Pour étudier le rôle de c-Jun dans les macrophages, nous avons développé deux modèles murins présentant une délétion spécifique de Jun dans la lignée de cellules myéloïdes. Le 1er modèle (JunΔCsf1r) a une délétion de c-Jun dans les cellules myéloïdes et leurs progéniteurs. Le 2nd modèle (JunΔLyz2) a une perte de c-Jun dans les cellules myéloïdes matures. L'analyse de ces modèles a montré que l'expression de c-Jun n'est pas nécessaire à l'homéostasie des macrophages tissulaires, mais est essentiel à l'expansion des macrophages de la MO et des progéniteurs de la rate. De plus, l'absence d'expression de c-Jun dans les macrophages inhibe leur prolifération induite par le facteur de croissance Csf1 in vitro. Et nous avons montré que ce défaut de prolifération était indépendant de JNK. Par ailleurs, mes travaux ont révélé que c-Jun est nécessaire pour inhiber l'expression basale des inhibiteurs du cycle cellulaire dans les macrophages, en particulier p16 qui joue un rôle important dans la sénescence cellulaire et dans la perte de la capacité de prolifération.Enfin, l'absence de c-Jun induit l'accumulation de cellules souches hématopoïétiques (HSC) dans la MO des souris JunΔCsf1r, mais aussi une diminution des progéniteurs myéloïdes (GMP/CMP). Des expériences effectuées sur des souris chimériques reconstituées avec un mélange de cellules précurseurs de MO de souris JunΔCsf1r et de souris contrôles en proportions égales ont montré que les HSCs n'exprimant pas c-Jun présentent un défaut de reconstitution de toutes les lignées hématopoïétiques, suggérant ainsi un rôle important de c-Jun dans l'homéostasie et dans la fonction des cellules souches hématopoïétiques. / To study the role of c-Jun in macrophages we have generated mice with a targeted deletion of Jun in the myeloid lineage. We used two transgenic mice: (JunΔCsf1r) where c-Jun is deleted in all myeloid cells and their progenitors, or the (JunΔLyz2) mice where c-Jun is deleted in mature myeloid cells. My analysis showed that c-Jun expression is not required for the maintenance of tissue macrophages, however it is critical for macrophage expansion from BM and splenic progenitors. There was a strong reduction in accumulation of monocyte-derived macrophages in mice where c-Jun expression was deleted in myeloid progenitors (JunΔCsf1r), compared to littermate control mice or the deletion of c-Jun in mature myeloid cells (JunΔLyz2). My further experiments showed that the deletion of c-Jun in macrophages inhibited Csf1-mediated proliferation in vitro. Activation of c-Jun is regulated by Jun N-terminal kinase (JNK), I showed in my experiments that the role of c-Jun in macrophage proliferation was JNK-independent. I show that c-Jun in macrophages is required to repress basal expression of cell cycle inhibitors, as p16, which is linked to cell senescence and loss of proliferative capacity.Finally, we observed that loss of c-Jun results in accumulation of hematopoietic stem cells (HSC) in the BM of JunΔCsf1r mice, but associated with a decrease in myeloid progenitors (GMP and CMP). Furthermore, competitive radiation-chimera experiments showed c-Jun deficient HSC are defective in their ability to repopulate all haematopoietic lineages, suggesting c-Jun has an important role in HSC homeostasis and function.

