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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Regulation of osteoclast activation and autophagy through altered protein kinase pathways in Paget's disease of bone

McManus, Stephen January 2016 (has links)
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.
2

Genetic Loci for Paget's Disease of Bone

Good, David Andrew, n/a January 2003 (has links)
Paget's disease of the bone is a skeletal disorder of unknown cause. This disease is characterised by excessive and abnormal bone remodelling brought about by increased bone resorption followed by disorganised bone formation. Increased bone turnover results in a disorganised mosaic of woven and lamellar bone at affected skeletal sites. This produces bone that is expanded in size, less compact, more vascular, and more susceptible to deformity or fracture than normal bone. Symptoms of Paget's disease may include bone pain, bone deformity, excessive warmth over bone from hypervascularity, secondary arthritis, and a variety of neurologic complications caused in most instances by compression of the neural tissues adjacent to pagetic bone. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. The identification of the molecular basis of Paget's disease is fundamental for an understanding of the cause of the disease, for identifying subjects at risk at a preclinical stage, and for the development of more effective preventive and therapeutic strategies for the management of the condition. With this in mind, the aim of this project is to identify genetic loci, in a large pedigree, that may harbour genes responsible for Paget's disease of bone. A large Australian family with evidence of Paget's disease was recruited for these studies (Chapter 3). This pedigree has characterised over 250 individuals, with 49 informative individuals affected with Paget's disease of bone, 31 of whom are available for genotypic analysis. The pattern of disease in these individuals is polystotic, with sites of involvement including the spine, pelvis, skull and femur. Although the affected individuals have a severe early-onset form of the disease, the clinical features of the pedigree suggest that the affected family members have Paget's disease and not familial expansile osteolysis (a disease with some similarities to Paget's disease), as our patients have extensive skull and axial skeletal involvement. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Due to the large size of this family and multiple affected members, this pedigree is a unique resource for the detection of the susceptibility gene in Paget's disease. The first susceptibility loci for Paget's disease of bone have been mapped by other investigators to chromosome 6p21 (PDB1) and 18q21.1-q22 (PDB2) in different pedigrees. Linkage analysis of the Australian pedigree in these studies was performed with markers at PDB1: these data showed significant exclusion of linkage, with LOD scores < - 2 in this region (Chapter 4). Linkage analysis of microsatellite markers from the PDB2 region excluded linkage with this region also, with a 30 cM exclusion region (LOD score < -2.0) centred on D18S42 (Chapter 4). This locus on chromosome 18q21.1-q22 contains a serine protease (serpin) cluster with similarities to chromosome 6p21. Linkage analysis of this region also failed to provide evidence of linkage to this locus (Chapter 4). These data are consistent with genetic heterogeneity of Paget's disease of bone. A gene essential for osteoclast formation encoding receptor activator of nuclear factor-kB (RANK), TNFRSF11A, has been previously mapped to the PDB2 region. Mutations in the TNFRSF11A gene have been identified segregating in pedigrees with Familial Expansile Osteolysis and early onset familial Paget's disease, however, linkage studies and mutation screening have excluded the involvement of RANK in the majority of Paget's disease patients. For the Australian pedigree, mutation screening at the TNFRSF11A locus revealed no mutations segregating with affected individuals with Paget's disease (Chapter 4). Based on these findings, our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree; this gene should be identifiable using a microsatellite genome-wide scan followed by positional cloning. A genome-wide scan of the Australian pedigree was carried out, followed by fine mapping and multipoint analysis in regions of interest (Chapter 5). The peak 2-point LOD scores from the genome-wide scan were LOD = 2.75 at D7S507 and LOD = 1.76 at D18S70. Two additional regions were also considered for fine mapping: chromosome 19p11-q13.1 with a LOD of 1.58 and chromosome 5q35-qter with a LOD of 1.57. Multipoint and haplotype analysis of markers flanking D7S507 did not support linkage to this region (Chapter 5). Similarly, fine mapping of chromosome 19p11-q13.1 failed to support linkage to this region (Chapter 5). Linkage analysis with additional markers in the region on chromosome 5q35-qter revealed a peak multipoint LOD score of 6.77 (Chapter 5). A distinct haplotype was shown to segregate with all members of the family, except the offspring of III-5 and III-6. Haplotype analysis of markers flanking D18S70 demonstrated a haplotype segregating with Paget's disease in a large sub-pedigree (descendants of III-3 and III-4) (Chapter 5). This sub-pedigree had a significantly lower age at diagnosis than the rest of the pedigree (51.2 + 8.5 vs. 64.2 + 9.7 years, p = 0.0012). Linkage analysis of this sub-pedigree demonstrated a peak two-point LOD score of 4.23 at marker D18S1390 (q = 0.00), and a peak multipoint LOD score of 4.71, at marker D18S70. An implication of these data is that 18q23 harbours a novel modifier gene for reducing the age of onset of Paget's disease of bone. A number of candidate Paget's genes have previously been identified on chromosome 18q23, including the nuclear factor of activated T cells (NFATc1), membrane-associated guanylated kinase (MAGUK) and a zinc finger protein. Candidate gene sequencing of these genes in these studies has failed to identify mutations segregating with affected family members in the sub-pedigree linked to chromosome 18q23 (Chapter 6). More recently, a mutation in the gene encoding the ubiquitin-binding protein sequestosome 1 (SQSTM/p62) has been shown to segregate with affected members of Paget's disease families of French-Canadian origin. In this study, a single base pair deletion (1215delC) was identified as segregating with the majority of affected members in the pedigree (Chapter 6). This deletion introduces a stop codon at amino acid position 392 which potentially results in early termination of the protein and loss of the ubiquitin binding domain. The three affected members of the family that do not share the affected haplotype do not carry a mutation in the coding region of SQSTM/p62. Screening of affected members from 10 further Paget's disease families identified the previously reported P392L mutation in 2 (20%) families. No SQSTM1/p62 coding mutations have been found in the remaining 8 families or in 113 aged matched controls. In conclusion, this project has identified genetic loci and mutations that segregate with individuals affected with Paget's disease. Further investigation of the functional significance of the genetic changes at these loci is expected to lead to a better understanding of the molecular basis of this disease.
3

