Pour augmenter l’affinité des anticorps sécrétés en réponse à un antigène, les gènes d’immunoglobulines subissent l’hypermutation somatique, une mutagénèse adaptative initiée par l’action de l’activation-induced cytidine deaminase (AID). L’uracile provenant de la désamination des cytosines par cette enzyme est réparé de façon erronée par la suite : si il est pris en charge par l’uracile N-glycosylase (UNG), enzyme à l’origine d’une réparation poursuivie habituellement par des composantes de la voie du "base-excision repair", il reste à sa place un site abasique franchissable par les ADN polymérases translésionnelles avec un taux d’erreur très élevé. Si le mésappariement U:G est reconnu par la voie du « mismatch repair », le brin d’ADN entourant le U est dégradé puis néo-synthétisé par une autre ADN polymérase translésionnelle particulièrement mutagène en face des bases T et A, la polymérase eta. Nous avons proposé que le choix entre ces deux voies de réparation mutagènes puisse être régulé en fonction du cycle cellulaire: les mutations des paires A:T seraient introduites dans les gènes d’immunoglobulines par la voie du mismatch repair en phase G1 alors que la voie erronée d’UN introduirait les autres mutations lors de la phase S. Nous sommes parvenus à restreindre l’activité de l’AID à deux parties distinctes du cycle, la phase G1 ou les phases S/G2/M, et nous avons établi le fonctionnement de ce système dans le modèle murin. De façon surprenante, nous avons détecté un taux de mutation proche du bruit de fond chez toutes les souris dont l’AID opérait uniquement dans les phases S/G2/M. Par contre, les souris dont l’AID a été restreinte en G1 présentaient un spectre de mutation diversifié sur les quatre bases et similaire au normal. A la lumière de ces résultats, nous proposons que les lésions introduites tout au long du cycle par l’AID soient diversifiées par les acteurs de l’hypermutation somatique pendant la phase G1, alors que les lésions seraient soit réparées de façon fidèle en dehors de cette phase-là, soit de faible impact. Afin d’expliquer le biais de brin dans l’hypermutation somatique observé pour les mutations sur les bases A :T, nous proposons pour l’ADN polymérase eta un rôle inhabituel de réparation du brin portant la « lésion », et non de synthèse translésionnelle classique en face de cette lésion. Nous avons analysé le profil, le taux et la distribution des mutations introduites par Pol eta sur un oligonucléotide cible pour l’hypermutation, qui a été inséré au locus des immunoglobulines et utilisé pour l’établissement des souris knock-in avec un fond génétique déficient ou non en UNG. Nos résultats, selon lesquels Pol eta continue de cibler le brin codant indépendamment de la localisation des « points d’entrée » en forme d’uraciles, contredisent les rapports déjà publiés sur ce sujet. De façon inattendue, nos résultats mettent en évidence une coopération entre les voies UNG et et les activités endonucléasique du mismatch repair, fournissant la cassure simple brin qui va permettre d'initier la resynthèse à fort taux d'erreur à l'origine de la mutagénèse A/T. Ces résultats résolvent aussi le paradoxe de la non-participation apparente du complexe effecteur du mismatch repair (Mlh1/Pms2) dans le processus d'hypermutation, en proposant qu'il fonctionne en redondance avec UNG, dans une distribution des tâches qui dépend du contexte de la séquence ciblée et de la densité du processus de deamination. / Somatic hypermutation is a localized mutagenesis, essentially targeted to the immunoglobulin V region, and occurring during the immune response. This process is triggered by AID (activation-induced cytidine deaminase) that deaminates cytosines into uracils at the Ig locus. This lesion is further processed by Ung or the Msh2-Msh6 complex, with an abnormal outcome for both pathways that results in an increased mutation load. The Msh2-Msh6 complex recruits Pol eta to generate a short patch DNA synthesis with mostly mutations at A and T bases. To get further insight into this error-prone repair process, we have generated hypermutation substrates consisting in an A/T oligonucleotide of 100 bases with or without 3 cytidines in its core region, inserted by knock-in at the heavy chain Ig locus. Our aim was to compare the mutation frequency, distribution and mutation profile of substrates with C on either the coding or the non-coding strand on WT or Ung-deficient background, taking into account that Pol eta is a preferred A to G mutator. Our results suggest that Pol eta resynthesis may proceed on the coding strand, whatever the strand localization of the uracil, thus contradicting previous reports. Unexpectedly, our results revealed a cooperation between the Ung pathway and the endonuclease activity of the mismatch repair, with both of them providing the single-strand nick that allows initiation of the error-prone process that generates mutations at A and T bases. These results resolve the apparent paradox of the non-involvement of the mismatch repair effector complex (Mlh1-Pms2) in hypermutation, by proposing that it works redundantly with UNG, in a distribution of tasks that will depend upon the sequence context and the intensity of deamination activity. We have also constructed cell cycle restricted mutants of AID, to study in which phase of the cell cycle this atypical, mismatch repair driven, error-prone synthesis is taking place. Using the Fucci restriction system (degrons based on Cdt1 or Geminin peptides), we have generated AID constructs with proper restriction in either G1 or S/G2/M phases. These retroviral constructs have been used to transduce mouse hematopoietic stem cells from either AID -deficient mice and to restore immunodeficient animals, in order to analyze their immune response. We report that restriction of AID expression in S/G2/M part of the cycle yielded only background mutation frequency, while AID operating in the G1 phase is able to generate an equal proportion of A/T and G/C mutations at the Ig loci, thus demonstrating that uracils generated in G1 are substrates for both Ung- and mismatch repair pathways.

