Dissertations / Theses on the topic 'Osteoclast'
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O'Brien, Elizabeth Ann. "Regulation of osteoclast activity : differential adhesion of osteoclasts to the bone surface." Thesis, University of Liverpool, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343930.
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Stephens, Sebastien. "Novel Osteoclast Signalling." Thesis, Griffith University, 2010. http://hdl.handle.net/10072/365823.
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When you say osteoporosis, people think of their grandma’s brittle bones, but scientists think of the osteoclast. When you say cancer, people think of death, but before this, many succumb to the osteoclast. The fact is all these things are true yet it is even truer to say that each disease in fact also has more of the other – osteoporosis and death, and cancer and brittle bones. However, the commonality is undeniably the osteoclast. Scratching the surface of the osteoclast reveals that it is the basis of a diversity of bone-related disorders yet the osteoclast itself is, even given the large amount of effort devoted to it to this day, an ill- defined cell. Not surprisingly, deaths related to osteoporotic fractures and skeletal pain due to metastases to bone remain far higher than ideal. Today’s dogma describes the osteoclast as a multinucleated cell derived from the haematopoietic cell lineage that is capable of bone resorption. It is suspected that it is through multiple signalling mechanisms, that this bone-resorbing activity is augmented in certain bone diseases eventually causing osteolysis or the break down of bone. Currently, it still stands that any way to advance our understanding of the osteoclast will provide stepping stones for eventual treatments and perhaps cures for these diseases. Thus the goal of this thesis was to better characterise the osteoclast. For this, four different projects were undertaken; analysis in the osteoclast of (1) the chemokine MCP-1, (2) the RhoGTPase family, (3) DMSO and (4) genes dependent on RANKL (by array). Project results are summarised in order.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Wilkinson, Debbie Isabelle. "Visualisation of osteoclast membrane domains." Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=158808.
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Osteoclasts polarise upon activation and form four distinct membrane domains; the basolateral domain, the sealing zone, the functional secretory domain and the ruffled border. The ruffled border is the resorptive organelle of the cell and provides a large surface area for the release of protons and enzymes into the space beneath the osteoclast. Defects in osteoclast formation or function can lead to diseases such as osteopetrosis. Ruffled border formation is a critical event in osteoclast function but the process by which it and other membrane domains form is only partially understood. Vesicular trafficking is essential for the tight regulation of the osteoclast membrane domains and it has been shown previously that treatment with pharmacological inhibitors causes disruption of trafficking. The aims of this PhD were to increase our understanding of vesicular trafficking in osteoclasts and to optimise ways of visualising osteoclast membrane domains. My studies of patients with osteoclast-poor osteopetrosis identified defects in RANKL as a cause of the defect. This in turn has identified a potential therapy of recombinant RANKL for patients with this form of the disease. Although purification of wild type or mutant RANKL was not completely successful, it did suggest that the mutant forms of RANKL were not functional. I have used pharmacological inhibitors to study osteoclast membrane domains, and found that transmission electron microscopy is an essential tool for studying membrane changes following pharmacological inhibition at the ultrastructural level. I also established that the study of vesicular trafficking to analyse formation of membrane domains can make excellent use of immuno-electron methods. Furthermore, genetic diseases associated with defective ruffled border formation such as XLA and osteopetrosis provide useful tools to further analyse the dynamics involved in the formation and maintenance of the ruffled border, as well as revealing more about the diseases themselves.
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Zaidi, Mone. "Novel mechanisms of osteoclast regulation." Thesis, University of London, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411434.
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Gu, Xiaomei Everett Eric T. "Physiopathology of osteoclast in bone." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1870.
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Thesis (M.S.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Master of Science in the Curriculum of Oral Biology." Discipline: Oral Biology; Department/School: Dentistry.
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Barnes, Calvin Langston Toure. "C-mpl Expression in Osteoclast Progenitors: A Novel Role for Thrombopoietin in Regulating Osteoclast Development." Yale University, 2006. http://ymtdl.med.yale.edu/theses/available/etd-06262006-123750/.
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A new paradigm has evolved in which multiple regulatory interactions between the skeletal and hematopoietic systems have been identified. Previous studies have demonstrated that megakaryocytes (MK) play a dual role in skeletal homeostasis by stimulating osteoblast proliferation and simultaneously inhibiting osteoclast (OC) development. Here we identify a novel regulatory pathway in which the main MK growth factor, thrombopoietin (TPO), directly regulates osteoclastogenesis. To study the role of TPO in OC development, spleen or bone marrow (BM) cells (2x10[exponent]6 cells/ml) or BM macrophages (BMM, 1x10[exponent]5 cells/ml) from C57BL/6 mice , as a source of OC precursors, were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) to induce OC formation. TPO (0.1-1000 ng/ml) and/or primary MK (0-0.5%), derived from C57BL/6 fetal livers, were titrated into these cultures and OC were identified as tartrate resistant acid phosphatase positive (TRAP+) giant cells with >3 nuclei. There was a significant, up to 15-fold reduction in OC formed when MK were added to all OC generating cultures, p < 0.001. Moreover, if OC generating cultures did not contain MK or MK progenitors, TPO treatment significantly enhanced OC formation up to six-fold, p < 0.01. This data demonstrates that MK are responsible for the inhibition of OC formation and that in cultures containing MK or MK progenitors such as BM or spleen cells, that TPO acts indirectly to inhibit OC formation by stimulating megakaryopoiesis, whereas in the absence of MK or MK progenitors TPO directly enhances OC formation. This conclusion is further supported by Real-Time PCR data which demonstrates that OC progenitors express c-mpl, the TPO receptor, albeit at low levels when compared to expression of c-mpl on MK. Finally, we have begun to dissect the c-mpl signaling pathway in OC progenitors. We have found that TPO induces tyrosine phosphorylation of several specific cellular proteins in the JAK/STAT pathway. Thus, TPO acts in a somewhat paradoxical manner by inhibiting OC formation through the stimulation of MK, while simultaneously playing a direct role in enhancing osteoclastogenesis.
