Dissertations / Theses on the topic 'Bone inhibition'

The most common cancer types have a high likelihood of metastasizing to the bone and can cause cancer-induced bone pain (CIBP). Current therapeutic options do not offer proper management and thus CIBP can severely affect a patient's quality of life. Dysregulation of the excitatory neurotransmitter, glutamate, may be involved in the complex and multifaceted mechanisms of CIBP. Because glutamatergic signaling promotes pain, a local rise in glutamate in the bone-tumor microenvironment may contribute to CIBP. Glutamate levels are regulated in part by the cystine/glutamate antiporter, system xc⁻. System xc⁻ is known to be expressed by many different cancer cell types. It functions by transporting cystine into cells and in return releasing glutamate into the extracellular space. Elevated glutamate levels driven by the upregulated expression of this antiporter may contribute to CIBP. Here we demonstrate that system xc⁻ is expressed on a spontaneously occurring murine mammary tumor cell line (66.1) and that treatment of these cells with the established inhibitor and anti-inflammatory agent, sulfasalazine, decreases glutamate secretion in a time and dose-dependent manner. Furthermore, in a novel model of breast CIBP, systemic sulfasalazine treatment not only reduces glutamate levels within the femur, but also significantly attenuates CIBP behaviors. Studies utilized 66.1 cells implanted into the femur intramedullary space of immunocompetent mice. Measurements of spontaneous and evoked pain were made 7 and 10 days post cancer cell inoculation. Systemic administration of sulfasalazine for 4 days (on days 7-10) significantly reduced spontaneous pain-related behaviors and glutamate in femur extrudate as compared to vehicle treated controls. In summary, we demonstrate that pharmacological inhibition of the system xc⁻ transporter attenuates CIBP related behaviors in mice. These data support a role for system xc⁻ in CIBP and validate it as an analgesic target. Further research is warranted to evaluate the potential repurposing of sulfasalazine as an antinociceptive agent for patients with CIBP.

The extensive infiltration of immune cells in the joints of patients affected by rheumatoid arthritis (RA), and the subsequent production of pro-inflammatory cytokines triggers bone erosion through the extensive stimulation of bone resorbing osteoclasts (OCs). The activity of γδ T cells has been implicated to influence the onset and severity of the disease pathology in murine models of human RA. With this study the effects of γδ T cells for influencing OC differentiation and resorptive activity were assessed in vitro. Activated γδ T cells exerted inhibitory effects on OC differentiation and resorptive activity, these effects were mediated by the release of soluble factors, since similar inhibitory effects were obtained using conditioned medium (CM) from activated γδ T cells. The primary mediator of such effects was determined to be IFN, since neutralisation markedly restored OC differentiation and resorptive activity. γδ T cell proliferation, activation and survival following culture with autologous mature OCs were assessed by flow cytometry. Interestingly, OCs and OC-derived CM induced activation of γδ T cells as determined by the expression of the early activation marker CD69. A mediator of this stimulatory effect on T cells was found to be TNF, since neutralisation of TNFα decreased the stimulatory effect of OCs on CD69 expression. Consistently, OCs, but not OC-derived CM, increased the proliferation of IL-2-stimulated γδ T cells and also supported the survival of resting γδ T cells. This study provides new insights into the in vitro interactions between human γδ T cells and OCs, moreover it defines osteoclasts as immune competent cells capable of influencing the activation status and the viability of T lymphocytes, and provide evidence for a novel stimulatory effect of OCs on γδ T cells.

Clinical observation suggest that bone formation is influenced by the environmental niche where it takes place and specifically that soft connective tissues may inhibit the bone healing process. The aim of the studies described in this thesis is to test the hypothesis that fibroblasts inhibit the differentiation and function of osteoblasts in vitro. To address this aim the ability of fibroblasts and their supernatants to inhibit osteoblast differentiation was investigated. In addition, the inhibitory effects of gingival and periodontal ligament fibroblasts were compared. Next Bone Morphogenetic Proteins (BMPs) and their antagonists were tested for their ability to modulate the activity of fibroblast supernatants. Finally the effects of fibroblast supernatants on osteoblast chemotactic responses were investigated. Primary fibroblasts were isolated from rat gingivae, skin, oral mucosa and periodontal ligament (PDL) using explant cultures. Primary osteoblast cultures were established by enzymatic digestion of neonatal rat calvariae. In other experiments the osteoblastic cell line ROS 17/2.8 was used. Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP). In co-culture experiments using 3-D collagen gels and diffusion chamber inserts fibroblasts strongly inhibited osteoblast differentiation. Furthermore conditioned medium from superficial connective tissues fibroblasts consistently inhibited osteoblast differentiation (greater than 50% inhibition of ALP expression. In contrast, PDL cells strongly stimulated ALP expression (greater than 100% increase). Stimulation of ROS 17/2.8 cells with BMP-2 increased ALP expression (more than 3 fold increase with 10ng/ml BMP-2), and this effect could be completely blocked by the BMP-antagonist 100ng/ml noggin. Similarly conditioned media from gingival, oral mucosal and skin fibroblasts totally suppressed the effects of BMP-2. In contrast, PDL conditioned media stimulated ALP expression in additively with BMP-2, and his effect could also be blocked by noggin. Using a micro-well Boyden Chamber both PDGF and BMP-2 caused a dose-dependent increase in chemotaxis. However fibroblast conditioned medium totally blocked the chemotactic effects on BMP-2, but had no effect on PDGF-induced chemotaxis. Overall these studies demonstrate that fibroblasts from superficial connective tissues (gingival, oral mucosa and skin) can inhibit osteoblast function by secretion of BMPantagonists and that superficial connective tissues and PDL are distinct in respect to their role in bone healing. Further studies are needed to identify the specific molecular identity of this inhibitory activity and to extend these observations to an in vitro model. However in the longer term it is proposed that information on the regional expression of BMP inhibitors may lead to novel therapeutic interventions to promote bone growth in periodontal and implant related bone regeneration procedures.

BACKGROUND:Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.RESULTS:In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.CONCLUSIONS:Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.

Background: Postmenopausal osteoporosis which results in a reduction of bone quality and bone density is one of the most prevalent diseases affecting people around the world. Cathepsin K (CatK) is one of the most potent proteases in lysosomal cysteine proteases family, of which main function is to mediate bone resorption. Currently, the Odanacatib (ODN) developed by Merck & Co. is the only Phase III CatK inhibitor candidate with high efficacy in treating postmenopausal osteoporosis. Unfortunately, the development of ODN was finally terminated due to the cardio-cerebrovascular adverse effects. In order to enhance the specificity of ODN to osteoclasts for suppression of bone resorption in postmenopausal osteoporosis, we have previously designed and synthesized (D-Asp8)-ODN conjugate by linking ODN with a promising osteoclast-targeted moiety D-Asp8. The data showed that D-Asp8 could facilitate the conjugated ODN specifically approaching osteoclasts, with reduced distribution in non-bone tissues, to inhibit the functional CatK activity within bone tissues in healthy rats. In this thesis, we hypothesized that the in vitro antiresorptive effects of (D-Asp8)-ODN conjugate were comparable with that of ODN. On the other hand, we also developed a QQQ-LC/MS method for quantitation of (D-Asp8)-ODN conjugate in plasma, which will be a valuable tool to support further pre-clinical studies. Aim: (1) To compare the antiresorptive effect between (D-Asp8)-ODN conjugate and ODN in vitro. (2) To develop and validate a practicable method for pharmacokinetic profile of (D-Asp8)-ODN conjugate in rats. Materials and Methods: The cytotoxic effect of (D-Asp8)-ODN conjugate and ODN were evaluated and compared by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of (D-Asp8)-ODN conjugate and ODN on Receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclasts formation and osteoclast function-related genes were evaluated and compared by Tartrate-resistant acid phosphatase (TRAP) staining and quantitative real time polymerase chain reaction (qRT-PCR). The effect of (D-Asp8)-ODN conjugate and ODN on osteoclast bone resorption activities were evaluated and compared by bone resorption pit assay. Moreover, the pharmacokinetic profile of (D-Asp8)-ODN conjugate in rat plasma was determined by using triple quadrupole liquid chromatography-mass spectrometry (QQQ-LC/MS) system. Result: The cytotoxicity of (D-Asp8)-ODN conjugate was significantly lower than that of ODN on the murine macrophage RAW 264.7 cell line. (D-Asp8)-ODN conjugate had no effect on RANKL-induced osteoclast formation, which was comparable with that of ODN. (D-Asp8)-ODN conjugate had no effect on the mRNA level of CTSK, but it could upregulate the mRNA levels of ACP5 and OSCAR, which was comparable with that of ODN. (D-Asp8)-ODN conjugate inhibited osteoclast bone resorption activity, which was comparable with that of ODN. The newly established QQQ-LC/MS protocol had good precision and accuracy for detecting (D-Asp8)-ODN conjugate in rat plasma. Finally, the pharmacokinetic profile of (D-Asp8)-ODN conjugate in rat plasma was determined. Following subcutaneous administration, the time to reach maximum concentration (Tmax) was 1.0 h, the antibiotics area under the concentration time-curves from time zero to infinity (AUC0-∞) was found to be 27.78 ug·mL-1·h and the terminal half-life (t½) was 1.4 h. Conclusion: (D-Asp8)-ODN conjugate had no effect on RANKL-induced osteoclast formation, which was comparable with ODN. The antiresorptive effect of (D-Asp8)-ODN conjugate was comparable with that of ODN. On the other hand, a new QQQ-LC/MS protocol has been established for the pharmacokinetic profile of (D-Asp8)-ODN conjugate in rat.