Para compreensão da biologia dos tumores adrenocorticais são utilizados marcadores moleculares que em geral são genes reguladores do ciclo celular. AP-1 é um fator dimérico composto principalmente pelas proteínas JUN (JUN, JUNB e JUND) e FOS. Nesse projeto tivemos como hipótese que as proteínas da família JUN, se correlacionam com proteínas reguladoras do ciclo celular em tumores adrenocorticais, e poderiam ser utilizados no diagnóstico e prognóstico desse tipo de tumor. O objetivo foi analisar o padrão de expressão gênica e proteica (PCR e Immunobloting). A análise por PCR Array em culturas de células de tumores adrenais, mostrou que os genes da família JUN e o gene FOS estão pouco expressos nessas culturas, o que foi confirmado em ensaios de qPCR. Não foi possível determinar um padrão de expressão que diferenciasse os tipos de culturas celulares estudados, ou mesmo tumores adultos e pediátricos. Os tratamentos com ACTH aumentam a expressão da proteina JUN e JUNB, e podem ter certa importância em tumores responsivos à esse hormônio, que merecem análises futuras. / In order to have a better comprehension of adrenocortical tumor biology, molecular markers are utillized because they are in general cell cycle regulators. AP-1 is a dimeric factor, compound mainly by JUN (JUN, JUNB, JUND) and FOS proteins. In the present study, our hypothesis is that JUN proteins are correlated with others cell cycle regulatory proteins and could be utilized as a prognose and diagnostic predictor for adrenocortical tumors. To confirm our hypothesi the aim was genic and protein profile analyzes (PCR and Immunobloting). The PCR Array analysis showed that JUN and FOS gene expression were down regulated in these adrenocortical tumors cells which were confirmed through qPCR analysis. The genes expression analysis wasn´t able to stablish a standard of expression between the studied cell cultures or even between adults and pediatric tumors. The ACTH treatments increased JUN and JUNB proteins that may have some significance in responsive tumors to this hormone, and deserve further analysis.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Para compreensão da biologia dos tumores adrenocorticais são utilizados marcadores moleculares que em geral são genes reguladores do ciclo celular. AP-1 é um fator dimérico composto principalmente pelas proteínas JUN (JUN, JUNB e JUND) e FOS. Nesse projeto tivemos como hipótese que as proteínas da família JUN, se correlacionam com proteínas reguladoras do ciclo celular em tumores adrenocorticais, e poderiam ser utilizados no diagnóstico e prognóstico desse tipo de tumor. O objetivo foi analisar o padrão de expressão gênica e proteica (PCR e Immunobloting). A análise por PCR Array em culturas de células de tumores adrenais, mostrou que os genes da família JUN e o gene FOS estão pouco expressos nessas culturas, o que foi confirmado em ensaios de qPCR. Não foi possível determinar um padrão de expressão que diferenciasse os tipos de culturas celulares estudados, ou mesmo tumores adultos e pediátricos. Os tratamentos com ACTH aumentam a expressão da proteina JUN e JUNB, e podem ter certa importância em tumores responsivos à esse hormônio, que merecem análises futuras. / In order to have a better comprehension of adrenocortical tumor biology, molecular markers are utillized because they are in general cell cycle regulators. AP-1 is a dimeric factor, compound mainly by JUN (JUN, JUNB, JUND) and FOS proteins. In the present study, our hypothesis is that JUN proteins are correlated with others cell cycle regulatory proteins and could be utilized as a prognose and diagnostic predictor for adrenocortical tumors. To confirm our hypothesi the aim was genic and protein profile analyzes (PCR and Immunobloting). The PCR Array analysis showed that JUN and FOS gene expression were down regulated in these adrenocortical tumors cells which were confirmed through qPCR analysis. The genes expression analysis wasn´t able to stablish a standard of expression between the studied cell cultures or even between adults and pediatric tumors. The ACTH treatments increased JUN and JUNB proteins that may have some significance in responsive tumors to this hormone, and deserve further analysis.