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Hale, Annette Julie. "The characterisation of the Pagetic osteoclast." Thesis, University of Nottingham, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359919.
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Lång, Johanna. "CCL11 and Effects on Pre-osteoclast Migration." Thesis, Umeå universitet, Institutionen för odontologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-143797.
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ABSTRACT Periodontitis is a chronic inflammatory disease due to dental bacteria, and the disease is highly prevalent worldwide. Both environmental factors and genetic variation are confounding factors. Characteristic for disease development is degradation of gingival tissue and resorption of the alveolar bone due to inflammation. The cells that are capable to resorb bone is named osteoclasts and those are recruited and activated by numerous cytokines. Cytokines are small signal proteins responsible for cell communication and cell recruitment. Cytokines with chemotactic capacity are called chemokines. Patients with periodontitis have increased levels of chemokine ligand 2 (CCL2) and chemokine ligand 11 (CCL11) in serum. The aim of this study is to investigate whether CCL11 increases pre-osteoclast migration. Bone marrow was isolated from mouse long bones to achieve pre-osteoclasts for migration experiments. A migration plate, with membrane pore size 8-μm was used for the experiments. The cells were added on top of the membrane with the medium underneath. The cells were incubated at 37 °C, 5 % CO2 and the incubation time 5 hours. Migrated cells were fixed and stained for the osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP). Migrated cells were counted using a light microscope. The result showed that CCL11 had a statistical significant chemotactic effect on pre-osteoclasts and increase cell migration. By identification of chemokines, it might be possible to test chemokine antibodies to stop bone resorption in inflammatory bone destructive diseases as periodontitis.
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Combs, Charlotte Emma. "The role of urocortin in osteoclast physiology." Thesis, St George's, University of London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517193.
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MacQuarrie, Robyn Melanie. "Arthroplasty-derived wear particles effect osteoclast differentiation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ63539.pdf.
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Sarma, Ushasri. "Regulation of human osteoclast formation 17β estradiol." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312178.
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Berger, Christine Elizabeth Marie. "Superoxide anion in osteoclast and osteoblast function." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265210.
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Rathod, Hersha. "Osteoclast-extracellular matrix interaction and intracellular signalling." Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387474.
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Muguruma, Yukari. "The origin and differentiation of the osteoclast /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5681.
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McManus, Stephen. "Regulation of osteoclast activation and autophagy through altered protein kinase pathways in Paget's disease of bone." Thèse, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8960.
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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.
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Tan, Jamie We-Yin. "The investigation of RANKL TNF-like core domain by truncation mutation." University of Western Australia. School of Surgery and Pathology, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0032.
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Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.
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Mattsson, Jan P. "The osteoclast H⁺-ATPase isolation and initial characterization /." Göteborg, Sweden : Dept. of Biochemistry and Biophysics, Dept. of Cell Biology, University of Göteborg and Chalmers University of Technology, 1995. http://books.google.com/books?id=_85qAAAAMAAJ.
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Davey, Tamara. "Functional characterisation of a novel osteoclast-derived factor." University of Western Australia. School of Surgery and Pathology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0219.
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[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.
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Moonga, Baljit Singh. "Studies on the intracellular mechanisms of osteoclast control." Thesis, St George's, University of London, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411462.
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Morrison, Matthew Sam. "Osteoclast function : role of extracellular pH and ATP." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369218.
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Tran, Anh Nhi. "Examining the role of autophagy in osteoclast function." Thesis, University of Aberdeen, 2018. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=238273.
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Osteoclasts are cells that degrade bone, by forming a ruffled border (RB) membrane, contained within an actin-rich attachment site (the sealing zone; SZ). Lysosomal vesicles fuse to the RB, and release their contents into the extracellular space to degrade bone matrix. LC3, a marker of autophagosomes, localises to the RB, implying that either canonical autophagy (i.e. autophagosomes) or non-canonical autophagy (a process where LC3 localises to non-autophagic membrane) is involved in the resorptive function of osteoclasts. To examine this in detail, this study used a model with reduced canonical autophagy (FIP200 conditional knockout mouse), and two non-canonical autophagy deficient models (Rubicon knockdown in RAW 264.7-cell derived osteoclasts and an Atg16L1 WD40 domain knockout mouse). Using advanced imaging and molecular techniques I examined whether impairing either process affected LC3 RB localisation and resorption. Reducing canonical autophagy through FIP200 deficiency did not significantly affect GFP-LC3 RB localisation or bone homeostasis. However, impairing non-canonical autophagy resulted in a trend towards increased resorption in vitro. In Atg16L1 WD40 domain-deficient osteoclasts, this may be due to the significantly larger SZs formed in the mutants, which were often stable and contained LysoTracker-positive acidic vesicles within them, putatively signalling increased resorption. As LC3 was frequently observed at the RB, I then examined the LC3-interacting lysosomal adaptor protein, PLEKHM1. I showed that in the PLEKHM1 functional knockout mouse model (R714 STOP), osteoclasts still form RBs but have impaired resorption in vitro. Detailed analysis of multiple aspects of resorption in PLEKHM1 deficient osteoclasts, was required to uncover these defects which may underlie the osteopetrotic phenotype observed in PLEKHM1 deficient mice. Overall this work reveals the potential role of non-canonical autophagy in osteoclast function. Additional dissection of this pathway in osteoclasts may uncover further new insights regarding the regulation of bone resorption and defects underlying bone disorders.
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Moreira, Mariana Matheus. "Efeito do alendronato sódico sobre a atividade clástica na periodontite experimental em ratos." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/23/23140/tde-18092014-133646/.