Distraction osteogenesis (DO) is a surgical technique widely used for the treatment of limb length discrepancies, limb deformities, long bone nonunions, and bone loss. The technique involves performing an osteotomy and then gradually distracting the two bone segments with an external fixator. This generates new bone within the distracted gap. Although very successful, one of the limitations of this technique is the long period of time the external fixator needs to be kept on, until the newly formed bone in the distracted zone consolidates. This prolonged process may lead to numerous social, psychological, and medical complications. Recent studies from our laboratory showed that the absence of signalling through the fibroblast growth factor receptor 3 (FGFR3) in DO leads to an increase in bone formation in mice deficient for FGFR3. Thus, we hypothesize that exogenous blocking of the FGFR3 pathway in wild-type mice may be efficacious in promoting bone formation in DO. We thus planned to block this pathway using the small molecule inhibitor, PD173074 or using anti-FGFR3 antibodies. In this study, we used 2-month old wild-type C57BL/6 mice divided in 5 groups. The mice have undergone a surgical osteotomy and installation of an external fixator (distraction apparatus). Following a 5-day latency period, distraction (0.2 mm/12 hours for 12 days) was initiated. The animals were sacrificed at day 33 post-surgery (mid consolidation). Micro-computed tomography (μCT) of the wild-type control group tibiae (not treated) was compared to the tibiae of mice receiving one of three increasing doses of small molecule inhibitors and mice receiving a blocking dose of anti-FGFR3 antibodies. Our results showed a trend towards increased bone volume in mice receiving the higher doses of the small molecule inhibitor as compared to those of the untreated wild-type mice. This dose ranging experiment represents a critical study in this translational research effort to accelerate bone formation in DO.
L'ostéogénèse par distraction (Distraction Osteogenesis, DO) est une technique chirurgicale largement utilisée pour le traitement des anomalies de la longueur des membres, des malformations, des fractures non-union, et de la perte osseuse suivant un traumatisme. La technique consiste à effectuer une ostéotomie puis à éloigner progressivement les deux segments osseux à l'aide d'un fixateur externe. Du nouveau tissu osseux se forme progressivement dans l'espace créé par la traction. Bien que montrant un taux de succès élevé, un des désavantages de cette technique est la longue période de temps où le fixateur externe doit être maintenu en place afin que l'os nouvellement formé se consolide. Cette période prolongée peut entraîner de nombreuses complications d'ordre social, psychologique ou médical. Des résultats récents de notre laboratoire montrent que l'absence de signalisation par le récepteur 3 des facteurs de croissance fibroblastiques (Fibroblast Growth Factor Receptor 3, FGFR3) au cours de la DO conduit à une augmentation de la formation osseuse chez les souris déficientes en FGFR3. Nous avons donc émis l'hypothèse que le blocage de la voie signalétique FGFR3 chez des souris de type sauvage pourrait être efficace afin d'augmenter la formation osseuse au cours de la DO. Afin de bloquer cette voie signalétique, nous avons utilisé un inhibiteur synthétique, PD173074, ou des anticorps anti-FGFR3. Des souris C57Bl/6 de type sauvage agée de 2 mois et divisée en 5 groupes. Les souris ont subi une ostéotomiechirurgicale et l'installation du fixateur externe (appareil de distraction). Après une période de récupération de 5 jours, la distraction a été amorcée (0,2 mm/12 heures pendant 12 jours). Les animaux ont été sacrifiés au jour 33 post-chirurgie (mi-consolidation). À l'aide de la tomographie haute résolution assistée par ordinateur (micro-computedtomography, μCT), nous avons comparé la formation osseuse dans l'espace créé par la traction entre le groupe contrôle (recevant seulement le véhicule) et les groupes traités avec une des trois doses croissantes d'inhibiteur ou avec l'anticorps anti-FGFR3. Nos résultats montrent une tendance dose-dépendante à l'augmentation de la formation osseuse chez les souris recevant l'inhibiteur, par rapport aux souris non traitées. Cette étude pilote est un jalon important dans la recherche translationnellevisant à accélérer la formation osseuse au cours de l'ostéogénèse par distraction.

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.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Periodontitis is an immunoinflammatory disease in that the involvement of chemical mediators culminates in destruction of alveolar bone. Long recognized regarding its pathogenesis, however, frequently some patients do not respond insatisfactorily to conventional treatments, which makes pharmacological alternatives are sought. In this context, Matricaria recutita (MTR), known as chamomile, stands out in the literature for its anti-inflammatory and a variety of constituents, especially apigenin (APG) flavonoid. Thus, the present study evaluated the involvement of cytokines in the anti-inflammatory and antiresorptive activities of MTR in alveolar bone resorption (ABR) induced by ligature in rats. For this, we used the dry extract of MTR (apigenin content 128.5Â0.99 mg/g). The ABR was induced in 90 wistar rats (199.3 Â 3.2 g) by ligation (nylon 3.0) of 2Â upper left molar, and contralateral was used as control. The rats received v.o. Tween 80 (TW) or MTR (10, 30 and 90 mg/kg) daily until 11 d, when they were killed. The hemiarcadas were processed for macroscopic (mm2) or histometric, histological and immunohistochemical analyzes for the ligand of the receptor activator of nuclear factor kappa B (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP). Blood samples were collected for measurement of bone alkaline phosphatase (BALP), while the gingival tissue was used for measuring of mieloperoxidase activity (MPO; mg/g) and of tumor necrosis factor-alpha (TNF-a) and interleukin-1β (IL-1β) levels (pg/mg) by ELISA. Systemically, serum bone alkaline phosphatase (BALP), AST/ALT, urea and creatinine, and white blood count were made, and we evaluated of macroscopic aspects of liver, kidneys and spleen, in addition to variation in body mass. Was set at p <0.05 (#) for Normal, (*) for TW and () for MTR 10 mg/kg; Ethical aspects: the Ethics Committee for Animal Use-UFC 70/13. It was found that ligation for 11 days caused intense ABR with furcation lesion pronounced, resorption of alveolar bone and cementum in the region between the first and second molars, reduction of serum BALP, intense leukocyte infiltrate in the periodontium these animals, increasing significant MPO, TNF-, IL-1β in challenged area underlying gingival tissue, and increased to RANKL and TRAP immunostaining, and reduced to OPG. Systemically, there was leukocytosis with a predominance of mononuclear cells. No major changes in organs and weight of animals were observed. MTR prevented, significantly, the ligature-induced ABR [TW=5.5Â0.2; MTR (10)=4.4Â0.1*; (30)=2.9Â0.1*; (90)=2.8Â0*], corroborating the reduction of furcation lesions [Normal=10.4Â0.8; TW=137.4Â23.3#; MTR (90)=81.0Â9.6*#] and the preservation of the alveolar bone and cementum [(Normal=0(0-0); TW=3(1-3)#, MTR (90)=1(1-3)#*] compared to the TW group while no bone anabolic activity was showed because MTR dit not prevent the reduction of serum BALP induced by ligature [Normal=99.4Â3.4; TW=61.3Â2.6#; MTR (10)=70.6Â3.6#; (30)=74.5Â3.7#; (90)=78.5Â2.8#); p>0.05]. However, MTR significantly prevented the leukocyte infiltration and the increase of MPO activity [Normal=3.6Â0.5; TW=9.4Â0.9#; MTR (10)=10.2Â3.3; (30)=4.5Â0.8*; (90)=4.2Â0.7*], of TNF-a [Normal=0.2Â0; TW=1.2Â0.2#; MTR (10)=0.4Â0.2*; (30)=0.2Â0.1*; (90)=0.1Â0*] and of IL-1β [Normal=1.5Â0.3; TW=8.0Â1.4#; MTR (10)=8.9Â1.9#; (30)=1.8Â1.0*; (90)=1.5Â0.9*] levels caused by ligature, and reduced immunostaining for RANKL and TRAP, and increased for OPG, comparing to TW group. Additionally, MTR prevented the leukocytosis caused by ligation and did not alter liver, kidney, spleen conditions or the variation of body mass. In short, the MTR prevented the ABR by reducing TNF-a and IL-1β, thus preventing the osteoclast activation due RANK-RANKL-OPG axis, without interfering with bone anabolism.
A periodontite à uma doenÃa imunoinflamatÃria onde a participaÃÃo de mediadores quÃmicos culmina em destruiÃÃo de osso alveolar. Muito se reconhece a respeito de sua patogÃnese, contudo, frequentemente alguns pacientes respondem de forma insatisfatÃria aos tratamentos convencionais, o que faz com que alternativas farmacolÃgicas sejam buscadas. Neste contexto, a Matricaria recutita (MTR), conhecida como camomila, destaca-se na literatura por sua propriedade anti-inflamatÃria e sua variedade de constituintes, especialmente o flavonoide apigenina. Assim, o presente estudo avaliou a participaÃÃo de citocinas nas atividades anti-inflamatÃria e antirreabsortiva da MTR na reabsorÃÃo Ãssea alveolar (POA) induzida por ligadura em ratos. Para isso, utilizou-se extrato seco de MTR (teor de apigenina de 128,5Â0,99 mg/g). A POA foi induzida em 90 ratos Wistar (199,3Â3,2 g) por ligadura (nÃilon 3.0) do 2 molar superior esquerdo, e contralateral como controle. Os ratos receberam v.o. Tween 80 (TW) ou MTR (10, 30 e 90 mg/kg) diariamente atà o 11 d, quando foram mortos. As hemiarcadas foram processadas para macroscopia (mm2) ou para anÃlises histomÃtrica, histolÃgica e imunohistoquÃmica para o ligante do receptor ativador do fator nuclear kappa-B (RANKL), a osteoprotegerina (OPG) e a fosfatase Ãcida resistente ao tartarato (TRAP). Amostras de sangue foram coletadas para dosagem de fosfatase alcalina Ãssea (FAO), enquanto que o tecido gengival foi utilizado para a dosagem da atividade de mieloperoxidase (MPO; mg/g), do fator de necrose tumoral-alfa (TNF-a) e da interleucina-1β (IL-1β) (pg/mg) por ELISA. Sistemicamente, foram feitas dosagens sÃricas AST/ALT, ureia e creatinina, avaliados leucograma, os aspectos macroscÃpicos de fÃgado, rins e baÃo, alÃm da variaÃÃo de massa corpÃrea. Considerou-se p<0,05 (#) para Normais, (*) para TW e () para MTR 10 mg/kg; Aspectos Ãticos: ComissÃo de Ãtica para Uso de Animais-UFC n 70/13. Verificou-se que a ligadura durante 11 dias causou intensa POA, com lesÃo pronunciada de furca e reabsorÃÃo de osso alveolar e cemento na regiÃo entre o primeiro e segundo molares, reduÃÃo dos nÃveis sÃricos de FAO, intenso infiltrado leucocitÃrio no periodonto destes animais, aumento significante de MPO, de TNF-a, de IL-1β no tecido gengival subjacente à Ãrea desafiada, alÃm de imunomarcaÃÃo aumentada para RANKL e TRAP, e reduzida para OPG. Sistemicamente, observou-se leucocitose com predomÃnio de mononucleares. NÃo foram observadas alteraÃÃes importantes de ÃrgÃos e peso dos animais. MTR foi capaz de prevenir, de forma significante, a POA induzida por ligadura [TW=5,5Â0,2; MTR (10)=4,4Â0,1*; (30)=2,9Â0,1*; (90)=2,8Â0*], sendo corroborada pela reduÃÃo das lesÃes de furca [Normal=10,4Â0,8; TW=137,4Â23,3#; MTR (90)=81,0Â9,6#*] e preservaÃÃo de osso alveolar e cemento [(Normal=0(0-0); TW=3(1-3)#; MTR (90)=1(1-3)#*] em comparaÃÃo ao grupo TW, embora nÃo demonstrou atividade anabÃlica Ãssea por nÃo prevenir a reduÃÃo dos nÃveis sÃricos de FAO induzida pela ligadura [Normal=99,4Â3,4; TW=61,3Â2,6#; MTR (10)=70,6Â3,6#; (30)=74,5Â3,7#; (90)=78,5Â2,8#); p>0,05]. Entretanto, MTR preveniu significantemente o infiltrado leucocitÃrio e o aumento da atividade de MPO [Normal=3,6Â0,5; TW=9,4Â0,9#; MTR (10)=10,2Â3,3; (30)=4,5Â0,8*; (90)=4,2Â0,7*], dos nÃveis de TNF-a [Normal=0,2Â0; TW=1,2Â0,2#; MTR (10)=0,4Â0,2*; (30)=0,2Â0,1*; (90)=0,1Â0*] e de IL-1β [Normal=1,5Â0,3; TW=8,0Â1,4#; MTR (10)=8,9Â1,9#; (30)=1,8Â1,0*; (90)=1,5Â0,9*], proporcionando imunomarcaÃÃo reduzida para RANKL e TRAP, e aumentada para OPG, em comparaÃÃo ao grupo TW. Adicionalmente, a MTR preveniu a leucocitose causada pela ligadura e nÃo alterou as condiÃÃes hepÃticas, renais, esplÃnicas e a variaÃÃo de massa corpÃrea. Em suma, a MTR preveniu a POA via reduÃÃo de TNF-a e IL-1β, prevenindo, assim, a ativaÃÃo osteoclÃstica decorrente do eixo RANK-RANKL-OPG, sem interferir no anabolismo Ãsseo.