As células Y-1 pertencem a uma linhagem clonal de células funcionais de córtex adrenal de camundongo, que respondem a ACTH. Em células Y-1, ACTH promove a esteroidogênese (função) e tem efeitos regulatórios complexos na transição G0→G1→S do ciclo celular. ACTH promove a transição G0→G1, mas inibe a transição G1→S. É possível que a regulação do ciclo celular por ACTH seja mediada pelo controle da expressão dos proto-oncogenes das famílias fos, jun e myc. Nosso laboratório mostrou, anteriormente, que ACTH induz a expressão dos genes fos e jun, mas inibe c-myc. O objetivo deste trabalho foi identificar pontos de controle na expressão dos genes fos, jun e myc e na atividade dos fatores de transcrição AP-1 (dímeros da proteínas Fos e Jun) por ACTH, derivados de cAMP (ativadores de PKA), PMA (ativador de PKC) e FCS (soro fetal bovino). ACTH, PMA e dcAMP aumentam a atividade de ligação de AP-1 a DNA, independentemente de síntese protéica. Ensaios de elongação de cadeia nascente de RNA (run off transcription) mostram que ACTH, PMA e FCS são fortes indutores de c-fos, c-jun e junB, enquanto dcAMP induz apenas c-fos e junB. Hibridizações Northern permitiram estimar a meia-vida dos mRNAs de c-fos e c-jun em 30 min, independentemente do tratamento com ACTH ou PMA. Diferentemente de c-fos, o mRNA de fosB é superinduzido por ActinomicinaD em células Y-1 tratadas com ACTH e PMA. / The Y-1 cells belong to a clonal lineage of functional mouse adrenocortical cells, which are responsive to ACTH. In Y-1 cells, ACTH promotes esteroidogenesis (function) and has complex effects on the G0→G1→S transition of the Y-1 cell cycle. ACTH induces the G0→G1 transition but inhibits the G1+S transition. Probably, the cell cycle regulation by ACTH is mediated by the expression control of the proto-oncogenes from the fos, jun and myc families. Our laboratory has previously shown that ACTH induces the fos and jun genes expression, but inhibits c-myc expression. The target of this work was to identify control points in the fos, jun and myc genes expression and in the AP-1 transcription factors (Fos and Jun proteins dimers) by ACTH, cAMP derivatives (PKA activators), PMA (PKC activator) and FCS (Fetal Calf Serum). ACTH, PMA and dcAMP raise the AP-1 DNA binding activity, independently of protein synthesis. Run off transcription assays show that ACTH, PMA and FCS are strong c-fos, c-jun and junB inducers, while dcAMP induces only c-fos and junB. Northern hybridisations allowed us to estimate the half life of the fos and jun mRNAs in about 30 min, independently of ACTH or PMA treatment. Differently of c-fos, fosB mRNA is superinduced by ActinomicinD treatment in Y-1 cells treated with ACTH or PMA.

O carcinoma adenóide cístico e o adenocarcinoma polimorfo de baixo grau de malignidade são neoplasmas de glândulas salivares. O carcinoma adenóide cístico pode apresentar-se em glândulas salivares maiores e menores, porém o adenocarcinoma polimorfo de baixo grau de malignidade acomete principalmente as glândulas salivares menores distribuidas na cavidade oral. Ambos os tumores compartilham muitas características comuns, como a alta propensão de invasão perineural e o padrão de infiltracão: sólido, tubular e cribriforme. Mas o carcinoma adenoide cístico e o adenocarcinoma polimorfo de baixo grau são tipos distintos de adenocarcinomas com prognóstico diferente, que ocasionalmente podem resultar em um diágnóstico errado. As proteínas c-jun e junB são membros da familia JUN, capazes de homodimerizar ou heterodimerizar com c-fos ou com outras proteinas bzip. Evidências das funcões específicas das subunidades do AP-1 foram mostradas por cjun e junB, que atúam antagónicamente no controle da transformação celular, diferenciação e expressão do AP-1 dependente do gene alvo. Mas a função de ambos é complexa e pode depender do tipo celular. O objetivo deste estudo foi determinar a expressão imunoistoquímica das proteínas c-jun e junB em 13 casos de carcinoma adenoide cístico e 12 de adenocarcinoma polimorfo de baixo grau de malignidade de glándulas salivares. Espécimes de mucosa normal foram incluídos e evidenciaram forte marcação nuclear e citoplasmática para junB e c-jun respectivamente. No presente estudo, independente da arquitetura histológica, ambos tumores mostraram muitas células tumorais com marcação nuclear e citoplasmática para a proteína c-jun e ausente para a proteína junB. As lesões do adenocarcinoma polimorfo de baixo grau de malignidade expressaram um maior número de células com marcação nuclear quando comparados ao do carcinoma adenoide císticode de mais baixo grau. De acordo com este estudo e com alguns estudos publicados na literatura, a c-jun é expressa em tumores de baixo grau e parece estar mais relacionada à diferenciação celular do que à proliferação celular. / Adenoid cystic carcinoma and polymorphous low grade are salivary gland neoplasms. ACC can arise in both, major or minor salivary glands. However, Polymorphous lowgrade adenocarcinoma, occurs specifically in minor salivary glands