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A periodontite é uma doença de natureza multifatorial e infecciosa, que resulta na inflamação e perda dos tecidos de suporte dos dentes. Essa inflamação é causada por bactérias associadas ao biofilme, causando a perda progressiva de inserção. Os bisfosfonatos são fármacos com capacidade de inibir a reabsorção óssea, atuando nas células clásticas. O presente estudo teve como objetivo investigar os efeitos do alendronato, um bisfosfonato nitrogenado com grande potência antireabsortiva, na evolução da doença periodontal induzida em ratos, bem como a possível presença de necrose óssea no processo alveolar. Foram utilizados 48 ratos Wistar albinos, do sexo masculino, com 3 meses de vida e peso médio de 250g. Os animais foram divididos aleatoriamente em dois grupos: Alendronato (ALN) e Controle (CON). A periodontite foi induzida com a inserção de um fio de seda 4.0 no sulco gengival do segundo molar superior. Os ratos do grupo ALN, receberam doses diárias de 2,5 mg/kg durante 7 dias antes e 7, 14, 21 e 30 dias após a indução da doença; o grupo CON recebeu solução salina estéril. Nos tempos citados as maxilas foram fixadas, descalcificadas e incluídas em parafina ou resina Spurr. Os cortes foram corados com HE, para análise morfológica, e histomorfométrica. Alguns cortes foram submetidos à imuno-histoquímica para detecção de RANKL e OPG. Foi utilizado o método TRAP, marcador de osteoclastos e microscopia eletrônica de transmissão para análise ultraestrutural. O ALN inibiu a reabsorção da crista alveolar de todos os grupos tratados. As células clásticas apresentaram-se em estado latente. No grupo controle a crista alveolar foi reabsorvida e o TRAP revelou clastos ativos, achados confirmados pela microscopia eletrônica de transmissão. A expressão de RANKL, molécula ativadora da célula clástica, não foi inibida pela droga. A expressão de OPG foi aumentada nos animais tratados. Os animais do grupo tratado durante 21 e 30 dias, apresentaram sinais de osteonecrose na crista alveolar, como lacunas de osteócitos vazias e regiões exposta de osso. Os resultados demonstraram que o uso de alendronato durante a doença periodontal inibe a reabsorção óssea e que durante tempos prolongados pode gerar osteonecrose na região da crista óssea.Periodontitis is an infectious disease of multifactor nature that results in the inflammation of the tissues supporting the teeth. This inflammation is caused by accumulation of biofilm and causes progressive insertion and bone loss. The bisphosphonates are drugs with the capability to inhibit the activity of clastic cells. The aim of this study was to investigate the effects of alendronate, a nitrogenated bisphosphonate with high antiresorptive power on experimental periodontal disease, and to analyze the possible presence of osteonecrosis in the rat alveolar process. Forty-eight male Wistar rats, three months old, with 250g weight were used. The animals were randomly divided into two groups: Alendronate (ALN) and Control (CON). The periodontitis was induced with a 4.0 silk wire inserted into the gingival sulcus around the right upper second molar. The ALN rats, received daily doses of 2.5 mg/kg alendronate (ALN) for 7 days before the induction of periodontitis; the treatment continued for additional 7, 14, 21 or 30 days. The CON rats, received sterile saline solution. In the time points cited, the maxillae were fixed, decalcified and embedded in Spurr resin or paraffin. The specimens were morphologically analyzed in HE stained sections, after which histomorphometry was carried out. Some stained sections were used for immunolabeling for RANKL and OPG. The osteoclasts were marker by tartrate-resistant acid phosphatase (TRAP) histochemistry. The ultrathin sections were examined in a transmission electron microscope. ALN reduced the activity of osteoclasts and significantly decreased the resorption of the alveolar crest. In the control group the alveolar crest appeared resorbed, while TRAP showed active osteoclasts, findings confirmed by transmission electron microscopy. The expression of RANKL, an osteoclast-activating molecule, was not inhibited by the drug. The expression of OPG was increased in the treated animals. The animals of the group treated for 21 and 30 days showed signs of osteonecrosis of the alveolar crest, as empty osteocyte lacunae in the exposed bone regions. The results showed that the use of ALN for periodontal disease inhibited bone resorption; when it was administered for prolonged periods it can cause osteonecrosis in the bone crest area.
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Wang, Ee Jen Wilson. "The effects of infection-related factors on bone resorption." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365291.
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Phan, Tuan (Tony). "Functional characterisation of an osteoclast-derived osteoblastic factor (ODOF)." University of Western Australia. School of Surgery and Pathology, 2004. http://theses.library.uwa.edu.au/adt-WU2005.0028.
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[Truncated abstract] Bone is a living tissue and is maintained by the coordinate action of osteoblasts and osteoclasts. The intercellular communication between these two cells is the quintessential mechanism in bone remodelling. Unfortunately, the importance of this interaction is often neglected and its significance is only realised when disruption of this “cross-talk” results in debilitating bone diseases. Additionally, the number of known proteins that are involved in this “cross-talk”, especially those that are osteoclast-derived, and act specifically on osteoblasts, is limited. This discrepancy leads to the question: Can osteoclasts directly control the growth and function of osteoblastic cells by expressing specific proteins that bind directly to osteoblasts? If so, is it possible to use these proteins to control and, possibly, treat bone disease? The objective of this thesis is to identify and characterise osteoclast-derived factors that can modulate bone homeostasis, as well as contribute to the intercellular communication between osteoblasts and osteoclasts ... Collectively, the data in this thesis culminates in one important conclusion: the identification of a novel paracrine secretory factor that has the potential to directly induce the formation of bone. These findings represent the first ever characterisation of a protein that allows the osteoclasts to directly control the growth and function of osteoblasts. Due to the potential function of ODOF to induce bone formation, this protein may be used therapeutically to treat bone disease.
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Jones, Gemma. "Optimisation and characterisation of osteoblast : osteoclast growth in biomaterials." Thesis, Keele University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505664.