Maladie de Paget : résistance à l'apoptose et défaut de l'autophagie / Paget's disease of bone : resistance to apoptosis and the defect of autophagy

Nazari, Shekeba January 2017 (has links)
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.
4

THE ROLE OF p62 IN OSTEOCLASTOGENESIS AND PAGET’S DISEASE OF BONE

Hadi, Tamer 20 November 2012 (has links)
Paget’s disease (PDB) is the second most common metabolic bone disease after osteoporosis, affecting up to 3% of adults over age 55. It is characterized by focal lesions of bone resorbed by hyperactive osteoclasts coupled with rapid formation of highly disorganized, low quality bone formed by osteoblasts. Such lesions cause skeletal deformity, fractures, and other symptoms that significantly decrease quality of life. In 2001, mutations in the SQSTM1/p62 gene were found in a subset of Paget’s patients. The work summarized in this dissertation sought to answer two broad questions: what is the function of p62 in normal bone homeostasis and how do PDB-associated mutations alter it? These studies took advantage of two mouse models: p62 knock-out (KO) mice, and p62P394L “knock-in” (KI) mice carrying the most common PDB-associated mutation. KO, KI, and wildtype (WT) controls were aged to one year for skeletal-histological characterization. No differences were observed in a variety of bone parameters between WT and KO bones, while bones from age-matched KI mice exhibited a 33% decrease in bone volume and a 25% increase in osteoclast formation. In vivo, TNF-α caused a potent induction of osteoclastogenesis in calvariae of WT and KI, but not KO, mice. In vitro, RANKL induced osteoclast formation in a dose-dependent manner in WT and KI, but not KO, cultures. Gene expression profiling of RANKL-treated osteoclast progenitors from WT, KO, and KI mice was then performed to identify the changes in signaling pathways responsible for these effects. Surprisingly, gene expression patterns from all three groups were consistent with robust activation of NFκB signaling in RANKL-treated samples, indicating that p62 is dispensable for RANKL activation of NFκB. Interestingly, gene expression patterns in KO cells suggested impaired proliferation and response to reactive oxygen species (ROS), a finding which was confirmed in cell culture experiments. In contrast, KI cells displayed enrichment for genes associated with the unfolded protein response, consistent with p62’s role in ubiquitin-mediated protein degradation via proteolysis and autophagy. These studies have therefore generated several novel hypotheses concerning the role of p62 in both normal bone homeostasis and Paget’s disease of bone.

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