Le locus des chaînes lourdes d’immunoglobulines (IgH) subit trois processus de remaniements géniques durant la lymphopoïèse B. Ces événements induisent des cassures de l’ADN potentiellement oncogéniques, d’où la nécessité d’une régulation extrêmement stricte. Ceci est dû aux deux principaux éléments cis-régulateurs du locus IgH. L’enhancer 5’Eµ régule les recombinaisons VHDJH qui établissent un répertoire antigénique fonctionnel lors des phases précoces. La région régulatrice en 3’ (3’RR) est essentielle aux hypermutations somatiques (SHM) et à la recombinaison de classe (CSR) aux stades tardifs, modifiant respectivement, l’affinité et les fonctions effectrices de l’Ig. La plupart des lymphomes B matures portent les stigmates de translocations d’oncogènes au locus IgH. Le but de ma thèse a été de mieux comprendre les interactions transcriptionelles entre les enhancers Eµ et 3’RR et évaluer si le ciblage de cette dernière pourrait se révéler une approche thérapeutique potentielle. Nous avons démontré que la 3’RR est l’élément essentiel qui contrôle la transcription du locus IgH dans les lymphocytes B matures. Elle est dispensable lors des phases initiales (recombinaisons VHDJH), mais agit comme silencer sur l’expression des segments DJH. L’analyse de la lymphomagenèse dans trois modèles murins porteurs d’une insertion de Myc en trois points du locus IgH a montré des différences dans les cinétiques d’émergence des lymphomes, leurs phénotypes et index de prolifération. L’effet de la 3’RR sur l’oncogène est suffisant pour l’émergence de lymphomes B. Son absence ne semble pas être préjudiciable au développement de réactions inflammatoires/immunes. Son ciblage pourrait donc se révéler une approche thérapeutique intéressante pour diminuer son activité transcriptionelle sur l’oncogène transloqué. Un rôle potentiel des inhibiteurs des histones désacétylases est à l’étude. / The immunoglobulin heavy chain locus (IgH) undergoes several changes along B-cell differentiation. VHDJH recombinations during the early stages give the diversity of the antigenic repertoire. Somatic hypermutation (SHM) and class switch recombination (CSR) during late stages allow affinity maturation and the acquisition of new effectors functions. These rearrangements are highly regulated and are under the control of the IgH locus cis-regulatory elements. The 5’ Eµ enhancer is important for VHDJH recombination. The 3’ regulatory region (3’ RR) is essential for both CSR and SHM. These events induce breaks into the IgH locus, making it a hotspot for oncogenic translocations. The aim of my thesis was to understand the transcriptional interactions between Eμ and 3'RR enhancers and to evaluate whether the targeting of the latter could be of a potential therapeutic approach. We have demonstrated that 3'RR is essential to control IgH transcription in mature B cells. It is dispensable during the initial stages of developement (VHDJH recombinations). At the pro-B cell stage, it has a silencer effect rather than a transcriptional one on the DJH segments expression. The analysis of lymphomagenesis in three mice models carrying an insertion of Myc in different locations at the IgH locus showed significant differences in lymphoma kinetics, phenotypes and proliferation index. 3'RR alone, as a major transcriptional activator of the IgH locus, is capable of leading to B-cell lymphomas. Its absence is not detrimental for the development of classical inflammatory/immune reactions. Its targeting may be of a potentially interesting therapeutic approach to decrease its transcriptional activity on the translocated oncogene. A potential role for histone deacetylase inhibitors is under study.