Skeletal development is a complex event that requires a delicate balance between bone formation and bone resorption. Multiple transcription factors expressed in the bone-forming cells, osteoblasts, play crucial roles during the process of bone formation. Among them, ATF4 (Activating Transcription Factor 4) is a basic domain-leucine zipper transcriptional activator that is responsible for osteoblast differentiation, osteoblast-specific genes expression, synthesis of type I collagen, and osteoclast differentiation. Mice deficient for ATF4 are runted and exhibit severe skeletal dysplasia. Our laboratory has discovered Factor Inhibiting ATF4-mediated Transcription (FIAT), whose name was coined for its interaction with ATF4 and subsequent repression of ATF4-mediated osteocalcin gene transcription. FIAT is a leucine zipper nuclear molecule lacking a basic domain for DNA binding. We hypothesize that FIAT suppresses the bone-forming activities of osteoblasts by interacting with ATF4 and thereby blocking ATF4 attachment to the DNA to mediate downstream signalling pathways. To prove this hypothesis, we monitored the expression profiles of FIAT in parallel with ATF4 during osteoblastogenesis. Mechanism of FIAT repression of ATF4 was investigated through structure-function and mutation analysis. The physiological significance of FIAT expression in osteoblasts was studied through silencing FIAT in osteoblasts by RNA interference, as well as through characterization of two genetic mouse models: FIAT transgenic mice which overexpress FIAT in osteoblasts, and osteoblast-specific FIAT knockout mice. These studies showed that FIAT and ATF4 are co-expressed in osteoblasts, and that FIAT inhibition of matrix mineralization requires dimerization with ATF4 through the second leucine zipper. Furthermore, transgenic mice overexpressing FIAT exhibited osteopenia whereas FIAT knockout mice showed enhanced bone formation. These results support our hypothesis and demonstrate that FIAT is a key transcriptional repressor that modulates osteoblast function.

Periodontal disease affects 47% of Americans over 30 and is a growing global concern. Current treatments for periodontal disease focus on the mechanical elimination of periodontal biofilms. Very few treatments are available that target the rampant, unregulated host immune response that is ultimately responsible for tissue degradation. BET proteins have been shown to play critical roles in inflammatory gene regulation and are therefore potentially ideal therapeutic targets for treating periodontal disease. RVX-208 is a selective BET-inhibitor with a high affinity for Bromodomain 2 (BD2) as compared to BD1 in BET proteins. Our previous studies have shown that RVX-208 inhibits inflammatory cytokine production and suppresses osteoclast differentiation. Cell culture assays have provided proof of concept for RVX-208 and its feasibility as a treatment for periodontal disease. As such, our long term goal is to develop RVX-208 as a front-line treatment for periodontitis. The objectives of this study were to determine the ability of RVX-208 to reduce bone loss in a ligature-induced periodontitis model, and to further investigate the mechanisms through which RVX-208 mediates its anti-inflammatory and osteoclastogenesis-suppressive effects. The specific aims of this study were: 1) To further validate the in vivo effects of RVX-208 on a ligature-induced periodontitis model in rats, and 2) To determine the molecular mechanisms of RVX-208 on preventing alveolar bone loss in periodontal disease. To investigate, a ligature-induced periodontitis model was created in rodents. Those rodents were treated with increasing dosages of RVX-208 (0-2.5 mM) by subgingival injection every other day. After 2 weeks, the maxillae were harvested and analyzed via a micro-CT protocol that had been created and validated through statistical analyses. To study the ability of RVX-208 to suppress osteoclastogenesis, RAW264.7 cells were induced into osteoclasts by RANKL and then treated with RVX-208. To ensure RVX-208 was not species specific, THP-1 cells were challenged with either E. coli-LPS or P. gingivalis bacteria and then treated with RVX-208. Linear and volumetric micro-CT analysis showed that RVX-208 could significantly ameliorate bone loss in a ligature-induced periodontitis model. RVX-208 was shown to prevent osteoclast differentiation by suppressing the expression of genes closely associated with osteoclast differentiation and maturation. RVX-208 was found to not be species specific, as it was able to mediate its effects on a human cell line, and had consistent anti-inflammatory effects regardless of whole pathogen or LPS-induced inflammatory response. Therefore, RVX-208 is a promising therapeutic for treatment of periodontal diseases.

Bone metastases are a frequent clinical complication associated with cancer. The aim of this PhD thesis was to set up a model system for the study of tumour cell – bone cell interactions in vitro, ex vivo and in vivo and to use this system to test the efficacy of a novel therapeutic agent for the treatment of osteolytic bone disease. Co-culture or conditioned medium studies using human or mouse cancer cell lines were used to develop an in vitro model system of tumour cell – bone cell interactions. This showed that osteolytic tumour cells enhance osteoclast formation, fusion and resorption through the production of various factors that act directly on osteoclasts and their precursors. And in addition, that osteolytic tumour cells also enhance osteoclastogenesis indirectly via increasing the production of RANKL in osteoblasts. Other effects on osteoblasts included reductions in differentiation, migration and adhesion. Successful ex vivo and in vivo models for the study of tumour – induced osteolysis were created using adapted organ cultures and intratibial injection techniques respectively. IGF-1 and its receptor are known to play important roles in both bone metabolism and breast cancer. Therefore a study of the effects of IGF-1 receptor inhibition on tumour cell – bone cell interactions was performed. In vitro studies showed that the novel IGF-1 receptor tyrosine kinase inhibitor PQIP significantly inhibited IGF-1 and breast cancer enhanced osteoclast formation. Western blot analysis suggested this may be due to the inhibition of both IGF-1 and cancer conditioned medium induced PI3k/Akt activation. Moreover, treatment of osteoblasts with PQIP inhibited cancer cell conditioned medium induced increases in RANKL production. Ex vivo studies using human MDA-MB-231 – mouse calvarial organ co-cultures demonstrated that MDA-MB-231 cells caused osteolysis and this was completely prevented by PQIP without affecting cancer cell viability. Furthermore, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following mouse 4T1 breast cancer cell intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in breast cancer-induced osteoclast number and activity. Consistent with the significant inhibition of osteoblast differentiation, spreading, migration and bone nodule formation observed in vitro, PQIP also inhibited osteoblast number and bone formation in vivo. No inhibition of in vivo tumour volume was observed. These findings clearly suggest that oral PQIP treatment reduced the rate of cancer associated bone turnover. In conclusion, this thesis successfully demonstrates a model system for investigating tumour cell-bone cell interactions in vitro, ex vivo and in vivo. Using this model system I showed that pharmacologic inhibition of IGF-1 receptor kinase activity using PQIP inhibits osteoclast and osteoblast changes induced by breast cancer cells in vitro and in vivo and prevents osteolysis ex vivo and in vivo. This indicates that PQIP and its novel derivatives which are now in advanced clinical development may be of value in the treatment of osteolytic bone disease associated with breast cancer.

Many common cancers, including breast, prostate and lung, have a predilection to metastasize to the bone, bringing not only bone destruction but severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain and drug-induced side effects; furthermore, recent findings suggest that patients are severely under-treated for their cancer pain. Strong analgesics, namely opiates, are the first-line therapy in alleviating cancer-related pain despite severe side effects including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which can bring highly undesirable side-effects including nephrotoxicity and osteonecrosis of the jaw. Thus novel therapeutics are needed to treat the pain of metastatic cancer patients. Animal models of cancer-induced bone pain (CIBP) have revealed that the neurochemistry of cancer has distinctive features from other chronic pain states. These include factors released from the cancer cells, tumor activated macrophages and increased osteoclast degredation of bone within the bone microenvironment, all acting to sensitize free nerve endings.One possibility of inhibiting cancer-mediated pain inducing factors includes agonism of the Cannabinoid 2 receptor agonists. Cannabinoid CB2 receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 both acutely and for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures. In addition, CB2 agonists significantly reduce breast cancer-induced bone pain, bone loss and breast cancer proliferation in part via cytokine/chemokine suppression. Studies utilized the spontaneously-occurring syngenic murine mammary cell line (66.1) implanted and sealed into the femur intramedullary space. Measurements were made of spontaneous pain, bone loss and cancer proliferation. The central and systemic administration of the CB2 agonist JWH015 for seven days significantly attenuates pain. Pharmacological characterization with cannabinoid 1 and 2 antagonists demonstrates that the effects JWH015 on pain were mediated by the CB2 receptor. We and others have found that bone induced cancer pain increases the expression of GFAP and Iba1 in the lumbar spinal cord which are markers of astrocytes and microglia respectively, compared to control animals. After administration of JWH015 (i.t), the release of spinal pro-inflammatory cytokines, IL-6 and TNFá, are reduced suggesting that modulation of glial cytokines may be one mechanism by which CB2 agonists can attenuate pain centrally. On the other hand, systemic administration of JWH015 reduces cancer-induced elevation of cytokines in the tumor microenvironment, suggesting a mechanism by which CB2 agonist is attenuating pain peripherally. Additionally, systemic administration improves bone modification, as demonstrated via micro-computed tomography and bone serum markers while decreasing femoral tumor burden. In vitro, JWH015 reduced cancer cell proliferation and other inflammatory mediators shown to promote pain, bone loss and proliferation. These results suggest CB2 agonists as a novel treatment for breast cancer-induced bone pain, where disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone-pain and increased survival without the major side effects of current therapeutic options. Another future therapeutic option for metastatic bone cancer pain may include cathepsin inhibitors. Cysteine cathepsins (B, C, F, H, K, L, O, L2/V, W, X/Z) are highly expressed in many human cancers and have been associated with poor patient prognosis. In the RIP1-Tag2 transgenic model of pancreatic cancer, mice treated with VBY-825, a reversible inhibitor of cathepsins S, B, V, L, K showed a significant reduction in tumor incidence and growth. Here we demonstrate the cathepsin inhibitor VBY-825 reduces cancer-induced pain behaviors. Additionally, tumor bearing animals treated with VBY-825 demonstrate a reduction in bone resorption, possibly mediated through a reduction in osteoclast activity. These results indicate that a cathepsin inhibitor targeting multiple cathepsins, such as VBY-825, could be a novel therapeutic for bone metastases.Part of the failure to palliate cancer pain is due to a poor understanding of the etiology of cancer pain. Preclinical studies have just begun to scratch the surface on how such cancers may interact with the bone microenvironment to result in pain and bone loss. Further studies are desperately needed at both the preclinical and clinical level to determine the unique molecular profile of cancer pain that may lead to the development of superior therapeutics for CIBP. The studies presented herein provide preclinical evidence that warrant the investigation of these compounds in the clinic as treatment for cancer-induced bone pain.

Metastatic bone cancer originates from breast malignancies causing severe pain and bone destruction in patients. Amongst the novel therapies under clinical development for the treatment of bone metastases are cathepsin inhibitors. Cysteine cathepsins (B, C, F, H, K, L, O, L2/V, W, X/Z) are highly expressed in many human cancers and have been associated with poor patient prognosis. In the RIP1-Tag2 transgenic model of pancreatic cancer, mice treated with VBY-825, reversible inhibitor of cathepsins S, B, V, L, K showed a significant reduction in tumor incidence and growth. In this study, we evaluate the efficacy of the cathepsin inhibitor, VBY-825 as treatment for cancer-induced bone pain. Breast cancer cells, 66.1, were injected within the intramedullary space of the femurs of female mice. After seven days of inoculation, the animals were treated with VBY-825 or vehicle (5% dextrose) subcutaneously for seven days. Spontaneous pain behaviors were significantly attenuated in cancer-induced mice treated with VBY-825, compared to vehicle treated animals. Additionally, cancer-induced animals treated with VBY-825 demonstrated both an improvement in bone integrity and reduction of tumor burden. These results indicate that a cathepsin inhibitor targeting multiple cathepsins, such as VBY-825, could be a novel therapeutic for bone metastases.

