Dissertationen zum Thema „Paget“

Il morbo di Paget e' una forma tumorale, a lenta crescita, localizzata al capezzolo e piu' raramente in regione ano-genitale, perianale, ascellare o inguinale. Si tratta di un adenocarcinoma epiteliale, che si associa spesso ad un sottostante carcinoma in situ o un carcinoma invasivo della mammella e in caso di localizzazione extramammaria, ad un carcinoma sottostante. La malattia si localizza tipicamente al capezzolo o all'areola mammaria, monolateralmente, sotto forma di una piccola chiazza eritematosa essudante o di un semplice ispessimento cutaneo, a lenta estensione centrifuga, spesso accompagnata da prurito o dolore. Ci sono due teorie sull'istogenesi del Paget: La teoria epidermotropica sostiene che le cellule di Paget siano cellule da carcinoma duttale migrate per continuita' di un sottostante carcinoma dei dotti galattofori nella forma mammaria e dalle ghiandole del glande, del retto, della cervice e della vescica nelle forme extramammaria. La teoria della trasformazione in situ e' stata proposta per spiegare lo sviluppo di questa alterazione in pazienti che non hanno un carcinoma mammario sottostante. Nei pazienti in cui il tumore e' clinicamente e radiologicamente negativo, puo' essere studiato attraverso l'istochimica e l'immunoistochimica. Studi di istochimica mostrano che le cellule di Paget sono PAS positive nel 30% dei casi ed alcian blu positive nel 20% dei casi.
Mammary and extramammary Paget's disease are rare neoplasm of epidermis and mucosal epithelium. Due to their nonspecific and variable clinical view, they have differential diagnosis with eczema, melanoma, Bowen's disease, etc. The histogenesis of Paet's disease has been hotly debated, and only recently has epidermotropic theory become widely accepted. With the evolution of our understanding of breast cancer, it became apparent that the prognosis of Paget's disease was more a reflection of that of the underlying carcinoma, be it intraductal or infiltrating. The standanrd treatment of Paget's disease remains mastectomy with or without axillary dissection. In this are of breast-conserving surgery, however, there is much evidence to suggest that conservative treatment of Paget's disease of the breast is possible.

Après un rappel des principaux aspects de la maladie osseuse de Paget chez le vivant, les 148 cas anciens de cette affection publiés dans la littérature paléoanthropologique sont analysés et critiqués. L'étude de trois cas personnels et la révision du premier cas publié (Pales, 1929) fournissent les éléments d'une description précise des lésions de la maladie de Paget sur l'os sec ; pour la reconnaître, nous proposons une liste de critères, avec une cotation pour chacun d'entre eux, permettant d'envisager ce diagnostic. La maladie apparaît d'abord au Néolithique, sur le rivage nord de la Méditerranée ; elle se répand ensuite lentement en Europe, au nord et à l'ouest de l'Empire romain, avant de devenir beaucoup plus fréquente dans l'Angleterre médiévale. Sa dissémination dans le monde contemporain semble liée à l'émigration anglo-saxonne. Sa présence dans l'Amérique préhistorique est contestée. Le rôle du chien, en tant que vecteur d'un éventuel agent pathogène, est discuté.

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Universidade Estadual Paulista (UNESP)
O objetivo deste trabalho foi estudar através do exame Imunoistoquimico, a correlação entre alguns fatores Anatomopatológicos (tipo histológico e grau nuclear) e alguns fatores biológicos (receptores hormonais de estrógeno e de progesterona, proteína c-erbB-2 e proteína p53, no carcinoma de Paget da mama, doença rara e especial. No período entre Janeiro de 1980 a Dezembro de 1998 foram tratados no Instituto Brasileiro de Controle do Câncer, 6.303 casos de câncer de mama, deste total 98 casos foram diagnosticados como carcinoma de Paget, cuja incidência foi de 1,55%. Estudamos retrospectivamente 60 casos, sendo que 38 deles foram excluídos da análise, devido a limitação e escassez da amostra no material disponível, a idade das pacientes variou entre 26 e 84 anos, com média de 55,4 anos, as informações clínicas e terapêuticas foram obtidas dos prontuários das pacientes considerando, idade na época do diagnóstico, tamanho do tumor quando palpável, estadiamento clínico, e o tipo de cirurgia realizada. As amostras de tecido mamário foram recuperadas dos arquivos do Departamento de Anatomia Patológica do I.B.C.C. / MATTOSINHO. Após a revisão histológica, realizada por dois patologistas os casos foram classificados em quatro grupos: Grupo A- Doença de Paget (forma pura) n = 09 Grupo B- Doença de Paget + neoplasia ductal “in situ”, n = 12 Grupo C- Doença de Paget, neoplasia ductal invasora, n = 30 Grupo D- Doença de Paget + neoplasia ductal “in situ”+ neoplasia ductal invasora, n = 09. Entre as variáveis anatomopatológicas, o grupo C prevaleceu com 30 casos (50%). O grau nuclear (GN-II) predominou com 45 casos (75%). Em relação as variáveis biológicas o receptor de estrógeno negativo predominou com 41 casos (68,3%), seguido pelo receptor de progesterona negativo com 40 casos (66,7%), podemos dizer que...
The objective of this paper was to study the immunohistochemical efects of Paget’s disease, a rare and special carcinoma of the breast, by correlating anatomopathological (histological and nuclear grade) and biological (estrogen and progesterone hormonal receptors, c-erbB-2 protein and p53 protein) factors. Between January 1980 and December 1998, 6303 cases of breast cancer were treated at the Brazilian Institute of Cancer Control; 98 of these were diagnosed with Paget’s carcinoma, an incidence of 1.55%. We retrospectively studied 60 of these cases; 38 were excluded due to lack of available sample material. Patient age varied between 26 and 84 years (mean 55.4), clinical and therapeutic information were obtained from patient’s medical records considering age at time of diagnosis, tumor size when palpable, clinical stage, and type of surgery performed. Samples of breast tissue were retrieved the Anatomical Pathology Department, I.B.C.C. / MATTOSINHO. After histological review, by two different pathologists, they were classified into four groups: Group A- Paget’s disease (pure form) n = 09 Group B- Paget’s disease + “in situ” duct neoplasia n = 12 Group C- Paget’s disease, invasive duct neoplasia n = 30 Group D- Paget’s disease + “in situ” duct neoplasia + invasive duct neoplasia n = 09.Invasive duct neoplasia was the most prevalent anatomopathological variable (Group C, n = 30, 50%). Nuclear grade (GNII) was found in 45 cases (75%). In relation to the biological variables, the negative estrogen receptor was predominant with 41 cases (68.3%), followed by the negative progesterone receptor with 40 cases (66.7%); this correlation had good concordance by the Kappa test. The c-erbB-2 protein was positive in 53 cases (88.3%) and p53 was negative in 47 cases (78.3%). From these results, we concluded that there was no statistical... (Complete abstract click electronic address below)

Orientadores: Cesar Cabello dos Santos, Marcelo Alvarenga
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Objetivos: Calcular a frequência, descrever aspectos clínicos, mamográficos, e histopatológicos, assim como formas do tratamento cirúrgico e o prognóstico de pacientes com doença de Paget de mama. Sujeitos e método: Foi realizado um estudo descritivo analítico entre os anos de 1988 a 2002 em mulheres tratadas de câncer de mama no Centro de Atenção Integral à Saúde da Mulher da Universidade Estadual de Campinas. Foram calculados os valores de frequência da doença, descritas características epidemiológicas, do exame clínico, das mamografias, além de formas de tratamento cirúrgico e o prognóstico destas pacientes. Para a análise estatística foram calculados valores de frequência e descritas as porcentagens das categorias das variáveis estudadas. Foram utilizados os testes qui-quadrado, t'student e exato de Fisher para análise de associação de algumas variáveis. Para avaliar o prognóstico da doença foram realizadas curvas de sobrevivência pelo método de Kaplan Mayer analisadas pelo teste LogRank. Resultados: Foram observados 4.536 casos de câncer de mama, sendo que destes, 80 apresentavam doença de Paget de mama. Isto correspondeu no período estudado a uma frequência média de 1,76%. As pacientes assintomáticas em relação ao complexo aréolo-papilar corresponderam a 46% dos casos estudados. Encontraram-se alterações no complexo aréolo-papilar ao exame clínico em 63% das pacientes e estavam associadas a nódulos mamários em mais da metade. A mamografia falhou em diagnosticar os tumores mamários em 30% e em 64% não mostrou alterações no complexo aréolo-papilar. Todos os casos associaram-se com carcinomas mamários subjacentes. Mulheres com sintomas de alterações de complexo aréolo-papilar apresentaram 44% de carcinoma in situ e 66% de axila negativa, sendo que entre aquelas sem sintomas, a freqüência foi de 3% e 34%, respectivamente (p=0,011 e p=0,000). A sobrevida global foi significativamente maior nas pacientes com sintomas no complexo aréolo-papilar do que naquelas sem sintomas (p=0,031). A presença de nodulação ao exame clínico associou-se à presença de carcinoma invasor em 89% e de axila comprometida em 61%, enquanto que a ausência de nodulação associou-se com 38% e 19% respectivamente (p=0,000). As recidivas locais ocorreram em 38% (5/13) das pacientes submetidas a quadrantectomias e em 7,7% (5/65) das mastectomizadas (p=0,002). Conclusão: A doença de Paget de mama foi uma entidade rara. Em quase metade das pacientes não manifestou sintomas no complexo aréolo-papilar. Grande parte das pacientes apresentou ao exame clínico alterações de complexo aréolo-papilar associadas a nodulações nas mamas, sendo que o achado clínico de nódulo esteve associado à doença mais avançada. O exame mamográfico foi inadequado para avaliação do complexo aréolo-papilar. As pacientes submetidas às quadrantectomias apresentaram alta taxa de recorrência local. Os exames clínicos e mamográficos não foram capazes de prever as recorrências locais no grupo das quadrantectomia. As pacientes com queixa de alterações de papila tiveram melhor sobrevida
Abstract: Objetive: To calculate the frequency, and describe the clinical, mamographics and the histopatologic aspects of Paget disease of the breast. Futhermore evalute the surgical treatment and the prognosis of these patients. Method: It was done a descriptive study in breast cancer women attended at Integral Women Attention Center (CAISM) of the University of Campinas between 1988 and 2002. It was calculated the disease frequency and It was performed the associated analyses of some clinical, variables with the Chi-square , t'student and e the FISHER's exat test. It was done Kaplan Mayer curves and LogRank analyse to evaluet the prognosis of the Paget disease of the breast with regards to some aspects. Results: The mean frequency was 1.76%. 46% of patients was assimptomatic about the nipple-areola complex. 63% presented nipple-areola complex disturbed associated to breast lumps generally. The mammography failed the breast tumor associated diagnosis in 30% and in 64% the nipple-areola complex was wrongly normal. All of the cases was associated to breast carcinomas beneth. The nipple-areola complex complains was associated to 44% of in situ ductal carcinomas and 66% of negative axila findings, while the noncomplains women with 3% and 34% respectivelly (p=0.011 and p=0.000). The women with nipple-areola complex complains were associated to better survival compared to the non-complains ones (p=0.031). The breast finding of lump was associated to 89% of invasive breast cancer and 61% of positive axilar node, compared to the non-breast lump patients with 38% and 19% respectivelly (p=0.000). The quandrantectomies was involved to 39% of local relapses compared to the mastectomies with 7.7% (p=0.002). Conclusion: The Paget disease of the breast was rare one. It was not observed nipple-areola complex symptoms in almost half of patients. In the majority of patients was observed finding of nipple-areola complex at clinical breast exam associated to breat lumpness. These clinical finding of breast lump was associated to more advanced breast cancer. The mammography was inappropiate to evaluate the nipple-areola complex. The patients submitted to the quandrantectomies presented high local relapse rate. The clinical and mammographic evaluation were inappropriate to identify the risk of local relapse in the conservative group. The women with nipple-areola complex complains was associated to better survival
Mestrado
Tocoginecologia
Mestre em Tocoginecologia