Recentemente, pesquisadores têm proposto o uso de Algoritmos Genéticos (AGs) para a determinação da estrutura tridimensional de proteínas. No entanto, este é um problema difícil para um AG tradicional, pois na maioria das vezes ocorre a convergência prematura das soluções para ótimos locais. Isto ocorre porque o uso de mecanismos de seleção no AG acarreta uma perda da diversidade das soluções. Assim, neste trabalho, são investigadas estratégias para controlar a diversidade da população do AG e evitar que a solução fique rapidamente presa em ótimos locais. São empregadas bases de dados de ângulos de torção para a cadeia principal, cadeia lateral e técnicas de controle de diversidade em AGs conhecidas como Hipermutação e Imigrantes Aleatórios. Além disso, um novo algoritmo baseado no AG com Imigrantes Aleatórios Auto-Organizáveis é proposto. Os resultados mostram que estas variações são efetivas no objetivo de não manter o conjunto de soluções preso a uma região apenas, além de melhorar o desempenho para o problema de determinação de estruturas terciárias de proteínas. / Recently, researchers have proposed the use of Genetic Algorithms (GAs) for the determination of the three-dimensional structure of proteins. However, this problem is considered a difficult problem for the standard GA, because most of the cases the convergence occurs early, into local minima instead of the global optimum. This occurs because the use of selection mechanisms in the GA leads to a loss of diversity of solutions. With this in mind, in this work, strategies to control the diversity of the population in the GA are investigated in order to avoid the solution subset to be early caught in local optima. Database sets of torsion angles for the main chain and the side chain are employed, and also modifications in the GAs, known as Hypermutation and Random Immigrants. Besides these approaches, a new algorithm based on the Self-Organizing Random Immigrants is proposed. Results show that these changes are effective in the goal of avoiding the results ensemble to be trapped in a region, and also help improve the performance for the protein structure prediction problem.

<p>B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (V_H) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load.</p><p>The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the V_H gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed V_H genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated V_H genes, thus questioning the cell of origin in HCL. Biased usage of particular V_H genes was detected in both HCL (V_H3-30) and MCL (V_H3-21 and V_H4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, V_H3-21⁺ MCLs showed a highly restricted V_λ3-19 gene use and they also had a superior outcome compared to other MCLs.</p><p>Rearrangement analysis of 67 V_H3-21⁺ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the V_λ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in V_H3-21⁺ CLLs. </p><p>In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse.</p><p>Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis. </p>

<p>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.</p><p>Our purpose was to examine the presence of clonality and somatic hypermutation of immunoglobulin (IgVн) of MALT lymphomas.</p><p>Paraffin-embedded tumour samples from13 MALT lymphoma were subjected to rearrangement analysis, by using PCR, heteroduplex gels and sequence analysis.</p><p>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.</p>

B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (VH) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load. The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the VH gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed VH genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated VH genes, thus questioning the cell of origin in HCL. Biased usage of particular VH genes was detected in both HCL (VH3-30) and MCL (VH3-21 and VH4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, VH3-21+ MCLs showed a highly restricted Vλ3-19 gene use and they also had a superior outcome compared to other MCLs. Rearrangement analysis of 67 VH3-21+ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the Vλ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in VH3-21+ CLLs. In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse. Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis.

No description available.