Neste trabalho foram analisados 30 casos de linfomas de células B da região oral, fixados em solução de formaldeído e incluídos em parafina, através da técnica de imuno-histoquímica para as proteínas c-Myc, Bcl-2, Bcl-6 e ciclina D1. Dos casos analisados 40% foram positivos para a marcação para c-Myc, 33,3% para a marcação para ciclina D1, 83,3% para a marcação para Bcl-2 e 53,3% para a marcação para Bcl-6. Todos os casos foram diagnosticados como linfomas difusos de grandes células B, o subtipo de linfoma com a maior casuística. A análise destas proteínas é de fundamental importância para o diagnóstico e direcionamento do tratamento de doenças hematopoiéticas, pois estão envolvidas em vários processos de controle da transcrição gênica, do ciclo celular e dos processos apoptóticos e o aumento do conhecimento sobre sua ação em diferentes subtipos de linfomas pode corroborar outros estudos. / In this study, 30 cases of formalin-fixed and paraffin-embedded B-cell lymphomas of the oral region were submitted to immunohistochemistry for the detection of proteins c-Myc, Bcl-2, Bcl-6 and cyclin D1. Fourty percent (40%) of the studied cases were positive for c-Myc, 10% for cyclin D1, 83.3% for Bcl-2 and 53.3% for Bcl-6. The analysis of these proteins has fundamental importance for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription and cell cycle. All cases were diffuse large B-cell lymphomas, the subtype with the highest incidence. The analysis of these proteins is very important for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription, cell cycle and apoptotic processes and an increase in the knowledge of their action in different subtypes of lymphomas can corroborate to other studies.

Orientador: Luis Otavio Zanatta Sarian / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-07T13:37:40Z (GMT). No. of bitstreams: 1 Peres_RaquelMaryRodrigues_D.pdf: 3497486 bytes, checksum: c509c5d08807f1a5bcd1b918eaf7d09d (MD5) Previous issue date: 2013 / Resumo: Introdução: A hipótese de que os efeitos do alumínio em células humanas podem ter implicações clínicas tem sido levantada há algum tempo, especialmente no que concerne ao câncer de mama. As evidências laboratoriais mostrando altos níveis de alumínio nos tecidos da mama e os efeitos biológicos conhecidos sobre esse metal não são suficientes para estabelecer uma relação causal entre a exposição ao alumínio e o risco aumentando para o desenvolvimento do câncer de mama. O objetivo deste estudo foi estabelecer a concentração de alumínio nas áreas centrais e periféricas de tumores de mama, assim como na área glandular normal da mama e correlacionar esses achados com a instabilidade dos genes ERBB2, C-MYC e CCND1 e a aneuploidia dos cromossomos que contêm estes genes. Métodos: Para este estudo foram incluídas 176 mulheres com diagnóstico de carcinoma invasor de mama, com tumores maiores de 1cm3, sem quimioterapia neoadjuvante, operadas enter 2008 e 2010 no Hospital da Mulher Prof. Dr. José Aristodemo Pinotti - Centro de Atenção Integral à Saúde da Mulher (CAISM) - UNICAMP. Para a análise da concentração de alumínio intracelular, amostras de 150 pacientes foram consideradas viáveis; para a análise da instabilidade genômica em função da concentração de alumínio, 118 amostras foram consideradas viáveis, definindo o espaço amostral de cada um dos artigos apresentados. As amostras das áreas centrais e periféricas dos tumores de mama e das áreas glandulares normais da mama foram obtidas. A quantificação do alumínio contido nos tecidos da mama foi feita através da técnica de Espectrometria de Absorção Atômica em Forno de Grafite (GFAAS). Uma lâmina de Tissue Microarray (TMA), contendo as amostras de tumor e tecido normal foi utilizado para a realização da técnica de FISH para acessar o status dos genes ERBB2, C-MYC e CCND1 e dos centrômeros dos seus respectivos cromossomos 17, 8 e 11. Os dados clínico-patológicos foram obtidos dos prontuários de pacientes. Resultados: A média da concentração de alumínio encontrada na mama foi de 1,88 mg/kg nas áreas centrais do tumor, 2,10mg/kg nas áreas periféricas do tumor e 1,68mg/kg nas áreas de tecido glandular normal. A amplificação e/ou aneuploidia para ERBB2/CEP17, C-MYC/CEP8 e CCND1/CEP11 foi encontrada em 24%, 36,7% e 29,3% dos tumores, respectivamente. A média da concentração de alumínio nas áreas tumorais (tanto centrais quanto periféricas) não foi significativamente diferente daquela nas áreas de tecido normal. A concentração de alumínio também não foi significativamente associada a nenhum status de amplificação e/ou aneuploidia para os genes/cromossomos em