Orientador: Laurival Antonio De Luca
Resumo: O objetivo deste trabalho foi estudar através do exame Imunoistoquimico, a correlação entre alguns fatores Anatomopatológicos (tipo histológico e grau nuclear) e alguns fatores biológicos (receptores hormonais de estrógeno e de progesterona, proteína c-erbB-2 e proteína p53, no carcinoma de Paget da mama, doença rara e especial. No período entre Janeiro de 1980 a Dezembro de 1998 foram tratados no Instituto Brasileiro de Controle do Câncer, 6.303 casos de câncer de mama, deste total 98 casos foram diagnosticados como carcinoma de Paget, cuja incidência foi de 1,55%. Estudamos retrospectivamente 60 casos, sendo que 38 deles foram excluídos da análise, devido a limitação e escassez da amostra no material disponível, a idade das pacientes variou entre 26 e 84 anos, com média de 55,4 anos, as informações clínicas e terapêuticas foram obtidas dos prontuários das pacientes considerando, idade na época do diagnóstico, tamanho do tumor quando palpável, estadiamento clínico, e o tipo de cirurgia realizada. As amostras de tecido mamário foram recuperadas dos arquivos do Departamento de Anatomia Patológica do I.B.C.C. / MATTOSINHO. Após a revisão histológica, realizada por dois patologistas os casos foram classificados em quatro grupos: Grupo A- Doença de Paget (forma pura) n = 09 Grupo B- Doença de Paget + neoplasia ductal "in situ", n = 12 Grupo C- Doença de Paget, neoplasia ductal invasora, n = 30 Grupo D- Doença de Paget + neoplasia ductal "in situ"+ neoplasia ductal invasora, n = 09. Entre as variáveis anatomopatológicas, o grupo C prevaleceu com 30 casos (50%). O grau nuclear (GN-II) predominou com 45 casos (75%). Em relação as variáveis biológicas o receptor de estrógeno negativo predominou com 41 casos (68,3%), seguido pelo receptor de progesterona negativo com 40 casos (66,7%), podemos dizer que... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The objective of this paper was to study the immunohistochemical efects of Paget's disease, a rare and special carcinoma of the breast, by correlating anatomopathological (histological and nuclear grade) and biological (estrogen and progesterone hormonal receptors, c-erbB-2 protein and p53 protein) factors. Between January 1980 and December 1998, 6303 cases of breast cancer were treated at the Brazilian Institute of Cancer Control; 98 of these were diagnosed with Paget's carcinoma, an incidence of 1.55%. We retrospectively studied 60 of these cases; 38 were excluded due to lack of available sample material. Patient age varied between 26 and 84 years (mean 55.4), clinical and therapeutic information were obtained from patient's medical records considering age at time of diagnosis, tumor size when palpable, clinical stage, and type of surgery performed. Samples of breast tissue were retrieved the Anatomical Pathology Department, I.B.C.C. / MATTOSINHO. After histological review, by two different pathologists, they were classified into four groups: Group A- Paget's disease (pure form) n = 09 Group B- Paget's disease + "in situ" duct neoplasia n = 12 Group C- Paget's disease, invasive duct neoplasia n = 30 Group D- Paget's disease + "in situ" duct neoplasia + invasive duct neoplasia n = 09.Invasive duct neoplasia was the most prevalent anatomopathological variable (Group C, n = 30, 50%). Nuclear grade (GNII) was found in 45 cases (75%). In relation to the biological variables, the negative estrogen receptor was predominant with 41 cases (68.3%), followed by the negative progesterone receptor with 40 cases (66.7%); this correlation had good concordance by the Kappa test. The c-erbB-2 protein was positive in 53 cases (88.3%) and p53 was negative in 47 cases (78.3%). From these results, we concluded that there was no statistical... (Complete abstract click electronic address below)
Doutor

Introducción y objetivos: La enfermedad ósea de Paget (EOP) puede cursar con hipoacusia. Con el objetivo de cuantificar, caracterizar, determinar los factores de riesgo de hipoacusia y analizar las posibles relaciones de la hipoacusia con los hallazgos tomográficos en un grupo de pacientes con EOP, se realiza el presente estudio. Métodos: Se realizó un estudio observacional, transversal del tipo casos y controles que incluyó una cohorte de 76 sujetos con diagnóstico de enfermedad ósea de Paget (EOP) en el grupo caso y un grupo control de 134 sujetos. Se analiza la información clínica, demográfica, audiométrica y radiológica, mediante una TC de hueso temporal, de los sujetos incluidos. Resultados: El análisis comparativo entre el grupo de sujetos con EOP y el grupo control determinó que el grupo caso presentaban un umbral medio auditivo mayor (39,51 dB) que en el grupo control (37,28 dB) (p=0,069) y que presentaba hipoacusia transmisiva con mayor frecuencia (22,76%) que el grupo control (12,05%) (p=0,0062). El análisis de los factores de riesgo de hipoacusia determinó que la afectación craneal en la gammagrafía ósea, la edad y la HTA entre otros, constituían factores de riesgo de mayor pérdida auditiva en EOP. El estudio tomográfico constató que los pacientes con EOP presentaban una menor densidad ósea en unidades Hounsfield (UH) en el peñasco del temporal respecto al grupo control. Así mismo se pudo observar que la densidad ósea en la cápsula ótica en pacientes con EOP se correlacionaba con el umbral auditivo tanto de vía aérea como de vía ósea (p<0,05) y que la presencia de un gap en la audiometría se correlacionaba con la afectación por hueso pagético del temporal. Conclusiones: - El 63,45% de los sujetos con enfermedad ósea de Paget (EOP) padecen hipoacusia, con un umbral auditivo medio de 39,51dB. - Los sujetos con enfermedad ósea de Paget (EOP) presentaron una pérdida auditiva más severa y con mayor frecuencia de tipo transmisivo que el grupo control. - Los sujetos con afectación de la calota craneal por EOP presentaron mayor pérdida auditiva que los sujetos sin afectación craneal. La afectación de la calota craneal por la EOP y la edad constituyeron factores de riesgo de hipoacusia. - En los sujetos con EOP el umbral auditivo tanto de vía aérea como ósea se relaciona con la disminución de densidad en la cápsula ótica.
Introduction and objectives: Paget’s disease of bone (PDB) may lead to hearing loss. The present study is conducted with the aim of measuring, characterizing, determining the risk factors for hearing loss and analyse the possible relation of hearing loss with tomography findings in a group of subjects with PDB. Methods: An observational, transversal, case-control study was conducted, a cohort of 76 subjects diagnosed of Paget’s disease of bone (PDB) in the case group and a control group of 134 subjects were included. Clinical, demographic, audiometric and tomographic data (by means of a CT scan study) were analysed. Results: The comparative analysis between the subjects in the PDB group and the control group found that the case group showed higher hearing thresholds (39,51dB) comparing with the control group (37,28 dB) (p=0,069) and presented a greater rate of conductive hearing loss (22,76%) than the control group (12,05%) (p=0,0062). The study of risk factors for hearing loss found that skull involvement in bone scintigraphy, age and high blood pressure were risk factors for higher hearing impairment in PDB. The CT scan data showed that subjects in the PDB group had lower temporal bone density (Hounsfield Units, HU) comparing with the control group. It was also found that the otic capsule bone density in the PDB group correlated with both the air and bone conduction hearing thresholds (p<0,05) and that the finding of an air-bone gap in the audiogram correlated with Paget’s disease temporal bone involvement. Conclusions: − The 63,45% of subjects with Paget’s disease of bone (PDB) suffer hearing loss with a hearing threshold of 39,51dB on average. − The subjects with PDB showed a deeper hearing loss and a higher proportion of conductive type than the control group. − The patients with PDB and skull involvement presented a more severe hearing loss compared with the subjects without skull involvement. Skull involvement and age were found to be risk factors for hearing loss. − In the PDB group both the air and bone conduction hearing thresholds correlated with the otic capsule bone density.

This thesis explores the late-Henrican polity through the archive and perspective of William Paget, Henry VIII's secretary at the end of his reign. Paget's papers as secretary (1543-1547), that form the basis of the thesis, are an extensive, unique and relatively under-used source. From this starting-point Paget's role as secretary is explored and he is revealed as the personal servant of the king, whose natural environment was the court. As such he was an influential source of counsel and perhaps the key patronage-broker at court. In this context Paget also had a significant influence over the operation of the dry stamp at the end of the reign. Equally, Paget's role in shaping the function of the secretary and his relations with the recently formed privy council was of considerable importance, providing the template for later Tudor secretaries. Diplomacy in the uncertain world of the 1540s was one of Paget's primary concerns and his priorities can be seen as trying to provide security and stability for the realm. This is revealed not only in his 'Consultation' of August 1546 but also in his diplomacy with the French, the Schmalkaldic League and the Papacy. In this he sometimes found himself at odds with the king and leading a privy council united in a desire for peace. Politically Paget has traditionally been cast as an ambitious politique, the 'master of practices' and part of the earl of Hertford's reform party. Whilst acknowledging Paget's close relations with Hertford this thesis questions the factional interpretation of the last years of the reign and argues that the predominant concern of Paget and his fellow privy councillors was a peaceful succession in which unanimity rather than conflict was the key-note.