Matrix Gla Protein (MGP) inhibits mineralization of arteries and cartilage. To determine its mineral-inhibiting potential in hard tissues, MGP was ectopically expressed in bones and teeth of mice using an osteoblast/odontoblast-specific 2.3kb proximal promoter for type 1 collagen. Mandibles and long bones of Col1a1-Mgp mice and wild-type littermates were analyzed by Faxitron(TM) radiography, PIXImus(TM) dual-energy x-ray absorptiometry (DEXA) and micro-computed tomography (mu-CT). In addition, light microscopy (LM) and transmission electron microscopy (TEM) were performed. While bone and tooth extracellular matrices (ECMs) appeared normally established in Col1a1-Mgp mice, examination of the mineral phase by radiography, DEXA and mu-CT, together with histological mineral localization by LM after von Kossa staining of undecalcified tissue sections, and by TEM, revealed massive hypomineralization of bone and tooth ECMs. In the skeleton of Col1a1-Mgp mice, alveolar bone of the mandible was most heavily affected (compared to long bones), showing a 50% increase in the unmineralized osteoid volume compared to wild-type littermates. For teeth, mineralization was virtually absent in root dentin of both incisors and molars, and absent in molar cellular cementum, whereas crown dentin showed localized "breakthrough" areas of mineralization. Acellular cementum formation and mineralization was absent in the Col1a1-Mgp mice. Immunohistochemical staining of bone and tooth ECM proteins in Col1a1-Mgp mice showed variations in staining relative to wild-type tissues, with immunostaining generally restricted to areas of mineralization. In conclusion, these results confirm in vivo that ECM proteins can act as inhibitors of bone and tooth mineralization.

Bibliography: leaves 86-103. Investigates the effect of zoledronic acid on myeloma cells and osteoblast-like cells to establish the molecular and cellular mechanisms responsible for the clinical effectiveness of bisphosphonates in the treatment of patients with myelomatosis. Concludes that zoledronic acid inhibits myelomatosis-induced osteolysis thorugh the mechanisms of myeloma cell death and proliferation and maturation of osteoblasts.

This observational study compared bone health biomarkers, bone mineral density (BMD),dietary habits, and physical activity levels of men (n=31) and non-estrogen supplementing postmenopausal women (n=23) divided according to duration of proton pump inhibitor (PPI) therapy; more than 5 years (n=16), less than 5 years (n=15), and no PPI therapy (n=23). The shortest duration of PPI therapy was 2 months and the longest duration of PPI therapy was 25 years with a mean duration of 7.5 years. No significant differences were found between measures of spine BMD, urinary deoxypyridinoline (bone resorption), urinary calcium and magnesium, serum osteocalcin (bone formation), serum parathyroid hormone, serum magnesium, serum 25 hydroxyvitamin D3, dietary and supplement intake, or physical activity levels. However, mean hip BMD was higher in females than in males in participants who took PPI therapy for any duration. In the no PPI therapy group, hip BMD was not significantly different between genders. These results suggest that there may be no measurable or clinically significant negative effects of long term PPI therapy on bone health. However, men may be at higher risk of hip fracture when taking long-term PPI therapy than women.

La Cathepsine K est parmi la plus efficace des collagénases de mammifère pour cliver la triple hélice de collagène de type-1. Nous avons développé une série d'azanitriles, (CKI-8 and CKI-13) inhibiteurs de cathepsine K. CKI-8 (un isomère de CKI-13) et CKI-13 ne sont pas toxiques sur les osteoblastes Saos-2 et les cellules RAW 264.7 jusqu' à une concentration de 1000 nM, tandis qu'ils ne le sont pas jusqu'à une concentration de 100 nM sur les osteoclastes. CKI-8 n'affecte pas l'activité de la phosphatase alkaline ainsi que la minéralisation induite par les Saos-2 et par les osteoblastes primaires. CKI-13 diminue de 35 % la minéralisation induite par les Saos-2 tandis qu'il n'affecte pas la minéralisation induite par les osteoblastes primaires. L'addition de CKI-13 diminue l'activité de la phosphatase alkaline d'environ 20% (Saos-2) et de 40 % (osteoblastes primaires). La résorption osseuse sur des tranches d'os d'origine bovine est diminuée avec 10 nM de CKI-13, 100 nM de CKI- 8 et 100 nM d'inhibiteur commercial E64. CKI-8 et CKI-13 diminuent la mobilité des osteoclastes. Nous avons développé un dosage d'hydrolyse de PPi par la phosphatase alkaline au moyen de l'IR, ayant l'avantage de fonctionner sur des vésicules matricielles et des cellules avec des substrats naturels à un pH physiologique. La bande de PPi localisée à 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) et celles de Pi localisées à 1076 cm-1 (∑= 1346 ± 116 M-1.cm- 1) et à 991 cm-1 (∑= 493 ± 49 M-1.cm-1) ont servis à mesurer les concentrations du substrat et du produit
Cathepsin K is among the most potent mammalian collagenase, capable of cleaving the triple helix in type-I collagen. We developed a series of azanitriles (CKI-8 and CKI-13) which are inhibitors of cathepsin K. CKI-8 (an isomer of CKI-13) and CKI-13 did not induce significant toxicity on osteoblasts Saos-2 and RAW 264.7 cells up to 1000 nM, while they were not toxic on mature osteoclasts up to 100 nM. Commercial E64 inhibitor was not toxic in primary osteoclast cells up to 1000 nM. CKI-8 did not affect alkaline phosphatase activity as well the mineralization induced by Saos-2 cells and by primary osteoblasts. CKI-13 decreased by 35% the mineralization induced by Saos-2 cells while it did not on mineralization induced by primary osteoblasts. Addition of CKI-13 decreased alkaline phosphatase activity by around 20% (Saos-2 cells) and 45% (primary osteoblasts). Bone resorption on bovine slices decreased significantly with 10 nM of CKI-13, with 100 nM of CKI-8 and commercial inhibitor E64. Our findings indicated that CKI-8 and CKI-13 inhibited bone resorption and affected the mobility of osteoclast. To monitor directly the PPi hydrolytic activity by alkaline phosphatase, we developed an infrared (IR) assay taking the advantage to use natural substrate under physiological pH in matrix vesicles and in living cells. PPi band located at 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) and Pi bands located at 1076 cm-1 (∑= 1346 ± 116 M-1.cm-1) and at 991 cm-1 (∑= 493 ± 49 M-1.cm-1) served to measure the substrate and the product concentrations

73-deoxychondropsin A (73-DOC), a natural product of the polyketide family sourced from the marine sponge Ircinia ramosa, has previously shown inhibition of vacuolar-type H+-ATPases. This enzyme is crucial for osteoclastic bone resorption and is important in the pathogenesis of metabolic bone diseases, which affect both osteoclasts and the bone-forming osteoblasts. Following extraction of 73-DOC from I. ramosa tissues, in vitro experiments were performed to establish the differential effects of 73-DOC on primary osteoclast and osteoblast differentiation and function. Dose-response and time course analyses, as well as confocal microscopy assessments of cellular acidification, showed that 73-DOC inhibited mouse and human osteoclast resorption at concentrations 5-10-fold lower than those causing inhibition of osteoblast activity. The inhibition of bone resorption whilst maintaining bone formation demonstrates a novel application for 73-DOC and potential as a therapeutic for osteolytic diseases such as osteoporosis. Symbiotic microorganisms have frequently been proposed as the true producers of sponge-sourced secondary metabolites. Whilst symbionts are typically resistant to culturing, metagenomic approaches allow for microbial biosynthetic genes from the sponge holobiome to be cloned and ultimately expressed in a heterologous host, leading to a sustainable supply of the compound. To clone and sequence the biosynthetic gene cluster responsible for 73-DOC, a metagenomic fosmid library was screened for conserved features of polyketide synthase biosynthesis. Metagenomic DNA was also directly sequenced and screened using in silico analysis tools, which led to the annotation of genes with putative partial involvement in 73-DOC biosynthesis. Further work required to identify the remaining biosynthetic components was suggested.

Activities associated with, or interacting with, leukaemic cell populations were assayed for the ability to influence in vitro haematopoiesis. The first of these, the glycoprotein leukaemia inhibitory factor (LIF), has a role in aspects of murine, non human primate and human haematopoiesis. It is thought to be particularly important in the development of megakaryocytes and is also known to induce the terminal differentiation of certain leukaemic cell lines. LIF was assayed both for direct and indirect effects on the proliferation of haematopoietic precursor cell populations in vitro. As a direct acting agent in semi-solid agar culture of haematopoietic cell populations derived from normal bone marrow or 15 day foetal liver, LIF was unable to support colony formation. In cultures of cells derived from normal bone marrow stimulated with single, or combinations of, growth factors, the addition of LIF had no statistically significant effect on the level of colony formation. In cultures of cells derived from foetal liver, stimulated with particular growth factor combinations (medium conditioned by the Wehi3B leukaemic cell line + medium conditioned by the lung fibroblast cell line, L929); GM-CSF + M-CSF; IL-la + IL-3 + M-CSF), LIF, was shown to decrease the level of colony formation. LIF did not directly alter the proportion of the population in DNA synthesis in cell populations derived from normal femoral marrow, 15 day foetal liver or y- irradiated femoral marrow. As an indirect acting agent LIF failed to block the synthesis of a stem cell stimulator, or it's action, on a population of high proliferative potential colony forming cells derived from normal femoral marrow, cloned in the presence of Wehicm+L929cm. (HPP-CFC (Wehicm + L929cm)) LIF's actions on clones of a murine myeloid leukaemia (SA2JMB1) were also assessed. LIF had no statistically significant effect on colony formation or the level of DNA synthesis in populations of SA2JMB1 leukaemic cells. A second group of associated activities was produced by the X- irradiation induced murine myeloid leukaemia (SA2JMB1). Medium conditioned by the leukaemic cells was assayed in vitro both for direct and indirect effects on the proliferation of haematopoietic cells derived from femoral marrow. As a direct acting agent in 7 and 14-day semi-solid agar culture of femoral marrow, leukaemic conditioned medium alone stimulated limited colony formation. In 7 and 14 day cultures stimulated with single and combinations of specific colony stimulating factors: (rmGM-CSF, rhM-CSF, rhIL-1a) a significant increase in colony number was noted in all cases when cultures were supplemented with leukaemic conditioned medium. SA2JMBlcm was shown to support the proliferation of an IL-3 dependent cell line (FDCP-A4 cells). The colony enhancing ability of SA2JMBlcm was shown to be blocked by pretreatment with antibodies to IL-3. This suggested that SA2JMB1 conditioned medium contained IL-3 or an IL-3 like activity, as one of its components. The conditioned medium failed to directly alter the level of DNA synthesis in a population of HPP-CFC (Wehicm+L929cm) derived from normal bone marrow or y- irradiated bone marrow. As an indirect acting agent the conditioned medium did block the action of a stem cell proliferation stimulator on normal bone marrow derived HPP-CFC (Wehicm+L929cm). This leukaemia associated activity was shown to be larger than 50KD, sensitive to heat treatment and able to act in a different manner to the stem cell inhibitor MIP-1-a. Thus this novel activity may be important in blocking stimulator action in haematopoietic stem cells and thus contribute to the haematopoietic insufficiency seen in leukaemia.