questão. Conclusões: Consideramos importante que estudos experimentais in vitro continuem sendo realizados para elucidar os possíveis efeitos do alumínio nos tumores de mama, quer sejam esses efeitos relacionados ao microambiente tecidual ou mesmo a outras vias de estabilidade genômica / Abstract: Introduction: It has long been hypothesized if the effects of aluminum on human cells may have clinical implications, especially regarding to breast cancer. The current laboratorial evidence showing higher levels of aluminum in breast tissues and the known biological effects of this metal, are not sufficient to establish a causal relationship between aluminum exposure and increased risk of developing breast cancer. The objective of this study was to establish the aluminum concentration in the central and peripheral areas of breast tumors as well as in normal glandular area of the breast and to correlate these findings with the instability of ERBB2, C-MYC and CCND1, and aneuploidy of chromosomes harboring these genes. Methods: This study included 176 women diagnosed with invasive breast carcinoma with tumors larger than 1cm3 without neoadjuvant chemotherapy, operated between 2008 and 2010 at the Women's Hospital Professor. Dr. José Aristodemo Pinotti - Centro de Atenção Integral à Saúde da Mulher (CAISM) - UNICAMP. To analyze the intracellular concentration of aluminum, samples from 150 patients were considered viable; for the analysis of genomic instability as a function of the concentration of aluminum, 118 samples were considered viable. These figures define the sample of each of the two articles that this PhD thesis comprises. Evaluation of tissue aluminum content was carried out using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). A TMA slide containing the tumor and normal samples was used in FISH assays to assess ERBB2, C-MYC and CCND1 and the respective chromosomes 17, 8 and 11 centromeres status. Clinicopathological data were obtained from patients' records. Results: The average aluminum content found in breast was 1.88 mg/kg in the central tumor areas, 2.10 mg/ kg in the peripheral tumor areas and 1.68 mg/ kg in the normal tissue areas. The amplification and/or aneuploid status for the ERBB2/CEP17, C-MYC/CEP8 and CCND1/CEP11 was detected in 24%, 36.7% and 29.3% of the tumors, respectively. The average aluminum content in tumor areas (either central or peripheral) was not significantly different from that in normal tissues. We found that aluminum concentration was not related to any of the gene status. Conclusions: We consider important that in vitro experimental studies continue to be done in order to elucidate the possible effects of aluminum in the development of breast tumors, whether it is influencing the tissue microenvironment or other genome stability pathways / Doutorado / Oncologia Ginecológica e Mamária / Doutora em Ciências da Saúde

Neste trabalho foram analisados 30 casos de linfomas de células B da região oral, fixados em solução de formaldeído e incluídos em parafina, através da técnica de imuno-histoquímica para as proteínas c-Myc, Bcl-2, Bcl-6 e ciclina D1. Dos casos analisados 40% foram positivos para a marcação para c-Myc, 33,3% para a marcação para ciclina D1, 83,3% para a marcação para Bcl-2 e 53,3% para a marcação para Bcl-6. Todos os casos foram diagnosticados como linfomas difusos de grandes células B, o subtipo de linfoma com a maior casuística. A análise destas proteínas é de fundamental importância para o diagnóstico e direcionamento do tratamento de doenças hematopoiéticas, pois estão envolvidas em vários processos de controle da transcrição gênica, do ciclo celular e dos processos apoptóticos e o aumento do conhecimento sobre sua ação em diferentes subtipos de linfomas pode corroborar outros estudos. / In this study, 30 cases of formalin-fixed and paraffin-embedded B-cell lymphomas of the oral region were submitted to immunohistochemistry for the detection of proteins c-Myc, Bcl-2, Bcl-6 and cyclin D1. Fourty percent (40%) of the studied cases were positive for c-Myc, 10% for cyclin D1, 83.3% for Bcl-2 and 53.3% for Bcl-6. The analysis of these proteins has fundamental importance for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription and cell cycle. All cases were diffuse large B-cell lymphomas, the subtype with the highest incidence. The analysis of these proteins is very important for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription, cell cycle and apoptotic processes and an increase in the knowledge of their action in different subtypes of lymphomas can corroborate to other studies.