La sérotonine est un neurotransmetteur qui semble jouer un rôle dans le métabolisme osseux. En partant de cette observation, nous avons étudié les récepteurs sérotoninergiques afin de déterminer leurs rôles éventuels dans l'ostéogénèse. En premier, l'importance du récepteur sérotoninergique 5-HT2B (5-HT2BR) au cours de la différenciation ostéogénique a été démontré par l'étude de la lignée mésoblastique C1. L'analyse des souris 5-HT2BR-/- ont permis de confirmer son activité in vivo. Seules les souris femelles âgées 5-HT2BR-/- présentent une ostéopénie qui s'intensifie avec l'âge. Ce phénotype traduit en fait un défaut de recrutement et de prolifération des ostéoblastes 5-HT2BR-/-. Ensuite, en cas de remodelage osseux excessif, des lignées lymphoblastoïdes de patients atteints de la maladie osseuse de Paget présentent une surexpression du 5-HT2BR et des taux élevés d'IL-6 et de son récepteur soluble. En conclusion, le 5-HT2BR apparaît comme un facteur physiologique de régulation de l'homéostasie osseuse
The serotonine is a neurotransmitter which seems to play a role in the osseous metabolism. So, we studied the serotoninergic receptors to determine their possible roles in the osteogenesis. First, the importance of the serotoninergic receptor 5-HT2B (5-HT2BR) during the osteogenic differentiation was demonstrated by the lineage mesoblastic C1 studies. The 5-HT2BR-/- mice analysis allowed to confirm in vivo its activity. Only old female mice 5-HT2BR-/- present an osteopenia which worsen with the age. This phenotype express recruitment and proliferation defects of 5-HT2BR-/- osteoblasts. Then, in case of excessive osseous remodeling, B cell lineages of paget's disease patients present a surexpression of the 5-HT2BR and the high rates of IL-6 and its soluble receptor. In conclusion, the 5-HT2BR appears as a physiological factor of regulation of the osseous homeostasis

Paget’s disease of bone (PDB) is an age-related metabolic bone disease characterized by focal lesions of increased bone resorption and formation, eventually leading to bone deformities. The cause of PDB and the mechanisms that give rise to focal lesions are yet to be understood, but findings suggest that the disease is driven by aberrant, highly nucleated, osteoclasts (OCs). In recent years evidences of a genetic involvement were found: mutations in UBA domain of SQSTM1, which encodes for p62, have been reported in both familial and sporadic cases of PDB (P392L most commonly). Although, their actual pathogenicity has been controversial in experimental studies. Moreover, mutations only involve a part of PDB cases and, although some novel genes have been more recently associated to PDB (e.g.ZNF687), the genetic background of PDB remains in part unknown. In an attempt to establish an experimental model of PDB and better understand p62 role in the disease, we compared two genetically modified murine models, systemic p62 knock-out (p62KO) and mutated p62-P394L (P394L) mice. To further characterize the genetic background of PDB we investigated PDB-associated genes and novel genes in SQSTM1-negative patients. In vitro bone marrow-derived macrophages (BMMs) showed a reduced RANKL-induced osteoclastogenesis (OCgenesis) in p62KO-mice, also seen by TRAP staining on bone sections. BMMs of P394L mice showed a higher sensitivity to RANKL and an increase in OC size and number of nuclei, resembling PDB. Such alterations did not result in a bone phenotype at 6 months of age in either model. However, we found that, with ageing, 47% of P394L mice do develop focal osteolytic lesions. Surprisingly, 78% of p62KO mice developed severe lesions. Although further histological characterization is needed, both animals showed focal, PDB-like, osteolytic/sclerotic features. In vitro analysis of aged p62KO BMMs no longer showed a reduction in OCgenesis potential. Taken together, our findings suggest that p62 mutations in UBA cause a loss of function mechanism in PDB, further exacerbated by total loss of the protein. In support of our hypothesis, proteomics showed that aged p62KO and P394L BMMs are primed for OCgenesis and both present similar expression profiles. Investigating possible molecular mechanisms, we found that UBA-dependent p62 functions of autophagy and NF-κB signalling are not altered in either p62KO and P394L cells. Genetic analysis of 34 patients was performed on genes SQSTM1, TNFRSF11A, VCP, ZNF687 and two variants on TM7SF4 and RIN3. The majority of our cohort was negative for rare mutations on such genes, apart from three cases, carrying TNFRSF11A_M566L, SQSTM1_S275N and ZNF687_P937R. Finally, taking advantage of a large pedigree of a severely affected PDB family we performed Exome NGS to identify novel causal gene. Analysis of impact, familial segregation and allelic frequency identified a novel mutation: PFN1_D107Rfs*3, that causes loss of C-terminal domain of PFN1. This gene encodes for profilin1, a regulator of actin polymerization and cell motility. Given the essential role of cytoskeleton reorganization in OCs biology and the previous findings of bone focal deformities in PFN1 OC-conditional knock-out mice, we started investigating its potential pathogenicity. Silencing of PFN1 in murine BMMs resulted in larger OCs with a higher number of nuclei and increased resorption activity. Screening of PFN1 mutations on other PDB cases is ongoing. Overall, our data demonstrated that both p62 depletion and P394L mutation and are sufficient to cause PDB-like disease in mice. Based on the available molecular data the likely role of p62 in PDB is UBA-dependent but autophagy and NF-κB independent. The genetic background of PDB remains largely unknown, as demonstrated by our screening. Finally, the gene discovery part of the project allowed to identify a likely novel gene for PDB, associated with an early onset and aggressive phenotype.

La maladie de Paget est une ostéopathie caractérisée par une augmentation multifocale du remodelage osseux, qui débute par un front de résorption osseuse, suivi d'une formation osseuse excessive, avec un remodelage anarchique et intense. Les ostéoclastes "OCs" impliqués dans la phase initiale sont les cellules responsables dans l'initiation du processus pagétique. Les OCs pagétiques sont caractérisés par une résistance à l'apoptose, et des anomalies du processus de l'autophagie "en particulier défaut d'induction"; afin de voir si ces deux caractéristiques étaient liées, nous avons émis l'hypothèse d’un rôle des complexes Bcl2-Beclin1. Beclin-1 est une protéine inductrice de l'autophagie qui peut lier les protéines anti-apoptotiques de la famille Bcl-2; Bcl-2 inhibe alors Beclin-1 "et donc l'induction de l'autophagie" en conservant ses fonctions anti-apoptotiques. Dans le but d'étudier l'impact de l'expression de Bcl2 sur l’autophagie dans les OCs humains, nous avons utilisé un modèle de différenciation in vitro à partir de monocytes dérivés de sang de cordon ombilical, cultivés en présence de RANKL et MCSF pendant 21 jours. Ces conditions permettent d'obtenir des cellules multinucléées au phénotype ostéoclastique. Pour augmenter l’expression de Bcl-2 dans les OCs et analyser son impact sur l’autophagie par interaction avec Beclin-1, les cultures ont été stimulées par TNFα ou RANKL dans le but d'induire une activation de NF-κB. L'expression de Beclin1 et Bcl2 a été confirmée par immunobuvardage dans les OCs. L’autophagie était induite dans les cultures réalisées en conditions stringentes "milieu pauvre en nutriments", sans variation de l'expression de Bcl2 ou Beclin 1 selon les conditions, et sans impact de TNFa ou RANKL. TNFa stimulait de manière significative l'activation de NF-kB dans les cellules HEK mais pas dans les OCs. Toutefois, et quelque soit les conditions, les immunoprécipitations ne permettaient pas de retrouver d'association entre Beclin1 et Bcl2. En revanche, le partenaire d'interaction classique de Beclin1, PI3K type III, était associé à Beclin1. En conclusion, notre travail n'a pas permis d'étudier la formation des complexes Beclin1/Bcl2 et les relations entre apoptose et autophagie, en partie du fait de la complexité du modèle "effets multiples de NF-kB et TNFa" ce qui n'exclut pas l'hypothèse initiale "à ré-évaluer par une méthodologie plus appropriée". En revanche les différentes techniques d'analyse sont maintenant au point pour la poursuite de l'étude.
Abstract : Paget's disease is an osteopathy characterized by a multifocal increase in bone remodeling, which begins with excessive bone resorption followed by increased bone formation. Osteoclasts "OCs" were incriminated in the initiation of the pagetic process. Pagetic OCs are characterized by a resistance to apoptosis, and abnormalities in the process of autophagy “in particular induction defect”. In order to define whether these two characteristics were linked, we hypothesized the role of Bcl2-Beclin1 complexes. Beclin-1 is an autophagy-inducing protein that can bind anti-apoptotic proteins of the Bcl-2 family; Bcl-2 then inhibits Beclin-1 "and thus the induction of autophagy" while retaining its anti-apoptotic functions. To study the impact of Bcl2 expression on autophagy in human OCs, we used an in vitro differentiation model that uses monocytes, which are derived from umbilical cord blood and grown in the presence of RANKL and MCSF for 21 days. These conditions make it possible to obtain multinucleated cells with an osteoclastic phenotype. To increase the expression of Bcl-2 in OCs and analyze its impact on autophagy due to its interaction with Beclin-1, cultures were stimulated with TNFα or RANKL in order to induce NF-κB activation. The expression of Beclin1 and Bcl2 was confirmed by immunoblotting of Ocs cell lysates. Autophagy was induced in cultures carried out under stringent conditions "nutriment-deprived mediun", but we did not observe any variation in the expression of Bcl2 or Beclin 1 according to the culture conditions or TNFα or RANKL stimulation. TNFα significantly stimulated the activation of NF-κB in HEK cells but not in OCs. However, whatever the conditions, results from immunoprecipitaion experiments did not reveal any association between Beclin1 and Bcl2. On the other hand, the classic interaction partner of Beclin1, PI3K type III, was associated with Beclin1. In conclusion, our work did not allow us to demonstrate the formation of Beclin1 / Bcl2 complexes and the relationship between apoptosis and autophagy, partly because of the complexity of the model "multiple effects of NF-κB and TNFα". Our initial hypothesis should thereby be re-evaluated using a more appropriate methodology. On the other hand, the different techniques are now ready for further study.