In vivo and in vitro degradation of resin-dentin interfaces can occur and accounts for poor clinical performance of adhesive dentin interfaces/restorations. Interfacial degradation results from several factors, to include, but not limited to: water sorption, hydrolysis of ester linkages of methacrylates resins, and activation of endogenous dentin matrix metalloproteinases (MMPs) in non-infiltrated collagen fibrils. Reduction of collagenolytic and gelatinolytic activity in dentin has been shown to be effective both in vivo and in vitro upon application of proteases inhibitors, such as chlorhexidine. This study compared the in vitro durability of resin-dentin bonds using microtensile testing over 12-month of water storage among five adhesive systems, including an experimental adhesive system, which had 2% chlorhexidine incorporated into the material. The results showed that all adhesive systems had a significant decrease in bond strength after 6 months, and that this reduction in bond strength was not significant different among the five adhesive systems studied. It is possible that chlorhexidine might have its inhibitory activity against MMPs lost or reduced due to chemical interaction with the adhesive system components. Also, to assess resin-dentin bonds degradation process, laboratorial studies use long-term water storage, which is labor-intense and time consuming process, therefore this study tested the possibility of accelerating the resin-dentin degradation process using water storage at 50° C. A significant reduction in microtensile bond strength occurred for specimens after 12-month storage at 50° C. The higher temperature may have increased the rate of water sorption and hydrolytic activity within the polymer network leading to adverse consequences to the interface.

Pain often accompanies cancer and most current therapies for treating cancer pain have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomyosin receptor kinase A (TrkA) has become an attractive target for attenuating chronic pain.In the present report, we use a mouse model of bone cancer pain and examine whether oral administration of a selective small molecule Trk inhibitor (ARRY-470, which blocks TrkA, TrkB and TrkC kinase activity at low nm concentrations) has a significant effect on cancer-induced pain behaviors, tumor-induced remodeling of sensory nerve fibers, tumor growth and tumor-induced bone remodeling. Early/sustained (initiated day 6 post cancer cell injection), but not late/acute (initiated day 18 post cancer cell injection) administration of ARRY-470 markedly attenuated bone cancer pain and significantly blocked the ectopic sprouting of sensory nerve fibers and the formation of neuroma-like structures in the tumor bearing bone, but did not have a significant effect on tumor growth or bone remodeling.These data suggest that, like therapies that target the cancer itself, the earlier that the blockade of TrkA occurs, the more effective the control of cancer pain and the tumor-induced remodeling of sensory nerve fibers. Developing targeted therapies that relieve cancer pain without the side effects of current analgesics has the potential to significantly improve the quality of life and functional status of cancer patients.

Post tensioning bridges is an efficient way of construction where segments of a bridge are strung together using small diameter high strength strands. The steel strands are passed through internal and/or external ducts within the concrete segments, jacked to high levels of stress, anchored using steel wedges, and then filled with a corrosion inhibiting filler. A cementitious material called grout has been widely used for over 50 years as this filler. The Federal Highway Administration design and construction manual provides recommendations in the properly filling techniques to ensure the tendons are full of grout. Despite the level of detail the manual provides, several Florida bridges that used this construction technique have seen severe corrosion related failures due to poor grout quality. Oil based products have been developed for the express purpose of reducing the corrosion rate in previously grouted tendons. Utilizing the interstitial spaces in the steel strands, low viscosity fluid is pumped through the strand which can also penetrate the surrounding grout and fill unintentional voids. However, by doing so, there is the concern that the bond strength could be compromised and increase the risk of failure. This study presents the findings of small scale grouted mono-strand tendons which determined the bond effects of defective grout, evaluated the injection capabilities of various penetrants, and determined whether or not the penetrant adversely affects bond after impregnation. No appreciable variation in bond was found.

Controlling lineage specific differentiation of stem cells is crucial for functional tissue engineering, and current methods have drawbacks and limitations. Histone deacetlyase (HDAC) proteins are key to cellular epigenetics, and a number of studies have shown that their inhibitors (HDACis) can control stem cell fate. The aim of this study was to investigate the effect of a novel HDAC2 and HDAC3 selective inhibitor, MI192, on human adipose derived stem cells (ADSCs), exploring the potential of utilising MI192 in controlling the osteogenic differentiation of ADSCs, with the long term goal of improving the efficacy of bone tissue engineering. MI192 was synthesised in house and characterised with NMR analysis. The effect of MI192 was explored on commercially (Life Technologies) available ADSCs. It was found that 1 µM to 100 µM MI192 was cytotoxic and reduced ADSC viability, with a dose response observed. Using propidium iodide staining, it was demonstrated that MI192 halted the cell cycle in the G2M phase. FACs analysis showed that MI192 altered the stem cell marker expression profile of ADSCs; increasing expression of some markers (CD34 and CD146) and decreasing some (CD29, CD44, CD73, CD105, CD166). When utilised in a pre-treatment strategy in 2D models, MI192 improved the osteogenic differentiation of ADSCs, strongly increasing production of alkaline phosphatase protein, with 2 days of 30 µM pre-treatment being the optimal concentration and treatment length. MI192 pre-treatment also increased ADSC mineralisation (calcium accumulation stained with Alizarin Red and mineral nodule formation stained with Von Kossa) and increased osteogenic gene expression (BMP2, RUNX2, COL1 and ALP). The adipogenic differentiation of the ADSCs was inhibited by MI192 pre-treatment, with reduced lipid droplet accumulation (stained with Oil Red O) and adipogenic gene (PPARγ and ADIPOQ) expression seen. Increased osteogenic differentiation was further demonstrated in 3D models, using Am silk scaffolds. Cell attachment was assessed with live cell labelling, and collagen production, mineralisation and protein production analysed with histology and immunohistochemistry. Finally, TaqMan® Gene Expression Array Cards were utilised to investigate how two days of MI192 treatment altered the expression of 96 different genes. Some key Wnt related genes, as well as other key osteogenic genes such as BMP2 were up-regulated, providing some mechanistic explanation for the increase in osteogenic differentiation potential seen with MI192 pre-treatment. This thesis demonstrates the potential of utilising MI192 to improve bone tissue engineering strategies.