Les lymphocytes B et T sont issus de cellules progénitrices lymphoïdes de la moelle osseuse qui se différencient grâce à l’action de facteurs de transcription, cytokines et voies de signalisation, dont l’interleukine-7 (IL-7)/IL-7 récepteur (IL-7R). Le facteur de transcription c-Myc est exprimé par les cellules lymphoïdes et contrôle leur croissance et leur différenciation. Cette régulation transcriptionnelle peut être coordonnée par le complexe c-Myc/Myc-Interacting Zinc finger protein-1 (Miz-1). Le but de ce projet était de comprendre les mécanismes qui impliquent Miz-1 et le complexe c-Myc/Miz-1 dans le développement des lymphocytes B et T. Pour réaliser ce projet, des souris déficientes pour le domaine de transactivation de Miz-1 (Miz-1POZ) et des souris à allèles mutantes pour c-MycV394D, mutation qui empêche l’interaction avec Miz-1, ont été générées. La caractérisation des souris Miz 1POZ a démontré que l’inactivation de Miz-1 perturbe le développement des lymphocytes B et T aux stades précoces de leur différenciation qui dépend de l’IL-7. L’analyse de la cascade de signalisation IL-7/IL-7R a montré que ces cellules surexpriment la protéine inhibitrice SOCS1 qui empêche la phosphorylation de STAT5 et perturbe la régulation à la hausse de la protéine de survie Bcl-2. De plus, Miz-1 se lie directement au promoteur de SOCS1 et contrôle son activité. En plus de contrôler l’axe IL-7/IL-7R/STAT5/Bcl-2 spécifiquement aux stades précoces du développement afin d’assurer la survie des progéniteurs B et T, Miz-1 régule l’axe EBF/Pax-5/Rag-1/2 dans les cellules B afin de coordonner les signaux nécessaires pour la différenciation des cellules immatures. La caractérisation des souris c-MycV394D a montré, quant à elle, que les fonctions de Miz-1 dans les cellules B et T semblent indépendantes de c-Myc. Les cellules T des souris Miz-1POZ ont un défaut de différenciation additionnel au niveau de la -sélection, étape où les signaux initiés par le TCR remplacent ceux induits par IL-7 pour assurer la prolifération et la différenciation des thymocytes en stades plus matures. À cette étape du développement, une forme fonctionnelle de Miz-1 semble être requise pour contrôler le niveau d’activation de la voie p53, induite lors du processus de réarrangement V(D)J du TCR. L’expression de gènes pro-apoptotiques PUMA, NOXA, Bax et du régulateur de cycle cellulaire p21CIP1 est régulée à la hausse dans les cellules des souris Miz-1POZ. Ceci provoque un débalancement pro-apoptotique qui empêche la progression du cycle cellulaire des cellules TCR-positives. La survie des cellules peut être rétablie à ce stade de différenciation en assurant une coordination adéquate entre les signaux initiés par l’introduction d’un TCR transgénique et d’un transgène codant pour la protéine Bcl-2. En conclusion, ces études ont montré que Miz-1 intervient à deux niveaux du développement lymphoïde: l’un précoce en contrôlant la signalisation induite par l’IL-7 dans les cellules B et T, en plus de l’axe EBF/Pax-5/Rag-1/2 dans les cellules B; et l’autre tardif, en coordonnant les signaux de survie issus par le TCR et p53 dans les cellules T. Étant donné que les thymocytes et lymphocytes B immatures sont sujets à plusieurs rondes de prolifération, ces études serviront à mieux comprendre l’implication des régulateurs du cycle cellulaire comme c-Myc et Miz-1 dans la génération des signaux nécessaires à la différenciation non aberrante et à la survie des ces cellules. Enfin, les modèles expérimentaux, souris déficientes ou à allèles mutantes, utilisés pour ce travail permettront de mieux définir les bases moléculaires de la transformation maligne des lymphocytes B et T et de révéler les mécanismes conduisant au lymphome. / Signaling pathways control the differentiation and proliferation of blood cells, like B