coupling proximity-dependent protein identification and genome editing (CRISPR/Cas9) to identify and compare the proteins recruited at individual genomic loci bound by AID. We performed the proof of principle of this innovative tool by identifying the proteome of abundant genomic loci. On the other hand, we functionally characterized Parp3, Parp9 and Med1, identified as AID partners, revealing novel mechanisms that tightly control AID activity and DNA repair during Ig diversification.