La maladie osseuse de Paget (MOP) est la deuxième maladie osseuse la plus fréquente après l’ostéoporose. Elle touche environ 3% de la population caucasienne après l’âge de 55 ans. La mutation p.Pro392Leu dans le gène appelé SQSTM1, liée à la MOP a été identifiée chez la moitié des formes familiales dans la population canadienne-française avec une transmission selon un mode autosomique dominant. Toutefois cette mutation n’explique pas complètement la maladie, notamment le phénotype cellulaire des ostéoclastes. Les objectifs de cette thèse étaient de mieux comprendre l’hétérogénéité clinique et moléculaire de laMOP, en étudiant les facteurs précoces contribuant au développement des phénotypes clinique et cellulaire de la MOP chez les apparentés porteurs de la mutation p.Pro392Leu, en recherchant un second gène modificateur de la mutation p.Pro392Leu dans certaines familles de notre cohorte et en étudiant l’effet modulateur de miR-16 sur l’expression du gèneSQSTM1.Nous avons examiné les données des descendants adultes porteurs de la mutationp.Pro392Leu âgés de <90 ans et leurs parents atteints. Les caractéristiques cliniques des parents comprenaient le sexe, l'âge au diagnostic, le nombre d'os affectés, les taux de phosphatase alcaline totale sérique (tALPs). L'évaluation du phénotype pagétique chez les descendants à l'inclusion entre 1996 et 2009 comprenait le dosage des tALPs, la scintigraphie osseuse et les radiographies du crâne et du bassin. Une réévaluation approfondie du phénotype de ces descendants par imagerie osseuse et dosage des marqueurs biochimiques a été effectuée entre 2016 et 2018. Le séquençage de l'exome entier a été effectué en utilisant un HiSeq 2500 chez quatre patients de deux familles différentes avec la MOP non porteurs d’aucune mutation dans le gène SQSTM1. Le phénotype clinique a été défini par l’âge au diagnostic, le taux de tALPs, le nombre d'os atteints chez les patients porteurs de la mutation p. Pro392Leu et / ou du variant p.Val45Ile dans deux familles (n = 14 participants). Les quantifications de miR-16 et de SQSTM1 ont été effectuées respectivement dans le plasma(97 participants) et dans le sang total (83 participants) par RT-PCR quantitative chez des patients pagétiques avec ou sans la mutation p.Pro392Leu, des porteurs sains de cette mutation et des témoins sains non mutés. L’expression de miR-16 a été corrélée avec l’expression de SQSTM1 et des associations avec l’âge au diagnostic, le sexe, le taux detALPs ainsi que le nombre d’os atteints ont été recherchées. L'impact du variant p.Val45Ilesur le phénotype ostéoclastique (ostéoclastogenèse, nombre de noyaux par ostéoclaste etrésorption osseuse) a été évalué par différenciation in vitro des monocytes du sang périphérique en ostéoclastes matures avec du RANKL et hMCSF pendant 21 jours, chez des patients atteints de MOP porteurs d’un et/ou l’autre variant génétique, des porteurs sains de ces variants, des patients pagétiques non mutés et des témoins sains non mutés. Le phénotypage clinique complet pour rechercher une MOP asymptomatique chez 36descendants sains porteurs de la mutation p.Pro392Leu a montré que parmi les 36descendants avec un phénotype mis à jour, quatre d'entre eux ont développé un phénotype clinique de la MOP caractérisé par une hyperfixation monostotique ou polyostotique associée à des lésions radiographiques typiques dans les sites affectés, représentant une incidence de1,83 par 1000 personnes- année. De plus, l'âge au diagnostic de la MOP a été retardé de 10ans en moyenne chez les descendants adultes porteurs de la mutation p.Pro392Leu par rapport à leurs parents affectés. Nos résultats soutiennent l'utilité d'une surveillance régulière des adultes porteurs de cette mutation. L'effet modulateur de miR-16 sur l'expression du gèneSQSTM1 par quantification de l'expression génique a conduit à une une corrélation négative non significative de l'expression du gène SQSTM1 par miR-16 chez les participants porteurs de la mutation p.Pro392Leu, chez les patients mutés et non mutés mais pas chez les contrôles sains (-0.17103, -0.11583 vs 0.05624, NS). Ce résultat suggère un rôle possible de miR-16comme modificateur épigénétique dans la MOP. Une analyse de l'exome entier chez deux denos grandes familles avec hérédité digénique a permis d'identifier un nouveau variantp.Val45Ile dans le gène DOCK6 qui pourrait être un gène modificateur de la mutationp.Pro392Leu. Ce variant rare p.Val45Ile pourrait atténuer la sévérité du phénotype clinique de la MOP causée par la mutation du gène SQSTM1 lorsque les deux variants sont portés parle même patient. Par contre, ce nouveau variant seul est suffisant pour donner lieu à un phénotype ostéoclastique pagétique mais ce dernier est moins sévère que celui observé avec la mutation p.Pro392Leu seule. Ce projet de thèse contribue ainsi à une meilleure compréhension des mécanismes physiopathologiques de la MOP qui pourraient conduire à une meilleure prise en charge clinique des patients avec la MOP et à l'identification de nouvelles possibilités diagnostiques ou thérapeutiques, notamment pour réguler le remodelage osseux.
Paget's disease of bone (PDB) is the second most common bone disease after osteoporosis. It affects approximately 3% of the Caucasian population after the age of 55 years old. Thep.Pro392Leu mutation in the gene called SQSTM1, linked to PDB has been identified in halfof the familial forms in the French-Canadian population with an autosomal dominant modeof inheritance. However, this mutation does not fully explain the disease, in particular the cellular phenotype of osteoclasts. The objectives of this study were to better understand the clinical and molecular heterogeneity of PDB, by studying the early factors contributing to the development of the clinical phenotype of PDB in relative carriers of the p.Pro392Leumutation, by looking for a second modifier gene of p.Pro392Leu mutation in some families in our cohort and by studying the modulating effect of miR-16 on the expression of the SQSTM1 gene We reviewed research records from adult offspring carriers of the p.Pro392Leu mutation aged <90 years and their affected parent. In parents, we collected data on sex, age at diagnosis, number of affected bones, total serum alkaline phosphatase levels (tALPs). PDB extended phenotype assessment relying on tALPs, total body bone scan and skull and pelvis radiographs, was performed in offspring at inclusion in 1996 to 2009. An extended phenotypereassessment of these offspring by bone imaging and biochemical markers assay was carriedout between 2016 and 2018. The quantification of miR-16 and SQSTM1 was carried out respectively in plasma (97 participants) and in whole blood (83 participants) by quantitativeRT-PCR in carriers of the p.Pro392Leu mutation (patients with PDB or healthy carriers), innot mutated patients with PDB and healthy controls not mutated. MiR-16 expression was correlated with SQSTM1 gene expression and associations with age atdiagnosis, sex, tALPs and the number of affected bones were searched. Whole exome sequencing was performedusing a HiSeq 2500 with the Agilent XT protocol on four patients from two different families with PDB not carriers of any SQSTM1 mutation in which at least one other sibling with PDB was carrier of the p.Pro392Leu mutation. The clinical phenotype was defined by the age at diagnosis, tALPs level, number of affected bones in patients carrying the p.Pro392Leu and/orp.Val45Ile variant in two families (n=14 participants). The impact of the p.Val45Ile varianton the osteoclastic phenotype (osteoclastogenesis, number of nuclei per osteoclast and bone resorption) was evaluated by in vitro differentiation of monocytes from peripheral blood intomature osteoclasts with RANKL and hMCSF for 21 days, in pagetic patient carriers of one and/or two variants, healthy carriers of these variants, pagetic patients not mutated and healthy controls not mutated. The complete clinical phenotyping to search for asymptomatic PDB in 36 healthy offspring carriers of the p.Pro392Leu mutation showed that among the 36 offspring with an updated phenotype, four of them developed a clinical phenotype of PDB characterized by monostoticor polyostotic increased bone uptake associated with typical radiographic lesions in the affected sites, representing an incidence of 1.83 per 1000 person-years. Moreover, the age at PDB diagnosis was delayed by 10 years in average in the adult offspring carriers thep.Pro392Leu mutation versus their affected parents. Our findings support the utility of a regular monitoring of the adult healthy offspring carriers of this mutation. The modulating effect of miR-16 on the expression of the SQSTM1 gene by gene expression quantificationled a non significant negative correlation of the expression of the SQSTM1 gene by miR-16in participant carriers of the p.Pro392Leu mutation, in patients mutated and not mutated butnot in healthy controls (-0.17103, -0.11583 vs 0.05624, NS). This result suggests a possible role for miR-16 as an epigenetic modifier in PDB. An analysis of the whole exome in two ofour large families with digenic inheritance allowed us to identify a new variant p.Val45Ile inthe DOCK6 gene which could be a modifier gene for the p.Pro392Leu mutation. This rare variant p.Val45Ile could reduce the severity of the clinical phenotype of PDB caused by the mutation in the SQSTM1 gene when the two variants are carried by the same patient. On the other hand, this new variant alone gives rise to a pagetic osteoclastic phenotype but lesss evere than the one observed with the p.Pro392Leu mutation alone. This thesis project thus provided a better understanding of the pathophysiological mechanisms of PDB which could lead to better clinical management of patients with PDB and to the identification of new diagnostic or therapeutic possibilities, to regulate bone remodeling