Hintergrund und Fragestellung: Seit der Zulassung des Tyrosinkinase-Inhibitors (TKI) Imatinib im Jahre 2001 hat sich die Therapie der chronisch myeloischen Leukämie (CML) grundlegend verändert. Imatinib inhibiert die konstitutiv aktive Tyrosinkinase BCR-ABL, welche die verstärkte Proliferation der leukämischen Zellen und die Entwicklung der CML bedingt. Das sehr gute klinische Ansprechen auf eine Imatinib-Behandlung resultierte in einer beschleunigten Zulassung dieses TKI auch bei pädiatrischen Patienten im Jahre 2003. Aufgrund von Punktmutationen und/oder strukturellen Änderungen innerhalb des BCR-ABL Fusionsproteins können sich Resistenzen gegenüber Imatinib entwickeln. Deshalb wurden Zweit- und Drittgenerations TKI wie Dasatinib und Bosutinib entwickelt. Imatinib wirkt nicht hoch spezifisch und hemmt neben BCR-ABL auch weitere Tyrosinkinasen, wie z.B. c-KIT, PDGF-R und c-FMS, welche am Knochenstoffwechsel beteiligt sind. Die Stimulation des Rezeptors c-FMS bewirkt die Differenzierung monozytärer Vorläuferzellen in knochenabbauende Osteoklasten. Zusätzlich unterliegt die Entwicklung der knochenaufbauenden Osteoblasten spezifischen Signalkaskaden an denen PDGF-R und c-Abl beteiligt sind. Als Nebenwirkung einer TKI-Therapie beeinträchtigt die Inhibition dieser Signaltransduktionswege somit das Knochen-“Remodelling“, indem die Entwicklung und funktionelle Aktivität von Osteoklasten reduziert wird. Gleichzeitig wird die Aktivität von Osteoblasten gestärkt, aber deren Proliferation inhibiert. Diese Dysbalance von Knochenaufbau und -abbau mit gestörter Kalziumhomöostase bedingt bei erwachsenen CML-Patienten veränderte endokrinologische Parameter des Kalziumhaushaltes, eine vermehrte Knochenmineralisation und eine erhöhte trabekuläre Knochendichte. Dagegen wurden bei pädiatrischen CML-Patienten unter Imatinib-Therapie Längenwachstumsstörungen beobachtet, welche bezüglich des Wirkmechanismuses von Imatinib auf den wachsenden Knochen bis heute noch nicht im Detail geklärt sind. Spekulativ ist auch, ob Zweit- und Drittgenerations-TKI ebenso wie Imatinib den Knochenstoffwechsel bei pädiatrischen Patienten stören. Angelehnt an ein erfolgreiches Applikationsschema bei erwachsenen CML-Patienten steht zusätzlich die Frage im Raum, ob eine intermittierende Gabe von TKIs (einen Monat Therapie, einen Monat Pause) eine Minderung der Nebenwirkung auf den Knochen bewirken könnte, ohne die Wirkung auf die CML-Behandlung zu beeinträchtigen. Vor diesem Hintergrund wurde ein Nagermodel etabliert, um Nebenwirkungen auf den Knochenstoffwechsel unter TKI-Exposition zu analysieren. Junge, wachsende Ratten wurden hierzu vom präpubertären Alter bis zur Adoleszenz kontinuierlich oder intermittierend mit den TKIs Imatinib, Dasatinib und Bosutinib exponiert und die Wirkung auf das wachsende Skelettsystem untersucht. Methoden: 4 Wochen alte männliche Wistar Ratten wurden über einen Zeitraum von 10 Wochen chronisch mit jeweils einem der drei im Trinkwasser gelösten TKIs exponiert. Neben einer unbehandelten Kontrollkohorte erhielt eine Gruppe jeweils eine Standarddosis und eine hohe Dosis (entsprechend der doppelten Standarddosis) des entsprechenden TKIs kontinuierlich. Eine weitere Gruppe erhielt die hohe Dosis intermittierend (an drei aufeinanderfolgenden Tagen TKI, danach vier Tage nur Wasser). Die Konzentrationen im Trinkwasser betrugen für Imatinib 1 mM und 2 mM und für Dasatinib und Bosutinib jeweils 50 µM und 100 µM. Nach zweiwöchiger (präpubertär), vierwöchiger (pubertäres Stadium) und zehnwöchiger Exposition (postpubertär) wurden die Tiere aller Gruppen nekropsiert und Röhrenknochen, Lendenwirbel und Blut asserviert. Zur Beurteilung des Knochenmetabolismus wurden folgende Parameter erhoben: Knochenlängen, Knochendichten mittels pQCT, trabekuläre Strukturen mittels µCT, Knochenfestigkeit mittels des 3-Punkt-Biege-Test und endokrinologische Parameter im Serum mittels ELISA. Zusätzlich wurde der jeweilige TKI Serum-Spiegel bestimmt. Ergebnisse: Die Gewichtsentwicklung, körperliche Entwicklung und das Sozialverhalten zeigten keine Unterschiede beim Vergleich von Kontrollkohorten mit exponierten Tieren. Die kontinuierliche Exposition mit Imatinib und Dasatinib bewirkte dosisabhängig eine Reduktion der Knochenlängen der Femura und der Tibiae. Bosutinib zeigte diesen Effekt nicht. Die intermittierende Exposition mit hoher Dosis resultierte in einer Knochenlängenreduktion, welche exakt dem Effekt der Standarddosis entsprach. Weiterhin resultierte aus der Exposition mit Imatinib oder Dasatinib eine Verminderung der trabekulären Knochendichten der Femura und Tibiae im präpubertären Stadium. Ratten, welche hoch dosiert Imatinib erhielten, zeigten diese Reduktion ebenfalls im pubertären Stadium, nicht jedoch unter Dasatinib- und Bosutinib-Exposition. Postpubertär unterschieden sich die trabekulären Dichten von Femura und Tibiae der exponierten Gruppen nicht von den Kontrollkohorten. Auf die kortikale Knochendichte und die kortikale Dicke dieser Röhrenknochen zeigte sich kein messbarer Effekt der TKI. Dennoch trat - nur nach Exposition der hohen Imatinibdosis - eine signifikant verminderte femorale Bruchfestigkeit postpubertär auf. Am Lendenwirbelkörper war pubertär und postpubertär die Höhe unter Imatinib-Exposition vermindert, während die Gesamt- und kortikale Knochendichte präpubertär erhöht war bei tendenziell erniedrigter trabekulärer Knochendichte. Die kortikale Dicke wurde durch alle TKI nicht beeinflusst. Dasatinib und Bosutinib bewirkten keinen Effekt auf die Wirbelhöhe, aber eine tendenzielle Minderung der trabekulären Knochendichte. Der serologisch erfassbare Knochenresorptionsmarker „tatrate resistant acidic phosphatase“ (TRAP) war unter kontinuierlicher Exposition mit hoher Dosis von Imatinib zu allen Zeitpunkten erniedrigt. Postpubertär zeigte sich dieser Effekt auch unter Standard- und Hochdosis von Bosutinib. Der Knochenformationsmarker Osteocalcin war unter Imatinib bei allen Kohorten zu allen Analysezeitpunkten erniedrigt, während Dasatinib und Bosutinib keinen Effekt auf diesen Parameter zeigten. Die erfassten Serum-Hormonparameter (Wachstumshormon, Parathormon) lagen unter der Exposition mit Imatinib als erhöhte Wachstumshormonspiegel pubertär und als verminderte Parathormonspiegel prä- und pubertär vor. Unter der Exposition mit Dasatinib kam es ebenfalls pubertär zu einer Erhöhung der Wachstumshormonspiegel und präpubertär zu einer tendenziellen Erhöhung der Parathormonspiegel. Postpubertär normalisierten sich beide Parameter unter der Exposition mit Imatinib und Dasatinib wieder. Unter Bosutinib konnte nur postpubertär erniedrigte Parathormonspiegel ermittelt werden. Eine intermittierende TKI-Exposition resultierte in einem Aufholwachstum und einer teilweise Normalisierung der knochenspezifischen Serumparameter. Als wichtige unerwartete Nebenwirkung zeigte sich unter Langzeitexposition mit Imatinib und Dasatinib eine Zunahme des Herzgewichtes. Unter Imatinib resultierten daraus keine klinischen Auffälligkeiten, während unter Dasatinib eine Herzinsuffizienz zum Tod eines Tieres führte. Bosutinib zeigte keine weiteren makropathologisch erfassbaren Nebenwirkungen. Bis heute sind keine kardialen Nebenwirkungen bei pädiatrischen Patienten nach mehrjähriger TKI-Therapie publiziert. Schlussfolgerung: Das etablierte juvenile Nagertiermodell ist gut geeignet, um die Nebenwirkungen einer Langzeitexposition von TKI auf den wachsenden Knochen zu erfassen. Bei Kindern und Adoleszenten klinisch beschriebene Wachstumsretardierungen unter Imatinib ließen sich zweifelsfrei bei Ratten verifizieren. Bei fehlenden klinischen Daten von Kindern zu Dasatinib präjudiziert das Modell, dass Dasatinib so wie Imatinib den gleichen, Bosutinib hingegen kaum einen Effekt auf den Knochen ausübt. Eine intermittierende Gabe der TKI scheint die Nebenwirkungen auf den Knochen abzumildern und könnte eine neue Möglichkeit der TKI-Therapie für pädiatrische Patienten darstellen. Aus dem Tiermodell der Langzeit-exponierten juvenilen Ratte lässt sich ableiten, dass beim wachsenden Kind unter jahrelanger TKI-Therapie klinisch sorgfältig der Knochenstoffwechsel und das Längenwachstum überwacht und unter Dasatinib zusätzlich kardiale Nebenwirkungen beachten werden sollten
Background: Since its approval in 2001 the tyrosine kinase inhibitor (TKI) imatinib has revolutionized the therapy of chronic myeloid leukaemia (CML). Imatinib inhibits the constitutively active tyrosine kinase (TK) BCR-ABL causing the increased proliferation of the leukemic cells and the progress of CML. According to improved survival rates imatinib has been licensed as frontline therapy also for paediatric CML in 2003. However, due to point mutations or structural changes within the BCR-ABL fusion protein resistance to imatinib occurs. Therefore 2nd and 3rd generation TKI like dasatinib and bosutinib have been developed. Beside BCR-ABL, Imatinib exerts also off-target effects on further TKs like c-KIT, PDGF-R, c-FMS which are involved in bone metabolism. Stimulation of the receptor c-FMS leads to the differentiation of monocytic progenitors to bone resorbing osteoclasts. In addition, the development of bone forming osteoblasts underlies specific signalling cascades involving PDGF-R and c-Abl. As a side effect of TKI therapy these specific signalling cascades are inhibited impairing bone remodelling by reducing the development and functional activity of osteoclasts. Simultaneously osteoblasts’ differentiation is promoted while their proliferation is inhibited. This dysbalance of bone formation and resorption results in altered endocrinological serum markers of the calcium homeostasis, increased bone mineralization, and increased trabecular bone density in adult CML patients. In contrast paediatric CML patients show longitudinal growth retardations under imatinib therapy, however, the detailed action of imatinib on the growing bone is not clarified yet. Additionally, it is unclear if 2nd and 3rd generation TKI will also disturb bone metabolism in paediatric CML patients. Based on an effective treatment strategy in adult CML patients, it is also questioned if intermittent TKI treatment (one month “on”, one month “off”) could minimise side effects on the bone without impairing CML therapy. On this background a rodent model was established to study side effects of TKI treatment on bone metabolism. Juvenile growing rats where exposed from prepubertal age till adolescence continuously or intermittently to imatinib, dasatinib, and bosutinib and the effects on the growing skeleton were analysed. Methods: Four weeks old male Wistar rats were chronically exposed to varying concentrations of one of the three TKIs via the drinking water for 10 weeks. Besides untreated controls a standard dosage group and a high dosage group (equalling the twofold standard dose) received every TKI continuously, while an additional group received the high dosage TKI in an intermittent fashion (3 days per week: “on” TKI; 4 days water without TKI). The concentrations applied were 1 mM and 2 mM for imatinib and 50 µM and 100 µM each for dasatinib and bosutinib, respectively. After 2 weeks (prepubertal), 4 weeks (pubertal stage), and 10 weeks (postpubertal) of exposure, respectively, animals were sacrificed and long bones, lumbar vertebra and blood were isolated. To evaluate bone metabolism the following parameters were analysed: bone length, bone mineral density (BMD) by pQCT, trabecular structure by µCT, bone strength by 3-point bending test, and endocrinological parameters by ELISA. Additionally, serum levels of TKIs were investigated. Results: In comparison to controls no alterations of exposed animals’ bodyweight, overall development and social behaviour were observed. Continuous exposure of imatinib and dasatinib led dose dependently to reduced femoral and tibial length. No such effect was observed under bosutinib. Intermitted exposure of high-dose TKIs resulted in reduced effects on femoral and tibial length identical to the effect observed in groups receiving just standard dose. Furthermore, exposure of imatinib and dasatinib lowered femoral and tibial trabecular BMD prepubertally. Rats receiving high dose imatinib showed reduced femoral and tibial trabecular BMD at pubertal stage, while this effect was not observed under dasatinib and bosutinib exposure. Postpubertally, femoral and tibial trabecular BMD of all exposed groups did not differ from controls. Femoral and tibial cortical BMD and cortical thickness were not affected by TKI exposure. However, under high dose imatinib exposure femoral mechanical breaking strength was reduced postpubertally. In vertebra the height was reduced under imatinib exposure pubertally and postpubertally, while the total and cortical BMD were increased prepubertally and trabecular BMD tended to be reduced. Cortical thickness was not affected by any TKI tested. Dasatinib and bosutinib exhibited no effect on the height of the vertebra but trabecular BMD tended to be reduced. The serum bone resorption marker ‘tartrate resistant acidic phosphatase’ (TRAP) was found reduced under continuous exposure of high dose of imatinib at all time points tested. Postpubertally, the same effect was detected after standard and high dosage of bosutinib. The bone formation marker osteocalcin was reduced in all groups and at all time points tested under imatinib exposure, whereas no such effect was observed for dasatinib and bosutinib. Serum bone related hormone markers (growth hormone (GH) and parathyroid hormone (PTH)) revealed under imatinib exposure increased GH levels pubertally whereas PTH was reduced pre- und pubertally. During dasatinib exposure GH levels were elevated pubertally and PTH levels were increased prepubertally. Postpubertally, both parameters normalised again under imatinib and dasatinib exposure. During bosutinib exposure reduced PTH levels were detected postpubertally only. Intermitted TKI exposure resulted in catch-up growth and partial normalisation of bone specific serum parameters. As major unexpected side effect during exposure increasing heart weights could be observed under long-time imatinib and dasatinib exposure. No clinical changes were observed under imatinib, whereas dasatinib led to cardiac insufficiency leading to death of one animal. Bosutinib showed no additional macrospathologic assessable side effects. To date no cardiac side effects were published in paediatric patients under prolonged TKI therapy. Conclusion: The established juvenile rat model is appropriate to examine side effects of long-term TKI exposure on the growing bone. Published longitudinal growth retardation in children and adolescents under imatinib treatment could be unequivocally mimicked in this rat model. Due to not yet available clinical experience with dasatinib in paediatric patients, this model predicts that dasatinib alters bone metabolism like imatinib whereas bosutinib shows less detectable effects. Intermitted TKI treatment may reduce side effects on the growing bone and therefore could represent a new opportunity of TKI therapy for paediatric patients. Summing up, TKI long-term exposure in this juvenile rat model challenges physicians to diligently monitor bone metabolism in not outgrown paediatric patients during long-term TKI treatment and additionally assess cardiac side effects under dasatinib exposure