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This investigation aims to utilise the cell-cell communications between osteoblasts and osteoclasts to create a functional tissue engineered construct that is closer to physiological remodelling than current single cell tissue engineered constructs. A ratio of osteoblasts:osteoclasts was optimised as well as a culture medium that supports both cell types. Four different materials, each with excellent properties for tissue engineering including biocompatible and biodegradable, were compared for their ability to support co-cultures. These materials are; silk fibroin, from Bombyx mori (water vapour and methanol stabilised), chitosan and Poly (1-lactin acid) (PLLA). Silk fibroin and chitosan were shown to support the growth and differentiation of both osteoblasts and osteoclasts in both mono and co-cultures, PLLA did not support osteoclast growth as determined by Tartrate resistant acid phosphatase stain and DNA concentration. The 2D films showed signs of degradation after 10 days culture according to differential scanning calorimetry, fourier transform infra red and gel permeation chromatography. Silk fibroin 3D sponges were manufactured to determine if the co-cultures adhere to, proliferate and differentiate in a 3D environment. Static and dynamic (rotary bioreactor) conditions were compared to determine if the bioreactor conditions enhanced the co-culture of osteoblasts and osteoclasts. The cells were shown to adhere to and proliferate on the sponges by scanning electron microscopy and DNA analysis. The sponges also showed early signs of degradation. The use of silk fibroin and a co-culture system appear to provide excellent potential for bone tissue engineering.
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O'Grádaigh, Donncha. "Osteoclast regulation in the erosive process in rheumatoid arthritis." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615638.
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Walsh, Catherine Ann. "The study of tumour stimulated osteoclast activity in vitro." Thesis, University of Liverpool, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317317.
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Luchin, Alexander I. "Regulation of Osteoclast differentiation by Microphthalmia-associated transcription factor /." The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486399451961539.
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Martin, Joanne. "In vitro osteoclast resorption of calcium phosphate bone substitutes." Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.695663.
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Resorption of calcium phosphate (CaP) biomaterials is traditionally assessed using an osteoclast (QC) resorption assay where resorption pits formed on the CaP surface are analysed by microscopy techniques and quantified on the basis of pit number, pit area or pit volume. Pit area measurements (20) have become common practice when assessing CaP biomaterial resorption in vitro. Apart from the time consuming nature of pit area analysis techniques it is not a precise indicator of resorption; variations in pit depth are not taken into consideration and it is unsuitable for use on porous materials where visualisation of internal structures is difficult or for use on materials with rough surfaces where determination of individual pits would be difficult. A 3D quantification of bioresorption is available but requires specialised, expensive equipment. An appropriate measure of resorption was required to be more efficient and more cost-effective than the current available in vitro methods, but most importantly, to directly correlate with pit area measurements and have the ability to be used in a broad range of in vitro experiments. An QC resorption assay was developed and optimised through a series of experiments. The established assay was used to assess several outcome measures as potential indicators of resorption in vitro, namely; the correlation of percentage area resorbed in vitro with Ca and P concentration in cell culture medium, QC number and QC activity. A dense substrate free from surface anomalies was used to accurately correlate pit area with the alternative outcome measures. This body of work has established two main outcome measures of bioresorption in vitro that correlate with pit area measurements; Ca and P ion concentration in culture medium and QC specific enzyme activity. These outcome measures will prove invaluable for improving the fundamental understanding of QC resorption of CaP biomaterials.
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Zhu, Min. "Regulation of osteoclast maturation and function by resolvin E1." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12701.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Innate and adaptive immunity actively interact with bone and play an important role in bone physiology and pathology. Acute inflammation is a physiologic response that represents the hosts' first line of defense. Failure to resolve the acute inflammatory lesion leads to a chronic pathologic lesion including bone destructive conditions such as periodontitis and arthritis. Resolution of inflammation is now known to be an active process with highly coordinated interaction of cells and soluble mediators leading to the return of tissues to homeostasis, rather than a passive decay of pro-inflammatory signals as previously supposed. The ω-3 polyunsaturated fatty acid derivative resolvin E1 (RvE1) is a novel lipid mediator shown to be actively involved in the resolution of inflammation. The goal of the current studies was to determine the cellular and molecular mechanism of RvE1 impact on osteoclasts. Investigation of the actions of RvE1 treatment on the specific stages of osteoclast maturation revealed that RvE1 targeted late stages of osteoclast maturation. Observations with time-lapse vital microscopy revealed that RvE1 inhibited migration and fusion of osteoclast precursors. Migration assays confirmed that chemotactic migration of osteoclast precursors was significantly inhibited by RvE1. To determine the molecular basis of RvE1 actions, fusion proteins that mediate the migration, fusion and function of osteoclasts and the essential transcription factor NFATc1 were examined. RvE1 specifically down-regulated the pivotal osteoclast fusion protein DC-STAMP (dendritic cell specific transmembrane protein) through the receptor BLT-1. RvE1 did not impact the induction of NFATc1 nor its nuclear translocation; however, NFATc1 binding to the DC-STAMP promoter was inhibited by RvE1 treatment. The results suggest that RvE1 inhibits migration and fusion of osteoclast precursors leading to decreased numbers of mature osteoclasts. On the molecular level, RvE1 binds to the cell surface receptor BL T-1 inhibiting the downstream binding of the transcription factor NFATc1 to the fusion protein DC-STAMP promoter, leading to a reduction in the number of functioning multinucleated osteoclasts and reduced bone resorption. These observations further establish a dual role for inflammation resolution in innate immunity as well as bone preservation through the direct regulation of bone cell function.
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Cheng, Tak Sum. "Molecular identification and characterization of novel osteoclast V-ATPase subunits." University of Western Australia. School of Surgery and Pathology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0068.
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[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.
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Zhao, Yuming. "Phenotypic analysis of osteoclast lineage in c-fos mutant mice." Thesis, King's College London (University of London), 2003. https://kclpure.kcl.ac.uk/portal/en/theses/phenotypic-analysis-of-osteoclast-lineage-in-cfos-mutant-mice(fafcec7f-6480-4f8c-87b6-3cca60a475fb).html.
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James, Ian Edward. "The production and characterization of human osteoclast-reactive monoclonal antibodies." Thesis, University of Bath, 1992. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303100.