La maladie osseuse de Paget (MOP) a changé de visage au cours des dernières années, augmentant le nombre d’individus atteints qui demeurent asymptomatiques. Étant donné le risque élevé de développer un ostéosarcome associé avec la MOP, cette maladie est une contre-indication à la prescription d’agents ostéoformateurs. Avec l’apparition prochaine de nouveaux agents ostéoformateurs pour le traitement de l’ostéoporose, il devient crucial de pouvoir dépister de façon fiable la présence de la MOP. Les objectifs de ce projet étaient (1) de mettre au point un test plus sensible permettant de détecter et d’évaluer la fréquence des mutations post-zygotiques SQSTM1/P392L chez les patients pagétiques, (2) de développer un test génétique de dépistage de la MOP incluant les mutations germinales et post-zygotiques dans SQSTM1, (3) et d’évaluer les performances diagnostiques de ce test intégré avec des marqueurs biochimiques dans une signature moléculaire spécifique à la maladie. Une technique de PCR sensible utilisant un acide nucléique bloqué (LNA) spécifique à la mutation SQSTM1/P392L a été développée, puis la présence de cette mutation a été recherchée dans les cohortes disponibles au laboratoire et dans différents tissus. Ensuite, le développement de la signature moléculaire a utilisé les données génotypiques et biochimiques disponibles dans les cohortes du laboratoire, puis des régressions logistiques ont été effectuées afin de déterminer la combinaison de marqueurs ayant la meilleure capacité à identifier correctement les patients avec la MOP. Des mutations post-zygotiques SQSTM1/P392L étaient présentes chez 4,8% des patients pagétiques, et 1,4% des individus sains dans les populations étudiées, cette mutation post-zygotique étant restreinte à la lignée monocytaire. Deux tests moléculaires sous forme d’algorithme en deux étapes ont ensuite été proposés. D’une part, un algorithme génétique pur pourrait être utilisé : des mutations germinales dans le gène SQSTM1 devraient d’abord être recherchées, et si elles sont absentes, le score génétique basé sur la combinaison de cinq marqueurs génétiques développée dans ce projet devrait être calculé. Cet algorithme génétique avait une sensibilité égale à 83,61% et une spécificité égale à 51,03% dans les cohortes étudiées. D’autre part, un algorithme génétique et biochimique pourrait être proposé: des mutations germinales dans le gène SQSTM1 devraient d’abord être recherchées, et si elles sont absentes, le score combiné basé sur la combinaison des marqueurs génétiques et biochimiques développée dans ce projet devrait être calculé. Dans les populations étudiées, cet algorithme avait une sensibilité égale à 93,88% et une spécificité égale à 54,00%. La découverte des mutations post-zygotiques confirme l’existence d’un spectre mutationnel de SQSTM1 dans la MOP et pourrait expliquer en partie son caractère focal. Ces résultats ont par la suite permis de développer deux tests moléculaires capables de dépister la MOP de façon plus fiable que les biomarqueurs actuellement disponibles en pratique clinique.
Paget’s disease of bone (PDB) has changed in recent years, increasing the number of affected individuals who remain asymptomatic. Given the high risk of developing an osteosarcoma associated with PDB, this disease is a contraindication to the prescription of bone anabolic agents. With the incoming introduction of new bone anabolic agents indicated for osteoporosis treatment, it will be crucial to screen accurately for the presence of PDB. The objectives of this project were (1) to develop a more sensitive test to detect and assess the frequency of SQSTM1/P392L post-zygotic mutations in pagetic patients, (2) to develop a genetic test of PDB, including germinal and post-zygotic SQSTM1 mutations, (3) and to assess the diagnostic performance of this test integrated with bone biomarkers in a molecular signature of PDB. A sensitive PCR method using a locked nucleic acid (LNA) specific to the SQSTM1/P392L mutation was developed, and the presence of this mutation was investigated in the cohorts available in the laboratory, and in different tissues. Then, the development of the molecular signature used genotypic and biochemical data available in the laboratory, and logistic regressions were performed to determine the combination of markers with the best ability to correctly identify PDB patients. SQSTM1/P392L post-zygotic mutations were present in 4.8% of pagetic patients, and in 1.4% of healthy individuals in the population studied, this mutation being restricted to the monocytic lineage. Two molecular tests relying on a two steps algorithm were then developed. Firstly, a pure genetic algorithm could be proposed: a screen in the SQSTM1 gene should first be performed to search for disease-causing germinal mutations, and if negative, the genetic score based on a combination of the five SNPs developed in this study should be calculated. In the populations studied, this genetic algorithm had a sensitivity of 83.61% and a specificity of 51.03%. On the other hand, a genetic and biochemical algorithm could be used: a screen in the SQSTM1 gene should first be performed to search for disease-causing germinal mutations, and if negative, the combined score based on a combination integrating both genetic and biochemical markers developed in this study should be calculated. This genetic algorithm had a sensitivity of 83.61% and a specificity of 51.03% in the populations studied. The presence of post-zygotic mutations confirms the existence of a mutational spectrum of SQSTM1 in PDB, and may explain its focal character. These results conducted to the development of two molecular tests which predicted the PDB phenotype better than bone biomarkers already available in clinical practice.

La malaltia òssia de Paget (MOP) es caracteritza per ser un trastorn crònic i focal del remodelat ossi que comporta a l’aparició de complicacions com deformitats òssies, artropatia i fractures. La MOP presenta una gran variabilitat en la seva distribució en funció de l’edat, gènere, ètnia i àrea geogràfica. Recents estudis han descrit canvis seculars pel que fa a la prevalença, incidència i gravetat al diagnòstic de la MOP, mostrant una disminució en les últimes dècades. La MOP es considera actualment una malaltia multifactorial amb participació de factors ambientals i genètics. El gen que ha mostrat major susceptibilitat és el Sequestosoma1 (SQSMT1), les mutacions descrites en ell no explicarien de manera completa la patogènia de la MOP. En l’actualitat, són escassos els estudis sobre variacions genètiques en els gens TNFRSF11B i TNFRSF11A associats a MOP clàssica així com l’existència d’interaccions genètiques entre els dos gens i el gen SQSMT1, que condueixi a un increment de risc de MOP i fins i tot que influeixen en el seu fenotip. Amb la finalitat d’investigar el patró de comportament clínic-epidemiològic com genètic de la MOP de la nostra població de baixa prevalença, ens plantegem els següents objectius. Els objectius es van centrar en avaluar canvis en l’evolució del percentatge de nous diagnòstics de la MOP durant el període 1970-2009 i descriure en aquests pacients diferències pel que fa a la severitat al diagnòstic. Així com analitzar la importància d’alteracions moleculars en el gen SQSTM1 i la presència de variacions al·lèliques en els gens TNFRSF11B i TNFRSF11A, tant en la susceptibilitat per al desenvolupament de la MOP com la influència en el seu fenotip. Pacients i Mètodes: Estudi descriptiu ambispectiu observacional de pacients procedents de l’Hospital de la Mar-Parc de Salut Mar (Barcelona), una àrea de baixa prevalença. Es van incloure 393 pacients, diagnosticats entre 1970 i 2009. L’estudi molecular es va dur a terme mitjançant el genotipatge de les 21 variants polimòrfiques per a l’estudi dels gens TNFRSF11B i TNFRSF11A, així com la seqüenciació de el gen SQSTM1 en una població de 200 pacients afectes de MOP i 200 controls hipernormales. Resultats: En l’estudi s’observa un descens progressiu del percentatge del nous diagnòstics de MOP en relació a la població de referència, entre 1994 i 2009, més marcat en el grup de ≥ de 65 anys. Així mateix, s’ha constatat que els pacients afectes de MOP presenten una disminució en l’activitat biològica de la malaltia, una menor extensió de la malaltia i una major edat de presentació al diagnòstic, en funció de l’any de naixement i any del diagnòstic. En contraposició, s’ha observat major severitat en el moment del diagnòstic en els pacients amb edats al diagnòstic de <45 anys i ≥75 anys. S’han identificat 5 factors independents de mal pronòstic al diagnòstic, que afavoriran pitjors desenllaços. Pel que podem concloure, que en el moment de diagnòstic de MOP, l’expressió de la malaltia és menys severa i esdevé de forma més tardana. Els resultats de l’estudi molecular destaquen l’associació significativa entre els SNPs rs3018362 i rs1805034 del gen TNFRSF11A i rs11573871 del gen TNFRSF11B amb l’increment de risc de desenvolupar MOP així com la influència en el seu fenotip. Així mateix es va observar que la presència de l’al·lel T en el polimorfisme rs6567274 del gen TNFRSF11A es va associar amb una disminució de risc de desenvolupar la MOP, així com a un fenotip més lleu. En l’estudi molecular del gen SQSTM1 es van identificar 6 variants genètiques “missense” de nova descripció, associades amb una major susceptibilitat de desenvolupament de la MOP.
La enfermedad ósea de Paget (EOP) se caracteriza por ser un trastorno crónico y focal del remodelado óseo que conlleva a la aparición de complicaciones como deformidades óseas, artropatía por vecindad y fracturas. La EOP presenta una gran variabilidad en su distribución en función de la edad, género, etnia y área geográfica. Recientes estudios han descrito cambios seculares en cuanto a la prevalencia, incidencia y gravedad al diagnóstico de la EOP, mostrando una disminución en las últimas décadas. La EOP se considera actualmente una enfermedad multifactorial con participación de factores ambientales y genéticos. El gen que ha mostrado mayor susceptibilidad es el Sequestosoma1(SQSMT1), las mutaciones descritas en él no explicarían de forma completa la patogenia de la EOP. En la actualidad, son escasos los estudios acerca de variaciones genéticas en los genes TNFRSF11B y TNFRSF11A asociados a EOP clásica así como la existencia de interacciones genéticas entre ambos genes y el gen SQSMT1, que conduzca a un incremento de riesgo de EOP e incluso que influya en el fenotipo. Con la finalidad de investigar el patrón de comportamiento clínico-epidemiológico como genético de la EOP de nuestra población de baja prevalencia, nos planteamos los siguientes objetivos. Los objetivos se centraron en evaluar cambios en la evolución del porcentaje de nuevos diagnósticos de EOP durante el periodo 1970-2009 y describir en dichos pacientes diferencias en cuanto a la severidad al diagnóstico. Así como así como analizar la importancia de alteraciones moleculares en el gen SQSTM1 y la presencia de variaciones alélicas en los genes TNFRSF11B y TNFRSF11A, tanto en la susceptibilidad para el desarrollo de la EOP como la influencia en su fenotipo. Pacientes y Métodos: Estudio descriptivo ambispectivo observacional de pacientes procedentes del Hospital del Mar-Parc de Salut Mar (Barcelona), un área de baja prevalencia. Se incluyeron 393 pacientes, diagnosticados entre 1970 y 2009. En base al estudio molecular se llevó a cabo el genotipado de las 21 variantes polimórfica para el estudio los genes TNFRSF11B y TNFRSF11A, así como la secuenciación del gen SQSTM1 en una población de 200 pacientes afectos de EOP y 200 controles hipernormales. Resultados: En el estudio se observa un descenso progresivo del porcentaje de nuevos diagnósticos de EOP en relación a la población de referencia, entre 1994 y 2009, más marcado en el grupo de ≥ de 65 años. Asimismo, se ha constatado que los pacientes afectos de EOP presentan una disminución en la actividad biológica de la enfermedad, una menor extensión de la enfermedad y una mayor edad de presentación al diagnóstico, en función del año de nacimiento y año del diagnóstico. En contraposición, se ha observado mayor severidad en el momento del diagnóstico en los pacientes con edades al diagnóstico de <45 años y ≥75 años. Se han identificado 5 factores independientes de mal pronóstico al diagnóstico, que van a favorecer peores desenlaces. Por lo que podemos concluir, que en el momento de diagnóstico de EOP, la expresión de la enfermedad es menos severa y acontece de forma más tardía. Los resultados del estudio molecular destacan la asociación significativa entre los SNPs rs3018362 y rs1805034 del gen TNFRSF11A y rs11573871 del gen TNFRSF11B con el incremento de riesgo a desarrollar EOP así como la influencia en su fenotipo. Asimismo se observó que la presencia del alelo T en el polimorfismo rs6567274 del gen TNFRSF11A se asoció con una disminución de riesgo a desarrollar la EOP, así como a un fenotipo más leve. En el estudio molecular del gen SQSTM1 se identificaron seis variantes genéticas "missense" de nueva descripción, asociadas todas ellas a una mayor susceptibilidad en el desarrollo de la EOP.
Paget’s Disease of Bone (PDB) is characterized by a chronic and focal disorder of bone remodeling that leads to the appearance of complications as bone deformities, osteoarthritis and fractures. The PDB presents a great variability in its distribution depending on the age, gender, ethnics and geographic area. Recent studies have described secular changes in the prevalence, incidence and severity at diagnosis of PDB, showing a decline in the last decades. PDB is currently considered a multifactorial disease involving environmental and genetic factors. The gene which has shown higher susceptibility is the Sequestosome-1 (SQSTM1), the mutations described in it would not be able to explain completely the PDB pathogenesis. Nowadays, there are few studies on genetic variations in the TNFRSF11B and TNFRSF11A genes associated with classic PDB, as well as the existence of genetic interactions between both genes and the SQSMT1 gene, leading to an increased risk of PDB or even influencing the phenotype. To investigate the pattern of clinical-epidemiological and genetic behavior of the PDB in our low prevalence population, we propose the following objectives. The objectives were focused on evaluating changes in the evolution of the percentage of new PDB diagnoses during the period 1970-2009 and to describing in those patients differences in terms of severity at diagnosis. As well as to analyze the importance of molecular alterations in the SQSTM1 gene and the presence of allelic variations in the TNFRSF11B and TNFRSF11A genes, both in the susceptibility for the development of PDB and its influence on the phenotype. Patients and Methods: Descriptive, ambispective observational study of patients from Hospital del Mar-Parc de Salut Mar (Barcelona), a low prevalence area. 393 patients, diagnosed between 1970 and 2009, were included. Based on the molecular study, the genotyping of the 21 polymorphic variants was carried out for the study of the TNFRSF11B and TNFRSF11A genes, as well as the sequencing of the SQSTM1 gene in a population of 200 PDB affected patients and 200 hypernormal controls. Results: The study observed a progressive decrease in the percentage of new PDB diagnoses in relation to the reference population, between 1994 and 2009, more marked in the group ≥65 years old group. Thereby, it has been confirmed that the PDB-affected patients show a decrease in the biological activity of the disease, a lower extension of the disease, and a higher age of presentation at diagnosis, depending on the year of birth and the year of diagnosis. In contrast, it has been observed higher severity at diagnosis in patients with ages at diagnosis <45 years and ≥75 years. Five independent factors of poor prognosis at diagnosis have been identified, which will favor worse outcomes. We can conclude, that at the time of diagnosis of PDB, the expression of the disease is less severe and occurs later. The results of the molecular study highlight the significant association between SNPs rs3018362 and rs1805034 of the TNFRSF11A gene and rs11573871 of the TNFRSF11B gene with the increased risk of developing PDB as well as the influence on its phenotype. Thereby, it was observed that the presence of the T allele in the polymorphism rs6567274 of the TNFRSF11A gene was associated with a decreased risk of developing PDB, as well as a milder phenotype. In the molecular study of the SQSTM1 gene, six newly described “missense” genetic variants were identified, all associated with greater susceptibility in the development of PDB.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