Recentemente, pesquisadores têm proposto o uso de Algoritmos Genéticos (AGs) para a determinação da estrutura tridimensional de proteínas. No entanto, este é um problema difícil para um AG tradicional, pois na maioria das vezes ocorre a convergência prematura das soluções para ótimos locais. Isto ocorre porque o uso de mecanismos de seleção no AG acarreta uma perda da diversidade das soluções. Assim, neste trabalho, são investigadas estratégias para controlar a diversidade da população do AG e evitar que a solução fique rapidamente presa em ótimos locais. São empregadas bases de dados de ângulos de torção para a cadeia principal, cadeia lateral e técnicas de controle de diversidade em AGs conhecidas como Hipermutação e Imigrantes Aleatórios. Além disso, um novo algoritmo baseado no AG com Imigrantes Aleatórios Auto-Organizáveis é proposto. Os resultados mostram que estas variações são efetivas no objetivo de não manter o conjunto de soluções preso a uma região apenas, além de melhorar o desempenho para o problema de determinação de estruturas terciárias de proteínas. / Recently, researchers have proposed the use of Genetic Algorithms (GAs) for the determination of the three-dimensional structure of proteins. However, this problem is considered a difficult problem for the standard GA, because most of the cases the convergence occurs early, into local minima instead of the global optimum. This occurs because the use of selection mechanisms in the GA leads to a loss of diversity of solutions. With this in mind, in this work, strategies to control the diversity of the population in the GA are investigated in order to avoid the solution subset to be early caught in local optima. Database sets of torsion angles for the main chain and the side chain are employed, and also modifications in the GAs, known as Hypermutation and Random Immigrants. Besides these approaches, a new algorithm based on the Self-Organizing Random Immigrants is proposed. Results show that these changes are effective in the goal of avoiding the results ensemble to be trapped in a region, and also help improve the performance for the protein structure prediction problem.

<p>B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (V_H) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. </p><p>Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by V_H3-21/V_λ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the V_H3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin.</p><p>In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases. </p>

B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (VH) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin. Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by VH3-21/Vλ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the VH3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin. In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases.

Pour augmenter l'affinité des anticorps sécrétés en réponse à un antigène, les gènes d'immunoglobulines subissent l'hypermutation somatique, une mutagénèse adaptative initiée par l'action de l'activation-induced cytidine deaminase (AID). L'uracile provenant de la désamination des cytosines par cette enzyme est réparé de façon erronée par la suite : si il est pris en charge par l'uracile N-glycosylase (UNG), enzyme à l'origine d'une réparation poursuivie habituellement par des composantes de la voie du "base-excision repair", il reste à sa place un site abasique franchissable par les ADN polymérases translésionnelles avec un taux d'erreur très élevé. Si le mésappariement U:G est reconnu par la voie du " mismatch repair ", le brin d'ADN entourant le U est dégradé puis néo-synthétisé par une autre ADN polymérase translésionnelle particulièrement mutagène en face des bases T et A, la polymérase eta. Nous avons proposé que le choix entre ces deux voies de réparation mutagènes puisse être régulé en fonction du cycle cellulaire: les mutations des paires A:T seraient introduites dans les gènes d'immunoglobulines par la voie du mismatch repair en phase G1 alors que la voie erronée d'UN introduirait les autres mutations lors de la phase S. Nous sommes parvenus à restreindre l'activité de l'AID à deux parties distinctes du cycle, la phase G1 ou les phases S/G2/M, et nous avons établi le fonctionnement de ce système dans le modèle murin. De façon surprenante, nous avons détecté un taux de mutation proche du bruit de fond chez toutes les souris dont l'AID opérait uniquement dans les phases S/G2/M. Par contre, les souris dont l'AID a été restreinte en G1 présentaient un spectre de mutation diversifié sur les quatre bases et similaire au normal. A la lumière de ces résultats, nous proposons que les lésions introduites tout au long du cycle par l'AID soient diversifiées par les acteurs de l'hypermutation somatique pendant la phase G1, alors que les lésions seraient soit réparées de façon fidèle en dehors de cette phase-là, soit de faible impact. Afin d'expliquer le biais de brin dans l'hypermutation somatique observé pour les mutations sur les bases A :T, nous proposons pour l'ADN polymérase eta un rôle inhabituel de réparation du brin portant la " lésion ", et non de synthèse translésionnelle classique en face de cette lésion. Nous avons analysé le profil, le taux et la distribution des mutations introduites par Pol eta sur un oligonucléotide cible pour l'hypermutation, qui a été inséré au locus des immunoglobulines et utilisé pour l'établissement des souris knock-in avec un fond génétique déficient ou non en UNG. Nos résultats, selon lesquels Pol eta continue de cibler le brin codant indépendamment de la localisation des " points d'entrée " en forme d'uraciles, contredisent les rapports déjà publiés sur ce sujet. De façon inattendue, nos résultats mettent en évidence une coopération entre les voies UNG et et les activités endonucléasique du mismatch repair, fournissant la cassure simple brin qui va permettre d'initier la resynthèse à fort taux d'erreur à l'origine de la mutagénèse A/T. Ces résultats résolvent aussi le paradoxe de la non-participation apparente du complexe effecteur du mismatch repair (Mlh1/Pms2) dans le processus d'hypermutation, en proposant qu'il fonctionne en redondance avec UNG, dans une distribution des tâches qui dépend du contexte de la séquence ciblée et de la densité du processus de deamination.