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McDermott, Emma. "Characterisation of the osteoclast ruffled border using advanced imaging techniques." Thesis, University of Aberdeen, 2018. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=236980.
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The osteoclast ruffled border is a highly convoluted, complex membrane that is necessary for bone resorption. It is thought to form following mass lysosomal fusion with the boneapposing plasma membrane and vesicular trafficking is vital for its formation and function. The aim of this PhD was to better understand the ultrastructure, formation and function of the ruffled border using TEM and advanced imaging techniques. Ruffled border reformation following calcitonin treatment was visualised and the stages of ruffled border formation were described. Ruffled borders in healthy and osteopetrotic osteoclasts were also imaged by TEM and characterised using a morphological grading system. The key findings of this thesis are as follows: (1) vacuoles, not lysosomes, are the primary contributors of membrane to the ruffled border and the membrane projections of the ruffled border form passively as a consequence of channel formation, not actively by membrane folding, (2) extracellular vesicles are located, and appear to be released, at the ruffled border. Various functional aspects of the ruffled border were also investigated. Vesicles near the ruffled border were identified and characterised by immunoelectron microscopy based on their content and morphology. We found no morphological defects in ruffled borders in mice deficient in Plekhm1. In osteoclasts derived from patients with a SNX10 mutation, we found that while the cells retained the capacity to form well-developed ruffled borders, they did so less often than healthy control osteoclasts. Importantly, we observed that even in a population of healthy osteoclasts, ruffled border morphology is highly heterogeneous because they are at different stages in the resorption cycle. In conclusion, the data in this thesis provide novel findings, previously unseen details regarding how resorbing osteoclasts interact with the bone surface, and have revealed unique insights into ruffled border morphology, formation and the vesicles with which it interacts.
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Tai, Victoria. "The effects of leukotriene B¦4 on osteoclast formation and osteoclastic bone resorption and the role of osteoblastic cells in these processes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28805.pdf.
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McMichael, Brooke Kristin Trinrud. "Tropomyosin 4, myosin IIA, and myosin X enhance osteoclast function through regulation of cellular attachment structures." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view.cgi?acc%5Fnum=osu1206052974.
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Materozzi, Maria. "Molecular biology of Paget’s Disease of Bone: role of p62 and novel genes." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1104964.
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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.
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Lees, Rita L. "Osteoclast heterogeneity, the importance of cell size and phase of activity." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/NQ53821.pdf.
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Brunton, Fiona. "Targeting the osteoclast alpha v beta 3 integrin by phage display." Thesis, Queen Mary, University of London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511761.
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Al-Hadi, Hadil. "The effect of Hyperbaric Oxygen Therapy on osteoclast and osteoblast function." Thesis, University of Plymouth, 2013. http://hdl.handle.net/10026.1/1614.
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Bone remodelling, the process by which the skeleton adapts to environmental changes, is dependent on the actions of osteoclasts that resorb bone and osteoblasts which make new bone matrix. Aberrant remodelling underpins bone loss in several debilitating skeletal diseases such as osteoporosis, metastatic breast cancer and multiple myeloma. Changes in remodelling activity can also arise as a consequence of therapeutic intervention for instance intravenous bisphosphonate treatment is associated with osteochemonecrosis of the jaw and localised osteoradionecrosis is a common side effect of radiotherapy. Hyperbaric oxygen is often used as an adjunctive therapy in the treatment of these disorders. HBO involves the administration of 100% oxygen at atmospheric pressures greater than one in sealed chambers. The following studies aimed to evaluate the effect of HBO, hyperoxia, and pressure on RANKL-induced osteoclast differentiation and bone resorption from RAW264.7 and human peripheral blood mononuclear cells (PBMC), and osteoblast differentiation in vitro. The study also aimed to further examine the effect of HBO on ex vivo osteoclast formation from peripheral blood monocytes obtained from patients undergoing HBO. Daily exposure to HBO for ninety minutes significantly suppressed osteoclast differentiation and bone resorption in mouse and human monocytes in normoxic and hypoxic conditions in vitro. The suppressive action of HBO on osteoclast formation was associated with a significant reduction in HIF-1α and RANK mRNA expression and HBO also caused a significant reduction in NFATc1 and DC-STAMP expression. This study has for the first time shown that HBO is able to reduce the ability of precursors to form bone resorbing osteoclast. HBO also suppressed the ability of peripheral blood monocytes to develop into RANKL-induced resorptive osteoclasts. In an ex vivo culture system the suppressive effect of HBO was meditated by an action prior to activation of osteoclast differentiation by RANKL and must therefore be an inhibitory effect on the ability of precursors to differentiate along the osteoclastic lineage. HBO also accelerates the rate of osteoblast differentiation and augments early stages of mineralization and has a more pronounced effect than hyperoxia or pressure alone. HBO enhanced bone nodule formation and ALP activity in human osteoblasts. Furthermore HBO promoted the expression of type I collagen and Runx-2 in both normoxic and hypoxic conditions. HBO had a greater effect on these key markers of osteoblast differentiation than hyperoxia or pressure alone. This study suggests that HBO suppresses osteoclast activity and promotes osteoblastic bone formation, which may at least in part mediate its beneficial effects on necrotic bone. This provides evidence supporting the use of HBO as an adjunctive therapy to prevent osteoclast formation in a range of skeletal disorders associated with low oxygen partial pressure. The study also provides further support for the use of HBO in the treatment of skeletal disorders associated with excessive resorption such as osteomyelitis, and also provides a potential mechanism through which short term HBO may help fracture healing.
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Huber, Dustin Michael. "ANDROGENS SUPPRESS OSTEOCLAST FORMATION INDUCED BY RANK LIGAND AND M-CSF." University of Cincinnati / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ucin999020063.
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Selinger, Christina Imanta. "Identification of RANKL-Regulated Genes Involved in Osteoclast Differentiation and Resorption." Thesis, Griffith University, 2008. http://hdl.handle.net/10072/367396.