CONTEXTE: La maladie osseuse de Paget (MOP) est transmise sur un mode autosomique dominant dans 30% des cas. Les mutations de SQSTM-1 expliquent 37% des formes familiales, ce qui suggère la présence d’autres loci. OBJECTIFS: Identifier des variants rares (VR) de gènes candidats situés aux nouveaux loci 1p13 et 8q22. Rechercher une association génétique de la MOP avec ces gènes candidats dans la population canadienne-française. MÉTHODES: Certains gènes candidats à la MOP aux loci 1p13 et 8q22 ont été sélectionnés, puis séquencés dans un échantillon de découverte. La fréquence de l’allèle mineur d’un VR devait être inférieure à 0,05. 4 VR ont été génotypés chez 240 cas et 297 témoins. RÉSULTATS: 74 VR ont été identifiés. Un VR (TM7SF4; rs62620995; Leu397Phe) était prédit dommageable par 2 outils d’analyse in silico. rs35500845 (CTHRC1) et rs62620995 (TM7SF4) étaient associés à la MOP de façon statistiquement significative. Mot clés : Maladie osseuse de Paget, Remodelage osseux, Variants rares, Polymorphismes nucléotidiques simples (SNPs), CTHRC1, TM7SF4 (DC-STAMP), Population canadienne-française, Population à effet fondateur.
BACKGROUND: Paget's disease of bone (PDB) is transmitted through autosomal dominant mode of inheritance in 30 percent of cases. Mutations of the SQSTM-1 gene account for 37 percent of familial forms of PDB, suggesting the involvement of other loci. PURPOSE: Identify rare variants (RV) of candidate genes located on new loci 1p13 and 8q22. Search for a genetic association of PDB with these candidate genes in the French-Canadian population. METHODS: We selected candidate genes on 1p13 and 8q22 loci and sequenced them in a discovery sample. RV was defined by a minor allele frequency less than 0.05. 4 RV were genotyped in 240 PDB patients and 297 healthy individuals. RESULTS: 74 RV were identified. One RV (TM7SF4; rs62620995; Leu397Phe) was predicted to be damaging by two in silico analysis tools. rs35500845 (CTHRC1) and rs62620995 (TM7SF4) were statistically associated with PDB. KEY WORDS: Paget’s disease of bone, bone remodelling, rare variants, polymorphisms, SNP, CTHRC1, TM7SF4, DC-STAMP, French Canadian population, founder effect population.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Ciências Médicas
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Introdução: As Glitazonas (rosiglitazona e pioglitazona) são fármacos utilizados para o tratamento do diabetes tipo II. Estes fármacos são agonistas do receptor ativados por proliferadores de peroxissomos gama (PPAR- ) e induzem à transcrição de genes relacionados ao metabolismo glicídico e lipídico e à expressão de proteínas produzidas e/ou liberadas no processo inflamatório. Objetivos: 1) Avaliar o efeito anti-inflamatório da rosiglitazona e da pioglitazona administrados por via intraperitoneal (i.p.), na primeira (4 h) e na segunda (48 h) fases da resposta inflamatória induzida pela carragenina (Cg) no modelo da pleurisia murina. 2) Avaliar o efeito antioxidante da pioglitazona utilizando-se o mesmo modelo experimental. Delineamento do estudo: Trata-se de um estudo experimental, prospectivo e controlado. Métodos: Neste estudo foram utilizados camundongos albinos Swiss de 1 mês de idade e pesando de 18-20 g. Diferentes grupos de animais foram tratados previamente com a rosiglitazona (0,5 - 10 mg/Kg) ou a pioglitazona (5 - 50 mg/Kg), em diferentes períodos de tempo (0,5 - 2 h) antes da Cg. Apenas para o grupo de animais destinado a avaliação da exsudação e a contagem total e diferencial dos leucócitos no lavado da cavidade pleural foi administrada 0,2 mL da solução de azul de Evans (25 mg/Kg, i.v.), 10 min antes da administração da Cg. Os parâmetros inflamatórios avaliados no lavado da cavidade pleural para ambos os fármacos foram: leucócitos, exsudação, atividade das enzimas mieloperoxidase (MPO), adenosina-deaminase (ADA), concentrações do fator de necrose tumoral alfa (TNF- ) e da interleucina-1 beta (IL-1 ). Além disso, para a rosiglitazona avaliou-se também as concentrações dos metabólitos do óxido nítrico (NOx), interleucina-17A (IL-17A) e fator de crescimento endotelial vascular-alfa (VEGF- ), no lavado da cavidade pleural. Para a pioglitazona os parâmetros estudados para avaliar o estresse oxidativo foram: atividade das enzimas superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa S-transferase (GST), no sangue total, bem como a peroxidação lipídica (TBARS) no lavado da cavidade pleural. A análise estatística utilizada foi o teste T de Student ou ANOVA (Newman Keuls). Além disso, para avaliar as possíveis correlações utilizou-se o teste de Pearson. Valores de P < 0,05 foram considerados significativos. Resultados: A rosiglitazona, em ambas as fases (4 e 48 h) da pleurisia induzida pela Cg, inibiu os leucócitos (P < 0,01), a exsudação (P < 0,01), a atividade das enzimas ADA e MPO (P < 0,05) e as concentrações de NOx, TNF- , IL-1 , IL-17A e VEGF- (P < 0,05). Já a pioglitazona, na pleurisia 4 e 48 h reduziu os leucócitos (P < 0,01), a atividade da ADA (P < 0,01) e as concentrações de TNF- e IL-1 (P < 0,01). A atividade da MPO foi inibida pela pioglitazona somente na pleurisia 4 h (P < 0,01). Na avaliação dos parâmetros relacionados ao estresse oxidativo, a pioglitazona na pleurisia 4 e 48 h reduziu a atividade das enzimas SOD, CAT, GPx e GST (P < 0,05) e as concentrações de TBARS (P < 0,05). Conclusão: A atividade anti-inflamatória da rosiglitazona e da pioglitazona parece estar relacionada com a redução do influxo de células ativadas e consequente redução da liberação e/ou produção de mediadores inflamatórios. Além disso, o efeito antioxidante observado para a pioglitazona deve-se em parte pela inibição de enzimas antioxidantes e marcadores de peroxidação lipídica.
Introduction: The glitazones (rosiglitazone and pioglitazone) are drugs used to treat type II diabetes. These drugs are receptor agonists of peroxisome proliferator-activated gamma (PPAR-) and induce the transcription of genes related to glucose and lipid metabolism and expression of inflammatory proteins produced and/or released in the inflammatory process. Objectives: 1) To evaluate the anti-inflammatory effect of rosiglitazone and pioglitazone administered intraperitoneally (i.p.) in the first (4 h) and second (48 h) phases of the inflammatory response induced by carrageenan (Cg) in the murine model of pleurisy. 2) To evaluate the antioxidant effect of pioglitazone using the same experimental model. Study desings: This is an experimental, prospective and controlled study. Methods: In this study it was used Swiss albino mice, 1 month old and weighing 18-20 g. Different groups of animals were pretrated with rosiglitazone (0.5 -10 mg/Kg) or pioglitazone (5 - 50 mg/Kg) in different periods of time (0.5 # 2 h) prior to Cg. To evaluate the exudation a group of animals received 0.2 mL of Evans blue (25 mg/Kg) 10 min before administration of Cg. The studied inflammatory parameters evaluated were: leukocytes, exudation, activities of myeloperoxidase (MPO) and adenosine deaminase (ADA), concentrations of tumor necrosis factor (TNF-) and interleukin-1 beta (IL-1). In addition for rosiglitazone it was evaluated concentrations of nitric oxide metabolites (NOx), interleukin-17A (IL-17A) and vascular endothelial growth factor-alpha (VEGF-) in the pleural fluid. For pioglitazone the following parameters of oxidative stress were evaluated: activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), gluthatione S-transferase (GST), whole blood, as well as lipid peroxidation (TBARS) in pleural fluid. The statistical analysis used was Student T test or ANOVA (Newman Keuls). In addition, to evaluate the correlations it was used Pearson. Values of P < 0.05 were considered significant. Results: Rosiglitazone in both phases (4 and 48 h) of pleurisy induced by Cg inhibited leukocytes (P < 0.01), exudation (P < 0.01), activities of ADA and MPO (P < 0.05), and concentration of NOx, TNF-, IL-1, IL-17A and VEGF- (P < 0.05). Pioglitazone in pleurisy 4 and 48 h also reduced leukocytes (P < 0.01), activity of ADA (P < 0.01), and concentration of TNF- and IL-1 (P < 0.01). The activity of MPO was inhibited by pioglitazone only in the pleurisy 4 h (P < 0.01). In the evaluation of parametes related to oxidative stress in pleurisy 4 and 48 h, pioglitazone reduced activities of SOD, CAT, GPx and GST (P < 0.05), and concentrations of TBARS (P < 0.05). Conclusion: The anti-inflamatory effects of rosiglitazone and pioglitazone appears to be related to reducing the influx of activated cells and consequent reduction of release and/or production of inflammatory mediators. The antioxidant effect observed for pioglitazone is may be due to in part by inhibiton of antioxidant enzymes and markers of lipid peroxidation.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências Médicas, Florianópolis, 2014.
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Introdução: Muitos aspectos da etiopatogênese da doença óssea de Paget ainda estão por ser elucidados, incluindo seus fatores causais, os motivos de acometer alguns ossos e poupar outros e os fatores determinantes da variabilidade de sua distribuição geográfica. Métodos: Portadores de doença óssea de Paget foram comparado com controles portadores de osteoartrite para avaliar diferenças nas concentrações séricas das citocinas: TNFa, IL1B, IL-6 e IL-17. Em outro protocolo, portadores de doença óssea de Paget foram comparados com controles portadores de osteoartrite para avaliar diferenças nas concentrações séricas dos biomarcadores de metabolismo ósseo: RANKL, osteoprotegerina, DKK-1, sFRP-1 e osteopontina. Para avaliar a presença de mutações genéticas associadas à doença óssea de Paget, portadores de doença óssea de Paget foram avaliados quanto à presença de polimorfismos genéticos dos genes: sequestossomo 1 (SQSTM1), VCP, TNFRSF11B e TNFRSF11A. A qualidade de vida e seus fatores determinantes foram avaliadas com as os questionários de qualidade de vida SF-36 e WHOQOL-bref. Resultados: Portadores de doença óssea de Paget apresentaram concentrações séricas aumentadas de IL-6 (p<0,05) e concentrações séricas reduzidas de IL-17 (p<0,05) quando comparados com o grupo controle. Os pacientes apresentaram concentrações séricas mais elevadas de osteopontina em comparação aos controles (p<0,05). Além disso, as concentrações de osteopontina e fosfatase alcalina óssea se correlacionaram à atividade e à extensão da doença (p<0,05). Quinze sujeitos (20,8%) apresentavam mutações no exon 8 do gene SQSTM1, oito (11,1%) apresentaram a mutação Pro392Leu, e os restantes apresentavam uma mutação ainda não descrita na literatura, Thr430Pro. Os únicos parâmetros significativamente associados à pior qualidade de vida foram: dor óssea, deformidades e estado civil (p<0,05). Conclusão: O presente estudo demonstrou pela primeira vez concentrações reduzidas de IL-17 e aumento das concentrações de osteopontina em portadores de doença óssea de Paget. Além disso, foi encontrada uma mutação do gene SQSTM1 até então inédita na literatura médica.