and T lymphocytes. They converge into regulating the activity of transcription factors that influence ultimately gene expression patterns. The transcription factor c-Myc is a central regulator of cellular proliferation and growth, and its deregulated expression has been demonstrated to be involved in many types of cancers, in particular lymphoma. Recent studies have shown that repression by c-Myc can be mediated by a complex formed with the BTB/POZ domain transcription factor Miz-1 (Myc Interacting Zinc finger protein-1). Given that both c-Myc and Miz-1 proteins are expressed in lymphoid precursors and since c-Myc has been shown to be important for B- and T-cell development, the aim of this thesis was to investigate the role of Miz-1 and the c-Myc/Miz-1 complex in regulating B and T cell survival, commitment and differentiation. To do so, mice expressing a non-functional Miz-1 protein lacking the BTB/POZ domain (Miz-1POZ) and knock-in mice expressing a mutant c-MycV394D allele that no longer interacts with Miz-1 were generated. B- and T-cell development requires the coordinated action of transcription factors and cytokines, in particular interleukin-7 (IL-7). The studies presented in this work demonstrated that mice deficient for the BTB/POZ domain of transcription factor Miz-1 almost entirely lack follicular B cells and T cells, since their progenitors fail to activate the JAK/STAT5 pathway and to up-regulate Bcl-2 upon IL-7 stimulation. Miz-1 exerts a dual role in the IL-7 receptor (IL-7R) pathway by directly repressing the JAK inhibitor SOCS1 and by activating Bcl-2 expression. In B cells, a functional form of Miz-1 is also required for the proper expression of early B cell genes like E2A and EBF. These data suggest that Miz-1 represents a new regulatory element of early B- and T-cell differentiation required for the regulation of the IL-7/IL-7R/STAT5/Bcl-2 axis by monitoring SOCS1 for survival and by regulating the EBF/Pax-5/Rag-1/2 axis for the proper commitment and differentiation of the B-cell lineage. The regulation exerted by Miz-1 in B and T cells is mostly likely independent of its interacting partner c-Myc, and seems specifically linked to the BTB/POZ domain of Miz-1. Mice deficient for the BTB/POZ domain of Miz-1 have additionally a severe differentiation block at the pre-T cell “-selection” checkpoint. Miz-1 deficient pre-T cells are highly apoptotic and do show cell cycle defects. This concurs with enhanced expression of p53-target genes such as p21CIP1, Bax, PUMA and Noxa, most likely induced by the DNA double-strand breaks generated during the V(D)J recombination of the TCR. Only the co-expression of rearranged TCR and Bcl-2 fully rescued Miz-1-deficient cell numbers and enabled them to differentiate into TCR+ cells. These data suggest that Miz-1 is required for both the regulation of the p53 response and proper expression of the pre-TCR to support the proliferative burst of pre-T cells. In conclusion, the studies presented in this thesis revealed the so far unknown implication of Miz-1 in B- and T-cell development. More specifically, Miz-1 exerts early regulatory functions by monitoring the IL-7/IL-7R signaling in B and T cells. It regulates later stages of differentiation by controlling the EBF/Pax-5/Rag-1/2 in B cells and the TCR expression and the p53 response in T cells. These studies and the generated mice model (conditional knock-out and knock-in) will help characterize the implications of transcription factors that have been causally implicated in the altered genetic programming found in hematopoietic malignancies due to their capacities to regulate cell cycle. Ultimately the characterization of Miz-1 and c-Myc functions in B and T cells will help better understand the mechanisms responsible for the emergence of leukemia and lymphoma.