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Peripheral blood mononuclear cells (PBMCs) are pluripotent for osteoclast and macrophage cell lineages. The differentiation of macrophages and osteoclasts from a common monocyte precursor is induced following exposure to macrophage-colony stimulating factor (M-CSF), or both M-CSF and receptor activator of nuclear factor B ligand (RANKL) respectively. Differential gene expression resulting from cytokine treatment of PBMCs was examined over time using differential display PCR (DD-PCR) and quantitative real-time PCR (Q-PCR). Q-PCR analysis verified the expression of a new chemokine, FAM19A1, in addition to Special AT-rich binding sequence 1 (SATB1), solute carrier family 16 member 6 (SLC16A6) and LIM kinase 1 (LIMK1) in primary human osteoclasts, however, only LIMK1 was significantly up-regulated by RANKL.
Highly efficient delivery of small interfering RNA (siRNA) transfection to primary human osteoclasts was developed, and represents a technical milestone due to the inherent phagocytic tendencies of the PBMC lineage. The development of RNA interference for use in primary human osteoclasts was conducted using siRNA synthesised by Dicer enzyme, to verify the role of candidate genes in osteoclast differentiation and osteoclast bone resorption. Cathepsin K (CTSK) is the key proteinase expressed by osteoclasts, and was used as a benchmark for the optimisation of siRNA inhibition in primary human osteoclasts. Transfection of primary human osteoclasts with siRNA to CTSK significantly diminished bone resorption, with a 60% reduction in area resorbed (P=1.3x10-2), and a 50% reduction in pit number (P=1.8x10-2). Normal bone remodelling is dependent on both the rate of osteoclast formation and resorption. A number of genes were examined for their contribution to osteoclast formation and resorption using siRNA. Nuclear factor of activated T cells, calcineurin dependant 1 (NFATc1) inhibition was found to significantly deplete osteoclast formation (P=4.0x10-3), confirming other NFATc1 inhibition studies, and the necessity of NFATc1 in osteoclast differentiation. In pre-differentiated osteoclasts, siRNA targeting NFATc1 did not reduce osteoclast bone resorption, rather it significantly increased area resorbed (P=1.0x10-3), with no significant difference in cell number. This result suggests that NFATc1 may act in accordance with its regulator calcineurin, which has been found to enhance osteoclast differentiation, but inhibit osteoclast resorption in mature cells.
The inhibition of LIMK1 by targeted small interferring RNA (siRNA) was found to significantly diminish osteoclast formation (P=1.0x10-3), pits resorbed (P=4.2x10-2), as well as area resorbed (P=4.0x10-3). LIMK1 is a signalling kinase, identified as RANKL-regulated in murine osteoclasts, notwithstanding, this is the first study that confirms LIMK1 involvement in osteoclast formation and activity. LIMK1/cofilin-mediated actin reorganisation is critical to progenitor cell migration to stromal cells, and also regulates the stability of F-actin formation. F-actin rings were analysed in LIMK1 depleted pre-differentiated osteoclasts, which seemed to have formed properly and did not appear dissimilar from controls.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Obaid, Rami Abdulhadi Abdulmajeed. "Investigating the role of optineurin in bone biology and Paget's disease of bone." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/23419.
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Paget’s disease of bone (PDB) is a common disease with a strong genetic component. Approaches such as linkage analysis and candidate gene studies have shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial cases and 10% of sporadic cases, however the majority of PDB patients have no mutations in this gene. Genome-wide association studies (GWAS) have recently identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A, OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be associated with PDB in various European populations. OPTN encodes optineurin, a widely expressed protein involved in many cellular processes but its role in bone metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3, the OPTN rs1561570 identified by previous GWAS was examined for its association with the severity and clinical outcome of PDB in patients without SQSTM1 mutations. The results showed that rs1561570 was significantly associated with total disease severity score so that carriers of the risk allele “T” had higher severity score compared to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36) was also associated with the rs1561570 risk allele, but the relationship was not statistically significant. In order to identify functional variants within OPTN, the coding regions as well as the exon-intron boundaries were sequenced in 24 familial PDB cases and 19 controls. No mutation was found that could be predicted as pathogenic suggesting that disease susceptibility could be mediated by regulatory polymorphisms that influence gene expression. In chapter 4, the role of OPTN was investigated in osteoclast development using in vitro knockdown experiments. Optn was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all stages of osteoclast development and it was significantly increased three days post RANKL treatment. Optn expression was knocked down in BMDMs and cells were induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted cells, Optn depleted cells formed significantly more and larger osteoclasts (P< 0.05). Optn knockdown was also found to enhance osteoclast survival as well as RANKL-induced NFκB activation. In chapter 5, the role of OPTN was investigated in vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn (OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts were significantly higher and larger in mutant mice compared to WT and the NF-B activity measured by luciferase reporter assay was significantly higher in cells from OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN from mutant and WT mice was co-precipitated with its CYLD binding-partner, which acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream signalling. The data from this immunoprecipitation (IP) experiment revealed that defective OPTN interacted less with CYLD from mutant mice compared to WT. This study also showed that OPTN was expressed in osteoblasts and the expression rate did not change during osteoblast development. The data obtained from the mineralization assay revealed no significant difference between OptnD477N/D477N and WT. In chapter 6, I investigated the effect of the D477N loss of function mutation in Optn on bone metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type (WT). Osteoid analysis showed evidence of increased bone formation parameters (OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a significant difference in mineral apposition rate (MAR) from mutant mice compared to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced bone turnover in mutant mice compared to WT. Micro computed tomography (μCT) analysis of 4 and 14 months old mice showed no significant differences in bone morphology between WT and OptnD477N/D477N mice of both sexes. In conclusion, this study has shown for the first time that OPTN plays a role in regulating bone turnover by acting as a negative regulator of osteoclast differentiation. The data obtained from this study strongly suggest the crucial role of OPTN in RANK signalling. The effect of OPTN on osteoblast activity may be direct or indirect compensation for increased osteoclast activity. Further detailed studies will be required to explore the underlying mechanism of OPTN including downstream RANK signalling and a complete knockout model to corroborate these findings.