Abstract : Many aspects of Paget's Disease of Bone (PDB) are still unknown, including its cause, the reasons for its bone distribution and the what determines is geographic distribution. Methods: Patients with PDB were compared with controls with primary osteoarthritis to evaluate possible differences in levels of pro-inflammatory cytokines: TNFa, IL1B, IL-6 and IL-17. In another protocol, patients with PDB were compared with controls with primary osteoarthritis to evaluate possible differences in levels of RANKL, osteoprotegerin, DKK-1, sFRP-1, osteopontin. Polymorphism of the following genes: SQSTM1, VCP, TNFRSF11B and TNFRSF11A were evaluated in PDB patients. To evaluate quality of life and determinant parameters, PDB patients answered to Brazilian versions of SF-36 and WHOQOL questionnaires. Results: Patients with PDB presented higher levels of IL-6 (p<0.05) and lower levels of IL-17 (p<0.05) in comparison to controls. PDB patients presented higher levels of osteopontin in comparison to controls (p<0.05). Moreover, osteopontin and BAP were correlated to disease activity and extension (p<0.05). Fifteen subjects (20.8%) carried mutations in exon 8 of ths SQSTM1 gene, eight (11.1%) carried the Pro392Leu mutation, while the remainders presented a previously undescribed mutation, Thr439Pro. The only parameters significantly associated to quality of life were: bone pain, deformities and marital status (p<0.05). Conclusion: The present study has demonstrated lfor the first time low levels of IL-17 and high levels of osteopontin in PDB patients. Besides, a previously not described mutation of the SQSTM1 gene were found.

Vernon Lee est une femme de lettres d'origine britannique, dont l'œuvre éclectique est située dans le contexte historique et socio-culturel de l'Angleterre victorienne de la fin-de-siècle dite "décadente" (1880-1914). La problématique de cette thèse est axée autour de deux fils conducteurs : d'une part, le regard que Vernon Lee porte sur la société de son époque, et d'autre part, son intérêt pour les récentes découvertes dans le domaine des Sciences humaines. Les années 1890 représentent une ère de transition entre le Romantisme et le Modernisme. On peut donc parler d'héritage romantique en ce qui concerne l'inspiration et l'imaginaire des écrivains victoriens de la fin du XIXème siècle. La littérature de la Décadence privilégie les thèmes mythiques, en particulier le mythe de la Femme fatale. L'esthétisme et l'éthique sont au cœur de l'œuvre de Vernon Lee. Écrivain et témoin de son époque, elle utilise son écriture pour défendre la condition féminine. C'est avant tout une écriture avant-gardiste, orientée vers le Modernisme. En effet, les récentes découvertes scientifiques (notamment la linguistique, la psychologie et la psychanalyse) influencent son écriture, lui permettant d'accéder au mieux à l'intériorité de ses personnages.

La maladie osseuse de Paget (MP) est une maladie métabolique de l’os. Bien que les facteurs génétiques jouent un rôle important dans la pathogénie de la MP, les facteurs environnementaux tels que la résidence rurale et l’exposition au chauffage au bois ont été associés avec la MP. Afin d’étudier le rôle des polluants de l'air extérieur et intérieur sur la MP, nous avons administré un questionnaire chez 140 patients canadiens-français avec la MP et 113 témoins sains. Ce questionnaire portait sur la pollution de l'air extérieur, comme la résidence près d'une autoroute, d’une station de bus, de train ou d’un aéroport, d’une station d'essence, et sur les polluants de l'air intérieur en mettant l'accent sur les combustibles de chauffage (charbon, bois, huile) et l'exposition au tabac. Dans un sous-groupe de patients, la concentration urinaire de 17 métaux lourds et de 11 hydrocarbures aromatiques polycycliques a été mesurée par spectrométrie de masse. À la lumière de ce que nous savions dès le questionnaire et les dosages urinaires, nous avons identifié certains toxiques pouvant être des facteurs de risque pour la MP. Pour explorer les effets in vitro de ces toxiques sur les ostéoclastes dans la MP, nous avons réalisé une différentiation in vitro de monocytes du sang périphérique provenant de plus de 40 participants, patients, porteurs sains de mutation dans le gène SQSTM1, et des témoins sains, en ostéoclastes traités avec ou sans les toxiques identifiés. La morphologie des ostéoclastes, le pourcentage de résorption osseuse, les niveaux d'expression génique, et les niveaux de stress oxydatif cellulaire ont été analysés. Les résultats ont montré un effet inhibiteur du condensé de la fumée de cigarette et des métaux lourds sur la morphologie et la fonction des ostéoclastes. De plus, des taux élevés de stress oxydatif chez les ostéoclastes des patients ont été observés, et un profil hétérogène des effets de métaux lourds sur l'expression des gènes a été identifié.
Paget's disease of bone (PDB) is a metabolic bone disease. Although genetic factors play an important role in the pathogenesis of PDB, environmental factors such as rural residence and the exposure to wood heating was associated with PDB. In order to study the role of outdoor and indoor air pollutants on PDB, we performed a survey in 140 French-Canadian patients with PDB and 113 healthy controls. The survey covered the outdoor air pollution such as the residence near a highway, a bus station, a train or an airport or a gas station, and indoor air pollutants by focusing on heating fuels (carbon, wood, oil) and exposure to tobacco smoke. In a subgroup of patients, urinary concentration of 17 heavy metals and 11 polycyclic aromatic hydrocarbons was measured by mass spectrometry. In light of what we knew from the survey and urinary assays, we identified certain toxics that could be risk factors for PDB. To explore the in vitro effects of these toxics on osteoclasts in PDB, we conducted in vitro monocytes differentiation from peripheral blood of more than 40 participants, patients, healthy carriers of p.Pro392Leu mutation, and healthy controls, which osteoclasts were treated with or without the identified toxic. The morphology of osteoclasts, the percentage of bone resorption, gene expression level, and cellular oxidative stress levels were assayed. The results showed an inhibitory effect of cigarette smoke condensate and heavy metals on morphology and function of patients’ osteoclasts. Further, high levels of oxidative stress in patients’ osteoclasts were observed, and a heterogenic profile of heavy metals effect on gene expression was identified.