Pancreatic cancer is nowadays one of the neoplasms with worst prognosis, so research towards the discovery of new molecular targets for therapy and diagnosis is more than urgent. In this direction, we have deeply evaluated the role of Galectin-1 (Gal-1) - a protein that is highly overexpressed in the tumor stroma- in pancreatic tumor progression. Interestingly, we have found that Gal-1 interacts with tissue plasminogen activator (tPA) and that this interplay seems to be involved both in pancreatic tumor epithelial cells and fibroblasts migration, Erk1/2 activation and invasion, suggesting its importance in the tumor/stroma crosstalk in vitro. We have also focused on the biochemical identification of tPA/Gal-1 interaction domains. Furthermore, we have studied Gal-1 role in pancreatic tumor progression in vivo, using murine (xenografts and transgenics) and zebrafish models. We have found that Gal-1 participates in proliferation, angiogenesis, stroma formation and necrosis in Ela-1-myc pancreatic tumors, as well as in the acinar to ductal metaplasia. Importantly, these effects result in an overall significant increase in the survival of Ela-1-myc mice with reduced Gal-1 levels. We have also analyzed Gal-1 role in mouse embryonic pancreatic development, finding interesting parallelisms with tumors. Finally, the molecular mechanisms involved in Gal-1 driving tumor pancreatic progression have been addressed through a transcriptome analysis. All together, our data support Gal-1 as a new molecular target to fight against pancreatic cancer. / Avui en dia, el càncer de pàncrees representa un dels tumors amb més elevats índex de mortalitat, per la qual cosa la recerca dirigida a la identificació de molècules per teràpia i diagnosi són més que necessàries. Amb aquest objectiu, hem avaluat el paper que juga Galectina-1 (Gal-1) - una proteïna altament sobreexpressada en l’estroma tumoral- en la progressió tumoral pancreàtica. Hem trobat que Gal-1 interactua amb l’activador tissular del plasminògen (tPA), participant en la migració, l’activació de Erk1/2 i la invasió, tant en cèl4lules tumorals pancreàtiques com en fibroblasts in vitro, suggerint una importància caudal d’aquesta interacció en la comunicació entre el tumor i l’estroma. Així mateix, ens hem centrat en la identificació bioquímica dels dominis d’interacció entre Gal-1 i tPA. A més, el paper de Gal-1 en la progressió tumoral pancreàtica ha estat adreçat in vivo, utilitzant models murins (xenografts i transgènics) i el peix zebra. Així doncs hem trobat que Gal-1 participa en la proliferació, angiogènesi, formació de l’estroma i la necrosi dels tumors pancreàtics dels ratolins Ela- 1-myc, així com en la metaplasia acinar-ductal. De forma significativa, aquests efectes es tradueixen en un increment important en la supervivència dels ratolins Ela-1-myc amb nivells reduïts de Gal-1. Hem també analitzat el paper que juga Gal-1 en el desenvolupament pancreàtic embrionari murí, trobant paral4lelismes interessants amb els tumors. Finalment, hem volgut ocupar-nos dels mecanismes moleculars involucrats en els efectes produïts per Gal-1 durant la progressió tumoral pancreàtica mitjançant microarrays. Les nostres dades presenten Gal-1 com una nova diana terapèutica per lluitar contra el càncer de pàncrees.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vi, 130 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 137-154).