dispersed in the oral cavity. They share many common histologic features, as infiltrating solid, tubular and cribiform patterns and also high propensity for perineural invasion. Nevertheless, adenoid cystic carcinoma and polymorphous low grade are distinct types of adenocarcinomas with different prognosis, which occasionally may result in a diagnostic pitfall. C-jun and junB are family JUN members that may form homodimerizes or heterodimerizes with c-fos or other bzip proteins. Evidence for specific functions of AP-1 subunits was shown for c-jun and junB, which act antagonistically to control cell transformation, differentiation and expression of AP-1 depending on the target genes. However, the role of both of them is complex and it depends on cell type. The aim of this study was to determine immunohistochemistry of c -jun and junB expression in 13 cases of adenoid cystic carcinoma and 12 cases of polymorphous carcinoma low-grade adenocarcinoma of salivary glands. Moreover slides of normal mucosa were included and there were strong nuclei and cytoplasmic for junB and c-jun respectly. In the present study, independent of the histologic architecture, in both tumors shown many tumoral cells presented nuclear and cytoplasmic staining of c-jun and were absent to the protein junB. In polymorphous low-grade adenocarcinoma lesions expressed in a greater number cells staining than in the adenoid cystic carcinoma of the most lowgrade. According with this study and with some studies of the literature, the c-jun is expressed in low-grade tumors and seems to be related to cell differentiation more than with cell proliferation.

Die Zellen des Hodgkin-Lymphoms sind, neben einer permanenten Aktivierung des NF-kB Signalweges, durch eine konstitutive AP-1 Aktivität gekennzeichnet. Die vorliegende Arbeit beschäftigte sich mit der Analyse der aberranten AP-1 Aktivität in Zellen des Hodgkin-Lymphoms. Von besonderem Interesse ist in diesem Zusammenhang der JUN Promotor, da c-Jun unter anderem die Fähigkeit besitzt seinen eigenen Promotor positiv zu regulieren (Autoregulation). Im Rahmen dieser Arbeit wurden Faktoren, die mit dem JUN Promotor in Zellen des Hodgkin-Lymphoms assoziiert sind, über verschiedene chromatographische Reinigungsschritte angereichert, mittels Massenspektrometrie identifiziert und hinsichtlich ihres Einflusses auf die c-Jun Expression analysiert. Dabei wurden die zwei Faktoren ATF-3, aus der Familie der AP-1 Proteine, und p52, aus der NF-kB Familie, hinsichtlich ihres Einflusses auf die Überexpression von c-Jun in Hodgkinzellen genauer untersucht. Die hier aufgeführten Ergebnisse tragen zu einem besseren Verständnis der Regulation der konstitutiven AP-1 Aktivität in den Hodgkinzellen bei. Im Rahmen dieser Arbeit konnten zwei Faktoren identifiziert werden, die maßgeblich an der Regulation der Expression von c-Jun in Hodgkinzellen beteiligt sind. Eine besondere Rolle kommt dabei dem Transkriptionsfaktor p52 zu, da dieser unter anderem auch die Expression anderer Mitglieder der AP-1 Familie reguliert. Ein weiterer Befund dieser Arbeit, dass p50 und p52 die zentralen Komponenten der konstitutiven NF-kB Aktivität sind, rückt p52 in das Zentrum zukünftiger Forschung. Die Befunde dieser Arbeit belegen, dass die aberrante Aktivierung von AP-1 im Hodgkin-Lymphom nicht auf ein einzelnes Ereignis in der Zelle zurückzuführen ist, sondern das Ergebnis eines komplexen Zusammenspiels vieler Faktoren ist. / The cells of Hodgkin''s lymphoma are characterized by a permanent activation of the NF-kB signaling pathway and a constitutive AP-1 activation. The present study focused on the analysis of aberrant AP-1 activity in cells of Hodgkin''s lymphoma. Of particular interest in this context is the JUN promoter, since c-Jun has the ability to regulate its own promoter (autoregulation). In this work different JUN promoter associated factors were enriched through various chromatographic purification steps, identified by Mass spectrometry and analyzed in terms of its influence on the expression of c-Jun. The focus was on specific factors in cells of Hodgkin''s lymphoma. The two factors ATF-3 and p52, were analyzed in terms of their influence on the overexpression of c-Jun in cells of Hodgkin''s lymphoma. Another interesting finding of this study is, p50 and p52 are central components of the constitutive NF-kB activity. This puts p52 in the center of future research. The results presented here contribute to a better understanding of the regulation of the constitutive AP-1 activity in the Hodgkin cells. In this work two Factors (ATF3 and p52) could be identified, which are are involved in the regulation of the expression of c-Jun in Hodgkin cells. A special role is played by the transcription factor p52, which also regulates the expression of other members of the AP-1 family. The findings of this study also show that the aberrant activation of AP-1 in Hodgkin''s lymphoma is not due to a single event in the cell. It is rather a result of a complex interplay of many factors.