The heart of this thesis is a very detailed reconstruction of the life of Thomas 3rd Baron Paget of Beau desert, Staffordshire (1544-1590), with particular emphasis between the years 1570 and 1590. A thorough analysis is made of his courtship and marriage to Nazareth Southwell (nee Newton) a member of the queen's privy chamber, as well as that of his household and family, which reveals that whilst his marriage helped advance his noble career, it was ultimately a failure which led to considerable social, religious and political difficulty. An exploration is made of Paget's involvement in Catholic circles in his native Staffordshire and surrounding area, as are his relationships with prominent local and national figures, both Catholic and Protestant. Paget elicited the support and loyalty of the local community and had solid friendships with members of the nobility centred on a shared love of music. In 1580 Paget was confined in a house in Windsor for approximately fourteen weeks, ostensibly because of his Catholicism. Paget indulged in multi-faceted discussions and arguments over the nature of religious and political loyalty; these arguments are analysed and contextualised. His release was secured. only after he agreed to satisfy a number of conflated personal, religious and political concerns, one of which was an apparent agreement to conform in matters of religion. Thomas made a shallow attempt to conform and after his release his Catholic resolve seemed only to harden. In 1583 Paget was drawn into the Throckmorton Plot conspiracy; finding himself implicated he fled abroad and settled for a time in Paris. The extent of his involvement in this plot and his divided loyalties are explored. Whilst abroad, finding himself exiled and ostracised, he worked on behalf of the Queen of Scots. A consideration is made of his motivations and a close exploration is made of his movements and dealings. After the death of the queen of Scots, Paget's loyalty was again in question.

Les ostéoclastes (OCs) sont responsables de la résorption de l’os normal. La différenciation, la maturation et la survie de ces cellules sont principalement stimulées par RANKL. Ces phénomènes se produisent dans les suites de l’interaction de RANKL avec son récepteur RANK situé à la surface de l’OC, via la transduction de signaux intracellulaires conduisant à l’activation de différentes cascades de signalisation. Les principaux facteurs de transcription induits par cette signalisation sont NF-?B et NFATc1, qui modulent l’expression de gènes d’importance majeure pour le bon fonctionnement des OCs. Dans la maladie de Paget, caractérisée par une augmentation multifocale du remodelage osseux avec hyperrésorption osseuse, les OCs ont une morphologie anormale, ils sont résistants à l’apoptose et leur activité est augmentée. Des mutations du gène codant pour la protéine p62 (séquestosome 1 ) ont été décrites dans cette maladie, identifiant p62 comme un facteur important de la signalisation de l’OC. Des travaux de notre laboratoire ont montré une augmentation de l’activation de NF-?B induite par RANKL dans les OCs pagétiques, avec même une activité constitutive de NF-?B en présence de la mutation p62 p.P392L. Notre étude a évalué l’expression et l’activation de NFATc1, et le rôle de PKC[C zêta] dans la signalisation NFATc1, cette kinase appartenant au complexe de signalisation TRAF6/p62 induit par RANKL. En culture primaire, des monocytes de sang périphérique de patients pagétiques et de contrôles (tous génotypés pour la mutation p62) ont été différenciés en OCs matures en présence de RANKL et M-CSF. Nous avons évalué l’effet de RANKL sur l’activation de NFATc1 (translocation nucléaire évaluée par immunofluorescence), en présence ou non d'un inhibiteur de PKC[C zêta], ainsi que l’expression protéique (immunobuvardage) et ARNm (RTPCR quantitative). Nous avons observé que RANKL stimulait l’activation de NFATcl dans les OCs pagétiques, porteurs ou non de la mutation p62 p.P392L, et que PKC[C zêta] intervenait dans cette signalisation. Dans un modèle de différenciation OC à partir de monocytes foetaux, nous avons par ailleurs mis au point l’étude de l’activation de NFATc1 par essai luciférase, et les conditions optimales pour évaluer son expression protéique et génique. Nos résultats démontrent pour la première fois dans des OCs humains que RANKL stimule l’activation et l’expression de NFATc1, et que l’activation de NFATc1 est augmentée dans les OCs pagétiques. De manière tout à fait originale, nous montrons également que la PKC[C zêta] contribue à l’activation de NFATc1. [symboles non conformes]

Résumé : La maladie osseuse de Paget (MP) est un désordre squelettique caractérisé par une augmentation focale et désorganisée du remodelage osseux. Les ostéoclastes (OCs) de MP sont plus larges, actifs et nombreux, en plus d’être résistants à l’apoptose. Même si la cause précise de la MP demeure inconnue, des mutations du gène SQSTM1, codant pour la protéine p62, ont été décrites dans une proportion importante de patients avec MP. Parmi ces mutations, la substitution P392L est la plus fréquente, et la surexpression de p62P392L dans les OCs génère un phénotype pagétique partiel. La protéine p62 est impliquée dans de multiples processus, allant du contrôle de la signalisation NF-κB à l’autophagie. Dans les OCs humains, un complexe multiprotéique composé de p62 et des kinases PKCζ et PDK1 est formé en réponse à une stimulation par Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), principale cytokine impliquée dans la formation et l'activation des OCs. Nous avons démontré que PKCζ est impliquée dans l’activation de NF-κB induite par RANKL dans les OCs, et dans son activation constitutive en présence de p62P392L. Nous avons également observé une augmentation de phosphorylation de Ser536 de p65 par PKCζ, qui est indépendante d’IκB et qui pourrait représenter une voie alternative d'activation de NF-κB en présence de la mutation de p62. Nous avons démontré que les niveaux de phosphorylation des régulateurs de survie ERK et Akt sont augmentés dans les OCs MP, et réduits suite à l'inhibition de PDK1. La phosphorylation des substrats de mTOR, 4EBP1 et la protéine régulatrice Raptor, a été évaluée, et une augmentation des deux a été observée dans les OCs pagétiques, et est régulée par l'inhibition de PDK1. Également, l'augmentation des niveaux de base de LC3II (associée aux structures autophagiques) observée dans les OCs pagétiques a été associée à un défaut de dégradation des autophagosomes, indépendante de la mutation p62P392L. Il existe aussi une réduction de sensibilité à l’induction de l'autophagie dépendante de PDK1. De plus, l’inhibition de PDK1 induit l’apoptose autant dans les OCs contrôles que pagétiques, et mène à une réduction significative de la résorption osseuse. La signalisation PDK1/Akt pourrait donc représenter un point de contrôle important dans l’activation des OCs pagétiques. Ces résultats démontrent l’importance de plusieurs kinases associées à p62 dans la sur-activation des OCs pagétiques, dont la signalisation converge vers une augmentation de leur survie et de leur fonction de résorption, et affecte également le processus autophagique.
Abstract : Paget’s disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover. In PDB, osteoclasts are larger, more active, more numerous, and resistant to apoptotic stimuli. While no single root cause has been identified, mutations to the gene encoding the p62 protein, SQSTM1, have been described in a significant population of patients with PDB. Among these mutations, the P392L substitution is the most prevalent, and overexpression of p62P392L in osteoclasts generates at least a partial pagetic phenotype in vitro. Normally this protein mediates a number of cell functions, from control of NF-κB signaling to autophagy. In human osteoclasts, a multiprotein complex containing p62 and protein kinases PKCζ and PDK1 (the principal kinase of Akt), form in response to stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL), the principal osteoclastogenic-signaling cytokine. We found that PKCζ is involved in RANKL-induced activation of NF-κB, and that it contributed to a basal activation of NF-κB observed in p62P392L mutants. This may be regulated in part by a PKCζ dependent increase in p65 phosphorylation at Ser536 which we characterized, independent of IκB. This could represent one alternative pathway by which mutant p62 leads to increased NF-κB activation. We observed increased basal phosphorylation of survival regulators ERK and Akt in PDB that was reduced upon PDK1 inhibition. The activity of 4EBP1 and Raptor, associated with mTOR activity, were also altered in pagetic osteoclasts and regulated by PDK1 inhibition. We then identified autophagic defects common to pagetic osteoclasts; with higher basal levels of LC3II (associated with autophagic structures), regardless of p62 mutation, and reduced sensitivity to autophagy induction in PDB. These results suggest an accumulation of non-degradative autophagosomes. Inhibition of PDK1 not only induced apoptosis in PDB and controls, but significantly reduced resorption in PDB, and with regards to autophagy, PDK1 inhibition was more potent in PDB than in controls. Therefore PDK1/Akt signaling represents an important checkpoint to PDB osteoclast activation. In sum, these results demonstrate the importance of several p62-associated kinases in the over-activation of pagetic osteoclasts, through increased survival and altered signaling. As p62 mutations alone do not account for most cases of PDB, the characterization of these pathways may identify a common factor linking pagetic osteoclasts. Therefore these studies represent a novel approach to osteoclast apoptosis, activation, and autophagy associated with PDB.