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Rezende, Eloiza de. "Estudo do efeito de bisfosfonatos nas células clásticas durante a ossificação endocondral do joelho de ratos e em cultura primária: abordagens morfológicas e moleculares." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-29052014-151314/.
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Na ossificação endocondral, osteoclastos (Oc) reabsorvem os remanescentes de cartilagem, e osteoblastos (Ob) depositam matriz óssea. Bisfosfonatos (Bps) inibem a ação dos Oc. Foi avaliado o efeito dos Bps alendronato (Aln) e etidronato (Etn) em joelhos de ratos jovens (in vivo) e na cultura primária de Oc (in vitro). O material in vivo foi analisado por MEV, MET e ML (morfologia e histoquímica para TRAP). RNA foi extraído para análise por RT-PRC e proteínas para análise por WB, que também foram extraídos após o tratamento da cultura com Bps. O tratamento com Etn revelou lâmina epifiseal desorganizada com extensa área de cartilagem; a MEV mostrou pouco osso trabecular com lacunas de reabsorção, que não foram observadas com Aln. O Aln revelou numerosos Oc TRAP-positivos latentes, confirmados por MET. In vivo os Bps diminuem a expressão dos genes analisados; In vitro o Aln diminui somente a expressão de Runx2, menos expresso com Etn, assim como Spp1. A expressão proteica variou entre os grupos. Aln é o mais potente em inibir os Oc enquanto o Etn atua sobre os Ob.In endochondral ossification, clastic cells (Oc) resorb the calcified cartilage, while osteoblasts (Ob) form new bone. Bisphosphonates (Bps) inhibit the action of Oc. The effect of the Bps alendronate (Aln) and etidronate (Etn) on the knees of young rats (in vivo) and in primary cultures of Oc (in vitro) was evaluated. The specimens were analyzed by SEM, TEM, and LM or TRAP histochemistry. RNA was extracted to analysis by RT-PRC and protein to analysis by WB. RNA and protein were also extracted after the treatment of cultures with Bps. Rats treated with Etn exhibited a disorganized epiphyseal plate containing large area of cartilage; SEM showed few bone trabeculae with resorption lacunae, which were not observed in Aln specimens. Aln showed numerous latent Oc by TRAP histochemistry and TEM. In vivo, the Bps decreased the expression of all analyzed genes; in vitro, Aln decreased only the expression of Runx2 as well as SPP1, which expression was less with Etn. Protein expression varied among the groups. Aln is more potent for inhibiting the Oc, while Etn acts on Ob.
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Marques, Natasha D'Andrea Mateus. "Estudo da expressão das moléculas reguladoras da remodelação do osso alveolar durante a movimentação ortodôntica com força contínua em ratos tratados com alendronato sódico." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/23/23140/tde-25022016-165617/.
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A movimentação dentária ortodôntica ocorre através de dois processos, nos quais o osso alveolar é reabsorvido nas áreas de pressão, enquanto que novo osso é formado na área de tração. O processo de reabsorção óssea ocorre pela ação de células multinucleadas, os osteoclastos. Os bisfosfonatos constituem um grupo de fármacos com propriedade de inibir a reabsorção óssea, foi utilizado no presente estudo com a finalidade de interferir na remodelação óssea induzida ortodonticamente. Para isso, força contínua de 15 cN foi aplicada aos primeiros molares superiores de ratos machos Wistar de 2 1/2 meses, utilizando uma biomecânica com fios superelásticos. Os animais foram divididos aleatoriamente em 4 grupos: 1) O grupo controle constituído por dezoito ratos, os quais foram injetados solução salina por 7 dias antes da instalação da biomecânica passiva, que permaneceu por 3, 10 e 18 dias; 2) Dezoito animais foram tratados com ALN (dose 2,5 mg/Kg) por 7 dias antes da instalação da biomecânica passiva que permaneceu por 3, 10 e 18 dias; 3) Dezoito animais foram tratados com alendronato com a mesma dose citada acima por 7 dias antes da instalação da biomecânica ativa que permaneceu por 3, 10 e 18 dias; 4) Dezoito animais foram injetados com solução salina 7 dias antes da instalação da biomecânica ativa que permaneceu por 3, 10 e 18 dias. As maxilas foram fixadas com 4% de formaldeído + 0,1% de glutaraldeído, descalcificadas em EDTA a 4,13% e incluídas em parafina ou resina Spurr. Os cortes foram corados com HE para análise morfológica. Alguns cortes foram submetidos à imuno-histoquímica para detecção de RANKL e OPG. Foi utilizado o método TRAP, marcador de osteoclastos e microscopia eletrônica de transmissão para análise ultraestrutural. Alguns espécimes tiveram a cortical óssea vestibular do primeiro molar superior congelada em nitrogênio líquido para análise da expressão de RANKL por Western Blotting. O ALN inibiu a reabsorção óssea e radicular de todos os grupos tratados. As células clásticas apresentaram-se em estado latente. No grupo da movimentação ortodôntica o osso alveolar foi remodelado e com 18 dias a superfície radicular apresentou-se reabsorvida e o TRAP revelou clastos ativos, achados confirmados pela microscopia eletrônica de transmissão. A expressão de RANKL, molécula ativadora de células clásticas, nao foi inibida pela droga. A expressão de OPG foi aumentada nos animais tratados. Os resultados demonstram que o uso de alendronato sódico na movimentação ortodôntica não interfere no recrutamento dos osteoclasto, ele aparentemente inibe sua ativação, o que pode interferir no processo de remodelação óssea e talvez diminua a quantidade de movimentação dentária.Orthodontic tooth movement occurs through two processes in which the alveolar bone is resorbed in the pressure areas, whereas new bone is formed in the tension area. The bone resorption occurs by multinucleated cell, the osteoclasts. The bisphosphonates are drugs with capability to inhibit clastic activity were used in the present study in order to interfere with the bone remodeling induced orthodontic. For this continuous force of 15 cN was applied to the first molars of Wistar male rats of 2 1/2 months, using a biomechanical with superelastic wire. The animals were randomly divided into 4 groups: 1) The control group consisted of eighteen mice, which received sterile saline solution saline for 7 days prior to installation of passive biomechanics, which remained for 3, 10 and 18 days; 2) Eighteen animals were treated with ALN (dose 2.5 mg / kg) for 7 days prior to installation of the passive biomechanical to remain for 3, 10 and 18 days; 3) Eighteen animals were treated with alendronate with the same dose