No Brasil, como no mundo, o carcinoma epidermóide bucal está entre os dez tipos mais comum de câncer e acomete mais de 13 mil pessoas por ano. Apesar de ser um sério problema devido a sua morbidade e mortalidade, alguns casos desta doença têm um comportamento biológico menos agressivo. A proteína ciclina D1, depois que forma complexos com as proteínas CDK4 e CDK6, tem como principal função fosforilar a proteína Retinoblastoma. Após sua fosforilação, a proteína libera um fator de transcrição, o E2F, que leva a célula à progressão da fase G1 para fase S do ciclo celular. A proteína c-jun, que faz parte do fator de transcrição AP-1, tem participação ativa no ciclo celular, principalmente durante a transcrição da fase G0 a G1. O gene retinoblastoma é um supressor de tumor. Este gene codifica uma fosfoproteína nuclear, que recebe o mesmo nome. Essa proteína regula o ciclo celular através de múltiplas funções. Também regula outros processos que afetam a proliferação celular, a diferenciação terminal e a apoptose. O HPV é um vírus de DNA que é encontrado em vários tipos de câncer e é o principal agente etiológico do carcinoma de colo uterino. Este trabalho comparou a expressão das proteínas ciclina D1, c-jun e do retinoblastoma em carcinomas epidermóides de baixo e alto grau de malignidade e tentou analisar se o HPV é um fator etiológico desta neoplasia. Apesar das lesões de baixo grau de malignidade expressarem as proteínas num maior número de células que as lesões de alto grau, só houve diferença estatística, entre os dois grupos estudados, para a proteína do retinoblastoma. Não foi encontrado o DNA do HPV em nenhum dos casos estudados. De acordo com este trabalho e com a literatura, a proteína do retinoblastoma é expressa em um número menor de células em carcinomas epidermóides bucais mais agressivos e o HPV não é um agente etiológico de todos os casos desta doença / In Brazil, as in the world, the oral squamous cell carcinoma is among the ten more common types of cancer e affects more than 13 thousand of people by year. Even though it is a serious problem due to its morbidity and mortality, some cases of this disease have a less aggressive biological behavior. The cyclin D1 protein after it forms complexes with the CDK4 and CDK6 proteins has as main function phosphorylate the Retinoblastoma protein. After its prosphorylation, the protein releases a transcription factor, the E2F, that leads the cell to the progression from the phase G1 to the phase S of the cell cycle. The c-jun protein, that is part of the transcription factor AP-1, has active participation in the cell cycle, mainly during the transcription from the phase G0 to G1. The retinoblastoma gene is a tumour suppressor. This gene codifies a nuclear phosphoprotein that receives the same name. This protein regulates the cell cycle through multiple functions. It also regulates other processes that affect the cell proliferation, the terminal differentiation and apoptosis. The HPV is a DNA virus that is found in many types of cancer and is the main etiological agent of the cervical cancer. This study compared the protein expression of the cilin D1, c-jun and retinoblastoma in low and high grade squamous cell carcinoma and tried to analyze if the HPV is a etiological factor for this neoplasm. In spite of the low grade of malignancy lesions express the protein in a greater number of cells than in the high grade lesion, there only was statistical difference, among the two studied groups, for the retinoblastoma protein. It was not found DNA of the HPV in any of the studied cases. According with this study and with the literature the retinoblastoma protein is expressed in a lower number of cells in the more aggressive oral squamous cell carcinomas and the HPV is not the etiological agent in all of the cases of this disease.