Abstract Bone remodelling occurs in humans throughout life, therefore bone is continuously renewed to better respond to changes in weightbearing circumstances. Bone remodelling is extremely vulnerable during fracture healing and integration of prostheses into the surrounding bone. Bone remodelling is a complex system in which many growth factors, cytokines and enzymes, which are essential for the differentiation of osteoblasts and osteoclasts, are involved. Some widely used drugs can affect this sensitive system of remodellation in unexpected manner. Painkillers such as cyclooxygenase (COX) inhibitors have been demonstrated in animal studies to interfere with fracture healing and a few retrospective clinical studies confirm these observations. Bisphosphonates (BP), main target of which is the bone resorbing osteoclast, have been suggested to be the drug of choice to improve periprosthetic bone density and thus prevent aseptic loosening of implants. The exact mechanism of action of clodronate (CLO), a non-amino-BP, which was selected for the study, has not been clarified thus far. In order to gain a deeper understanding of the role of the COX enzyme in the differentiation of osteoblasts we studied human mesenchymal stem cell (hMSC) cultures in the presence of different COX-inhibitors; indomethacine, parecoxib and NS398, a specific COX-2 inhibitor. We used the liposome encapsulated CLO metabolite (AppCCl2p) to study in detail the mechanism of BP induced apoptosis in osteoclast. The effects of different BPs CLO, pamidronate (PAM) and zoledronic acid (ZOL), on the differentiation of osteoblasts and osteoclasts were tested in vitro. The optimal concentration for in situ CLO rinsing in clinical study was found. Finally, the effects of in situ and per oral CLO on the periimplant bone density and integration of prostheses were studied in vivo. All tested COX-inhibitors significantly inhibited osteoblast differentiation from hMSCs and stimulated the differentiation of adipocytes. It was also demonstrated that AppCCl2p inhibits mitochondrial function by a mechanism that involves competitive inhibition of ADP/ATP translocase. In the comparison of BPs, ZOL seemed to posses the properties of both non-amino- and amino-BPs and it thus belongs to a new class of BPs. Peroral and in situ CLO seemed to have different mechanisms of action. Peroral CLO delayed the integration of prosthesis to the bone and increased peri-implant osteolysis while is situ CLO accelerated integration. In conclusion, we can alter normal bone remodellation during fracture healing and prosthesis integration. On the other hand, we can also improve the circumstances for the integration of implant to the surrounding bone by in situ BP rinsing, thus creating a better environment for bone ingrowth
Tiivistelmä Läpi elämän luustossa tapahtuu uudelleenmuotoutumista, remodelaatiota, jonka seurauksena luu pystyy paremmin vastaamaan muuttuneisiin kuormitusolosuhteisiin. Remodelaatioprosessi on hyvin haavoittuvainen murtuman luutumisen aikana sekä proteesin kiinnittyessä ympäröivään luuhun. Luun remodelaatioon osallistuvat kasvutekijät, sytokiinit ja entsyymit, jotka puolestaan ovat välttämättömiä osteoblastien ja osteoklastien erilaistumiselle. Monet lääkeaineet voivat yllättävällä tavalla vahingoittaa tätä herkkää remodelaatiosysteemiä. Kipulääkkeet, kuten syklo-oksygenaasi (COX) estäjät, voivat häiritä murtuman luutumista aikaisempien eläintöiden ja muutamien retrospektiivisten potilastutkimusten mukaan. Lisäksi bisfosfonaatit, joiden päävaikutuskohde on luuta hajoittava osteoklasti, voisivat olla lupaavia lääkkeitä myös parantamaan proteesia ympäröivän luun laatua ja siten estämään aseptista implantin irtoamista. Tutkimuksen yhtenä tarkoituksena oli selvittää klodronaatin, ensimmäisen polven typpi-ryhmää sisältämättömän bisfosfonaatin tarkka vaikutusmekanismi. Viljelemällä ihmisen luuytimen kantasoluja indometasiinia, parekoksibia tai spesifistä COX-2 estäjää NS 398:a, sisältävässä kasvatusliuoksessa selvitettiin COX-entsyymin merkitys osteoblastien erilaistumiselle. Liposomien sisälle pakattua klodronaatin metaboliittia (AppCCl2p) käytettiin tutkittaessa millä vaikutusmekanismilla klodronaatti aiheuttaa osteoklastien apoptoosin. Bisfosfonaattien; klodronaatin, pamidronaatin ja tsoledronaatin vaikutusta osteoklastien ja osteoblastien erilaistumiseen tutkittiin soluviljelmämallissa ja määritettiin kliinisessä potilastyössä paikallisesti käytettävän klodronaattiliuoksen pitoisuus. Lopuksi potilastyössä selvitettiin paikallisen klodronaattihuuhtelun ja suun kautta annostellun klodronaatin vaikutus proteesia ympäröivän luun tiheyteen ja proteesin kiinnittymiseen ympäristöönsä. Tutkimukseen valitut COX-estäjät vähensivät ihmisen kantasolujen erilaistumista osteoblasteiksi ja lisäsivät erilaistumista rasvasoluiksi. Lisäksi todettiin, että AppCCl2p estää mitokondrioissa tapahtuvaa hengitystä estämällä ADP/ATP-vaihtajan toiminnan, saaden aikaan solukuoleman. Vertailtaessa bisfosfonaatteja, tsoledronaatilla vaikutti olevan sekä ensimmäisen, että kolmannen polven (sisältää typpi-ryhmän) bispfosfonaattien vaikutuksia, joten tsoledronaatti kuuluu aivan uuteen bisfosfonaattiryhmään. Potilastutkimuksessa suun kautta ja paikallisesti reisiluun ytimeen annostellulla klodronaatilla oli täysin erilainen vaikutus. Suun kautta syötynä klodronaatti hidasti proteesin kiinnittymistä ja aiheutti osteolyysiä. Sen sijaan paikallinen klodronaatti nopeutti merkittävästi proteesin kiinnittymistä ympäröivään luuhun. Näiden tutkimustulosten perusteella voidaan olettaa, että COX-estäjät, samoin kuin peroraalinen bisfosfonaatti, voivat tahattomasti häiritä luun remodelaatiota

The Ni-Fe-S C-cluster of carbon monoxide dehydrogenases (CODH), which catalyzes the reversible oxidation of CO to CO2, can be stabilized in four redox states: Cox, Cred1, Cint, and Cred2. The best-supported mechanism of catalysis involves a one-electron reductive activation of Cox to Cred1 and a catalytic cycle in which Cred1 binds and oxidizes CO, forming Cred2 and releasing CO2. Recently reported experiments appear to have disqualified this mechanism, as activation was concluded to require reduction to a C-cluster state more reduced than Cred1. The results presented in this dissertation suggest that the activation potential was milder than that required to reduce these clusters. The results support a mechanism in which Cred1 is the form of the cluster that reacts with CO. The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans (CODHCh) includes a ??2-sulfide ion bridged to the Ni and unique Fe, while the same cluster in enzymes from Rhodospirillum rubrum (CODHRr) and Moorella thermoacetica (CODHMt) lack this ion. This difference was investigated by exploring effects of sulfide on activity and spectral properties. Sulfide partially inhibited CO oxidation activities of CODHRr and CODHMt. Adding sulfide to CODHMt in the Cred1 state caused the gav = 1.82 Electron Paramagnetic Resonance spectroscopy (EPR) signal to decline and new features to appear. Sulfide did not affect the gav = 1.86 signal from the Cred2 state. A model was developed in which sulfide binds reversibly to Cred1, inhibiting catalysis. The results also suggest that the substrate hydroxyl group bridges the Ni and unique Fe. A cysteine residue recently found to form a persulfide bond with the C-cluster was characterized. The Ser mutant of the persulfide-forming Cys316 was inactive and displayed no C-cluster EPR signals. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

Current hydrophilic resin adhesives undergo hydrolytic degradation and show a decrease in bond strength over time. Nanoleakage and ultrastructure studies suggest that inadequately infiltrated collagen leads to enzymatic degradation and resin-dentin bond failure. Adequate degree of conversion (DC) of resin adhesives is also critical to resin-dentin bond strength and durability. The long-term goal of this dissertation is the realization of durable resin-dentin bond. It is hypothesized that ethanol-wet bonding technique (EW) may effectively facilitate the infiltration of hydrophobic monomers into hydrophilic acid-etched dentin by maintaining interfibrillar spacing, stiffening collagen matrix, and improving adhesive resin-demineralized dentin matrix miscibility. Chlorhexidine (CHX), Matrix Metalloproteinase-inhibitor (MMP-inhibitor), should further preserve collagen integrity and resin-dentin bond strength. Moreover, efficient photoinitiator systems that broaden light absorptivity and provide more reactive radicals may enhance polymerization. In this dissertation, a clinically-relevant EW protocol, 3×15s absolute ethanol rinsing, provided significantly higher microtensile bond strength (πTBS) of a hydrophobic resin (70%BisGMA/30%TEGDMA) to dentin as compared to water-wet bonding (WW). All groups showed no significant drop of πTBS after 1-year storage except EW without CHX application, showing marginally significant reduction in πTBS (p=0.0558) suggesting MMP-inhibition by CHX in EW. These results were consistent with subsequent experiments. EW maintained interfibrillar width and hybrid layer thickness for resin infiltration and retention. Monomer molar concentration across the hybrid layer was significantly higher in EW than WW. An application of 2% CHX diacetate further preserved collagen banding in EW. WW showed more generalized spotted nanoleakage, while EW presented localized reticular nanoleakage. The use of Irgacure 819 (BAPO) alone and in combination with benzoyl peroxide (BPO) or camphorquinone (CQ) increased DC of hydrophobic and hydrophilic resins over resins containing the CQ/amine (4E) control. Only BAPO and BAPO/BPO demonstrated significantly higher immediate shear bond strength than CQ/4E. Within the limitations of these studies, EW improved resin-dentin bond durability by maintaining collagen interfibrillar spaces for efficient infiltration of a hydrophobic BisGMA/TEGDMA resin resulting in significantly higher πTBS and monomer molar concentrations with less nanoleakage distribution within the hybrid layer than WW. CHX further maintained collagen integrity and πTBS in EW. BAPO is a potential alternative photoinitiator of dental resins.

As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis. Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis. In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro. This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo. As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.

The aim of this study was to evaluate the histologic and cellular response to A.actinomycetemcomitans (A.a) infection, and how diabetes-enhanced TNF-α production and diabetes-enhanced apoptosis contribute to the periodontal disease progression and bone coupling. Conclusion. The results link A. a infection with important characteristics of periodontal destruction and provide new insight into how diabetes aggravates A. a induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection. In addition, diabetes prolongs inflammation and osteoclastogenesis in periodontitis and through TNF limits the normal reparative process by negatively modulating factors that regulate bone.
El objetivo de este estudio fue evaluar la respuesta histológica y celular a la infección por A.actinomycetemcomitans (A.a) y como la diabetes exacerba la producción de TNF- α y la apoptosis que contribuye a la progresión de la enfermedad periodontal y al acoplamiento del hueso. Los resultados enlazan la infección de A. a con características importantes de destrucción periodontal y ofrece una nueva visión de cómo la diabetes agrava la destrucción periodontal con A. a mediante un aumento significativo de la respuesta inflamatoria, lo que lleva al aumento de pérdida ósea y produce un aumento de apoptosis en el epitelio gingival y en las células del tejido conectivo a través de un mecanismo de caspasa-3 dependiente. Los antibióticos tuvieron un efecto más pronunciado en mucho de los parámetros evaluados en las ratas diabéticas que en las normoglucémicas, sugiriendo una deficiencia en la capacidad de los animales diabéticos en combatir la infección. Además la diabetes prolonga la inflamación y la osteoclastogénesis en la periodontitis y a través de TNF limita el proceso normal de reparación modulando negativamente factores que regulan el hueso.