quoted above for 7 days prior to the biomechanical installation that remains active for 3, 10 and 18 days; 4) Eighteen animals were injected with sterile saline solution 7 days prior to the biomechanical installation that remains active for 3, 10 and 18 days. The maxillae were fixed with 4% formaldehyde + 0.1% glutaraldehyde, decalcified in EDTA 4.13% and embedded in paraffin or Spurr resin. The specimens were morphologically analyzed in HE stained sections. Some stained sections were used for immunolabeling for RANKL and OPG. The osteoclasts were marked by tartrate-resistant acid phosphatase (TRAP) histochemistry. The ultrathin sections were examined in a trasnmission electron micrsocpe. Some specimens were frozen in liquid nitrogen for protein extraction and Western Blotting protein expression analyzes. The ALN inhibited bone resorption and root of all the treated groups. The clastic cells present in a latent state. In the orthodontic movement group alveolar bone was remodeled with 18 days to root surface presented itself reabsorbed and the TRAP revealed clasts assets, findings confirmed by transmission electron microscopy. Expression of RANKL activating molecule clastic cells was not inhibited by the drug. The OPG expression was increased in treated animals. The results demonstrate that the use of alendronate in the orthodontic movement does not interfere with osteoclast recruitment, it apparently inhibits their activation, which can interfere in the bone remodeling process and may reduce the amount of tooth movement.
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Hu, Rong. "Regulation of osteoclast differentiation by transcription factors MITF, PU.1 and EOS." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1166644761.
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Dossa, Tanya. "Osteoclast-specific inactivation of the Integrin-Linked Kinase (ILK) inhibits bone resorption." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=99336.
Full textAbstract:
Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the alphavbeta 3 integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the Integrin-Linked Kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast-specific ILK gene ablation. Mice with one inactivated ILK allele (ILK+/-) were mated with TRAP-Cre transgenic mice. Progeny from this cross (TRAP-Cre;ILK+/-) was bred to mice homozygous for a floxed ILK allele (ILKfl/fl) to yield mutant mice with ILK-deficient osteoclasts (TRAP-Cre;ILK+/fl ). The mutant animals thus had one ILK allele inactivated in all tissues, and both alleles disrupted in osteoclasts. Mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclastogenesis, assessed by TRAP staining of bone sections or in vitro cultures, was not affected. Indeed, osteoclast-specific ILK ablation was associated with an increase in osteoclast number both in vitro and in vivo. Primary cultures of osteoclasts were generated on synthetic calcium phosphate discs, as well as dentin, and the mutant cells displayed a decrease in resorption activity. We also measured decreased serum concentrations of the C-terminal telopeptide of collagen, a marker of osteoclastic activity, in mice with ILK-deficient osteoclasts. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts. The characterization of the molecular mechanisms responsible for the observed phenotype will identify novel pathways regulating bone resorption.
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Mellis, David. "The study of RANK mutations associated with the diseases of osteoclast dysfunction." Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=166647.
Full textAbstract:
Osteoclasts are the cells that resorb bone to maintain a healthy skeleton. Receptor activator of NFkB (RANK) is a receptor that is critical for the formation, activity and survival of osteoclasts. A number of mutations have been identified within RANK that cause bone diseases with opposite osteoclast phenotypes. The aim of this thesis was to study the downstream consequences of these disease-associated mutations for RANK protein processing and activation of the RANK signalling pathway. Early onset Paget’s disease of bone (ePDB), Familial Expansile Osteolysis (FEO) and Expansile Skeletal Hyperphosphatasia (ESH) are conditions featuring focal areas of increased bone resorption driven by overactive osteoclasts. These conditions are caused by heterozygous insertion mutations in the signal peptide region of the RANK gene, but the mechanisms underlying the development of overactive osteoclasts are not known. In this thesis, in vitro study of the mutant RANK proteins demonstrated that homozygous overexpression caused inactivation of RANK signalling due to intracellular accumulation of RANK within an extended form of the endoplasmic reticulum. By contrast, when expressed in a heterozygous manner, the mutant proteins were found at the plasma membrane and caused prolonged ligand-dependent signalling. Taken together and as predicted by the clinical situation, these data strongly suggest that heterozygous expression of the mutant RANK proteins hold the key to the hyperactive osteoclast phenotype associated with these diseases. Osteoclast-poor osteopetrosis is a disease in which osteoclasts do not form leading to a high bone mass phenotype. Single base pair mutations within RANK have been identified in some patients with this condition. These mutant RANK proteins were studied and the findings related to regions within RANK in which the mutations occur that have been shown to be critical for its function. In addition, osteoclast formation was assessed in cultures of peripheral blood mononuclear cells isolated from patients with osteopetrosis with unidentified genetic background in order to further characterise the osteoclast phenotype for each patient. In summary, the findings presented in this thesis begin to elucidate the molecular mechanisms leading to diseases of bone with opposite osteoclast phenotypes that, paradoxically, are all caused by inactivating mutations in the RANK gene.
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Wani, Mohan Ramchandra. "Regulation of osteoclast formation and activation by TRANCE and prostaglandin Eâ†2." Thesis, St George's, University of London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341938.
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Althnaian, Thnaian Ali. "Factors that regulate osteoclast formation and bone resorption in regenerating deer antlers." Thesis, Royal Veterinary College (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439832.
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