O carcinoma adenóide cístico e o adenocarcinoma polimorfo de baixo grau de malignidade são neoplasmas de glândulas salivares. O carcinoma adenóide cístico pode apresentar-se em glândulas salivares maiores e menores, porém o adenocarcinoma polimorfo de baixo grau de malignidade acomete principalmente as glândulas salivares menores distribuidas na cavidade oral. Ambos os tumores compartilham muitas características comuns, como a alta propensão de invasão perineural e o padrão de infiltracão: sólido, tubular e cribriforme. Mas o carcinoma adenoide cístico e o adenocarcinoma polimorfo de baixo grau são tipos distintos de adenocarcinomas com prognóstico diferente, que ocasionalmente podem resultar em um diágnóstico errado. As proteínas c-jun e junB são membros da familia JUN, capazes de homodimerizar ou heterodimerizar com c-fos ou com outras proteinas bzip. Evidências das funcões específicas das subunidades do AP-1 foram mostradas por cjun e junB, que atúam antagónicamente no controle da transformação celular, diferenciação e expressão do AP-1 dependente do gene alvo. Mas a função de ambos é complexa e pode depender do tipo celular. O objetivo deste estudo foi determinar a expressão imunoistoquímica das proteínas c-jun e junB em 13 casos de carcinoma adenoide cístico e 12 de adenocarcinoma polimorfo de baixo grau de malignidade de glándulas salivares. Espécimes de mucosa normal foram incluídos e evidenciaram forte marcação nuclear e citoplasmática para junB e c-jun respectivamente. No presente estudo, independente da arquitetura histológica, ambos tumores mostraram muitas células tumorais com marcação nuclear e citoplasmática para a proteína c-jun e ausente para a proteína junB. As lesões do adenocarcinoma polimorfo de baixo grau de malignidade expressaram um maior número de células com marcação nuclear quando comparados ao do carcinoma adenoide císticode de mais baixo grau. De acordo com este estudo e com alguns estudos publicados na literatura, a c-jun é expressa em tumores de baixo grau e parece estar mais relacionada à diferenciação celular do que à proliferação celular. / Adenoid cystic carcinoma and polymorphous low grade are salivary gland neoplasms. ACC can arise in both, major or minor salivary glands. However, Polymorphous lowgrade adenocarcinoma, occurs specifically in minor salivary glands dispersed in the oral cavity. They share many common histologic features, as infiltrating solid, tubular and cribiform patterns and also high propensity for perineural invasion. Nevertheless, adenoid cystic carcinoma and polymorphous low grade are distinct types of adenocarcinomas with different prognosis, which occasionally may result in a diagnostic pitfall. C-jun and junB are family JUN members that may form homodimerizes or heterodimerizes with c-fos or other bzip proteins. Evidence for specific functions of AP-1 subunits was shown for c-jun and junB, which act antagonistically to control cell transformation, differentiation and expression of AP-1 depending on the target genes. However, the role of both of them is complex and it depends on cell type. The aim of this study was to determine immunohistochemistry of c -jun and junB expression in 13 cases of adenoid cystic carcinoma and 12 cases of polymorphous carcinoma low-grade adenocarcinoma of salivary glands. Moreover slides of normal mucosa were included and there were strong nuclei and cytoplasmic for junB and c-jun respectly. In the present study, independent of the histologic architecture, in both tumors shown many tumoral cells presented nuclear and cytoplasmic staining of c-jun and were absent to the protein junB. In polymorphous low-grade adenocarcinoma lesions expressed in a greater number cells staining than in the adenoid cystic carcinoma of the most lowgrade. According with this study and with some studies of the literature, the c-jun is expressed in low-grade tumors and seems to be related to cell differentiation more than with cell proliferation.