Das Bone Morphogenetic Protein 2 (BMP2) gehört zur TGF-beta Superfamilie und findet seinen Fokus in der osteogenen Aktivierung und in der Anwendung bei der Frakturheilung. Es wird angenommen, dass weitere, bisher unbekannte Verbindungen existieren, die die BMP2-Signalübertragung und die osteogene Aktivität verbessern und somit zu einer verbesserten klinischen Wirksamkeit von BMP2 führen. Für den Stickstoffoxid (NO)-Signalweg ist bereits bekannt, dass im endothelialen Kontext eine Verbindung zum BMP2-Signalweg existiert. Ziel dieser Arbeit war es daher, eine Verbindung zwischen dem NO- und BMP2-Signalweg bezüglich der Regulierung des BMP2-abhängigen Signalwegs und der Osteoinduktion aufzuzeigen. Dies erfolgte durch Anwendung von Inhibitoren (LNAME, ODQ und LY83583) und Aktivatoren (L-Arginin, Deta NONOate, SNAP und YC-1) des NO-Signalwegs, in Kombination mit BMP2. Eine mögliche Verbindung zwischen dem BMP2- und NO-Signalweg, über eine Protein Kinase A (PKA) Brücke, wurde durch die Anwendung des PKA Inhibitors H89 untersucht. Zusammenfassend zeigen diese Ergebnisse, dass der NO-Stoffwechselweg den BMP2-vermittelten Signalweg und die osteoinduktive Aktivität modulieren kann, wobei PKA beide Signalwege im Rahmen der BMP Signalübertragung verbindet, jedoch nicht zu einer BMP2-vermittelten Osteoinduktion führt.
Bone Morphogenetic Protein 2 (BMP2) is a TGF-beta superfamily member, with a major focus on osteogenic activity and application in fracture healing. In order to improve efficiency of BMP2 in the clinic, it is assumed that additional, yet unknown compounds can improve BMP2 signalling and osteogenic activity. The Nitric Oxide (NO) pathway has previously shown to be connected with the BMP2 pathway in an endothelial context. Therefore, it was the aim of this study to unravel connections between the NO and BMP2 pathway in regulating BMP2 mediated signalling and osteoinduction. This was carried out through the application of inhibitors (LNAME, ODQ and LY83583) and activators (L-Arginine, Deta NONOate, SNAP and YC-1) of the NO pathway in combination with BMP2. A proposed connection between BMP2 and NO pathways via a Protein Kinase A (PKA) bridge was investigated by application of H89 inhibitor. In summary, these results show that the NO pathway can modulate BMP2 mediated signalling and osteoinductive activity. The PKA bridge connects NO and BMP2 only for the process of BMP2 signalling, but not for BMP2 mediated osteoinduction.

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

L’électrolyse de l’aluminium est un processus consommateur en énergie et en ressources (matières premières, personnel qualifié, temps, etc.). De nombreux projets de recherche sont en cours à travers le monde dans le but d’améliorer l’efficacité du procédé de fabrication de l’aluminium, de diminuer les rejets toxiques de gaz (CO2, CO, CF4, C2F6…) et d’en réduire les coûts de production. Un des problèmes actuels de l’électrolyse de l’alumine est la consommation excessive des anodes en carbone. En effet, ces anodes, lorsqu’elles sont chauffées à haute température, sont attaquées par l’air ambiant entre 400 et 600 °C, et par le CO2 à 960 °C, ce qui a pour conséquence d’entraîner une surconsommation de carbone, réduisant ainsi la capacité de fabrication de l’aluminium métallique par kg de carbone consommé. Actuellement, la durée de vie moyenne d’une anode est entre 20 et 30 jours. L’objectif de ce projet est de diminuer la vitesse de réaction à l’air de l’anode. Différentes méthodes ont été élaborées afin d’obtenir une protection efficace et économique contre le phénomène d’oxydation à l’air et au CO2 réduisant ainsi la surconsommation en carbone de l’anode. L’oxyde de bore étant connu comme inhibiteur de la réaction carbone/oxygène, des essais ont été réalisés dans le but de produire un revêtement sur l’anode et ont permis de confirmer l’effet inhibiteur de l’oxyde de bore sur la réaction d’oxydation à l’air, permettant ainsi la protection des anodes de carbone. L’influence de chacun des paramètres (température, concentration, durée d’imprégnation dans la solution, etc…), ont été également étudiés. La tomographie par rayons X a démontré que l’anode est principalement attaquée sur la surface et que le revêtement d’oxyde de bore créé une barrière physique empêchant l’accès de l’oxygène à l’anode. Des études plus approfondies ont été réalisées afin de comprendre le mécanisme de protection de l’oxyde de bore avec la réaction carbone-oxygène. Selon la littérature, l’oxyde de bore et l’acide borique peuvent agir de deux façons : soit en se fixant sur la surface de l’anode en bloquant les sites actifs du carbone ou encore en créant une couche vitreuse qui sert de barrière pour l’oxygène. Une étude cinétique a été établie et confirme que le nombre de collisions entre l’oxygène et les sites de carbone diminuent en présence du bore. La technique ToF-SIMS a également démontré que le bore se trouve sous forme d’oxyde sur la surface de l’anode, mais aussi sous forme de liaison carbone bore. Il s’agit donc d’une protection physique d’une part et une protection chimique en bloquant les sites actifs du carbone par les atomes de bore. La consommation de l’anode en carbone dans la cuve d’électrolyse est contrôlée par les impuretés, par le niveau de graphitisation mais également par le transport de masse à travers sa structure poreuse. La protection des particules de coke avec de l’oxyde de bore pourrait avoir un impact physique sur la porosité et la distribution de celle-ci. Des particules de coke (allant de 4 000 μm à 4 760 μm de diamètre) ont été imprégnées par de l’oxyde de bore afin de révéler la sélectivité des porosités. Les surfaces et les volumes spécifiques différentiels de ces trois tailles de particules gazéifiées à 3 pourcentages (0, 15 et 35%) déterminés par adsorption d’argon et par infiltration de mercure ont permis d’évaluer les contributions des gazéifications sous-critiques et sur-critiques sur la gazéification totale des anodes sous air à 525 °C. La détermination de la taille critique des pores (TC) pour le coke traité et non-traité et la mesure des contributions sous-critique et sur-critique ont permis de révéler que les pores ayant une taille supérieure à cette taille critique jouerait un rôle prépondérant dans la réactivité à l’air du coke. Dans cette thèse, une nouvelle méthode de protection des anodes par l’oxyde de bore a été développée. Ceci consiste à traiter les matières premières, avant la fabrication de l’anode. En utilisant une faible concentration d’oxyde de bore (de l’ordre de ppm) dans le but de limiter le niveau d’impureté dans l’aluminium produit. Les résultats montrent que la réactivité à l’air de l’anode diminue de 15%, le charbonnaille de 90% et le dégagement gazeux (CO2 et CO) de 30%. L'influence de chacun des paramètres (température, concentration, etc.) sur la protection de l’anode a également été optimisée.
Aluminum electrolysis is a process that consumes energy and resources (raw materials, qualified personnel, time, etc.). Several research projects are underway around the world to improve the efficiency of the aluminum manufacturing process, to reduce toxic gas emissions (CO2, CO, CF4, C2F6 ...) and to reduce production costs. One of the current problems of alumina electrolysis is the excessive consumption of carbon anodes. Indeed, these anodes, when they are heated at high temperatures, are attacked by ambient air between 400 and 600 °C, and by the CO2 at 960 °C which results in an over-consumption of carbon, thereby reducing the manufacturing capacity of metallic aluminum per kg of carbon consumed. Currently, the average lifetime of an anode is between 20 and 30 days. The objective of this project is to reduce the reaction rate of anode oxidation under ambient air. Different methods have been developed to obtain an effective and economical protection which would reduce the over-consumption of the carbon anode against the phenomenon of air oxidation. Since boron oxide is known as an inhibitor of carbon/oxygen reaction, several attempts have been made to make a coating on the anode, confirming the inhibitory effect of boron oxide on this reaction, thus allowing protection of the carbon anodes. The influence of each of the parameters (temperature, concentration, duration of impregnation in the solution, etc.) were studied, as well. X-ray tomography showed that the anode is mainly attacked on the surface and that the boron oxide coating creates a physical barrier preventing access of oxygen to the anode. Further studies have been carried out to understand the inhibitor mechanism of boron oxide on carbon-oxygen reaction. According to the literature, boron oxide and boric acid can act in two ways: either by fixing on the anode surface resulting in blocking the active carbon sites or by creating a vitreous layer which serves as a physical barrier to oxygen. A kinetic study has been established which confirms that the number of interactions between oxygen and carbon sites decreases in the presence of boron. ToF-SIMS has revealed that boron is present as an oxide on the anode surface and also in the form of carbon-boron bond (BC-). Therefore, this acts like a chemical protection while boron atoms block the carbon active sites, preventing oxidation. The consumption of the carbon anode in the electrolysis cells is controlled by the impurities and the graphitization level as well as the mass transport through its porous structure. The impregnation of coke particle could have an effect on the porosity and its distribution. Coke particles (from 4000 μm to 4 760 μm in diameter) was impregnated with boron oxide in order to reveal its effect on the porosity. The specific surface area and the volumes of 3 conversion rates of particles (at 0, 15 and 35%) were determined by argon adsorption and mercury infiltration in order to evaluate the contributions of subcritical gasification on the total gasification of the anodes under air at 525 °C. To determine the critical pore size (TC) for the treated and untreated coke, the measurement of internal and external contributions of pores was used. It was revealed that the pore sizes of 0.1-10 μm and larger were the most active pores for the gasification under air. In addition, the volume of only very small pores (0.0004-0.001 μm) was slightly decreased by boron impregnation. However, the contribution of the size range of these small pores to anode gasification is negligible. In this thesis, a new method for the protection of anodes by boron oxide has been developed. This involves treating the raw materials before anode is formed by using a low concentration of boron oxide (in ppm) in order to limit the level of impurities contained in the produced metal. The results performed with standard equipment showed that the air reactivity of the anode decreased by 15%, the dusting by 90% and CO2/CO loss by 30%. The electrical resistivity of the anode was not affected by boron oxide at this low level. The influence of each of the parameters (temperature, concentration, etc.) on anode protection was optimized, as well.

Vascular remodelling due to excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (ECs) has been attributed to the pathogenesis of pulmonary arterial hypertension (PAH). It is an incurable cardiovascular disorder, which leads to right heart failure and death, if left untreated. Heterozygous germline mutations in the bone morphogenetic protein receptor type II (BMPR2) have been linked with the majority (~75%) of the familial form of the disease (HPAH). Mutations in the BMPR2 gene impinge upon the BMP signalling which perturbs the balance between BMP and TGF-β pathways leading to the clinical course of the disease. Current therapies were discovered prior to the knowledge that PAH has substantial genetic components. Hence, this study aims to identify novel therapeutic intervention and provide novel insights into how the dysfunctional BMPRII signalling contributes to the pathogenesis of PAH. This work demonstrates that cryptolepines and FDA approved drugs (doxorubicin, taxol, digitoxin and podophyllotoxin) inhibit the excessive proliferation and induce apoptosis in BMPR2 mutant PASMCs by modulating the BMP and TGF-β pathways. Moreover, established drug PTC124 has also been tested but has failed to promote translational readthrough. I have also shown that dysregulated apoptosis of PASMCs and HPAECs is mediated through the BMPRII-ALK1-BclxL axis. Finally, the siRNA screen targeting approximately 1000 genes has identified novel proteins including PPP1CA, IGF-1R, MPP1, MCM5 and SRC each capable of modulating the BMPRII signalling. Taken together, this study for the very first time has identified novel compounds with pro-BMP and anti-TGFβ activities which may provide therapeutic intervention prior to or after the onset of PAH.
Commonwealth Scholarship Commission in the UK
The full text will be available at the end of the embargo period, 31st July 2024.

NF1 ist eine autosomal dominante Erbkrankheit, die durch inaktivierende Mutationen im Neurofibromin-Gen verursacht wird. NF1 manifestiert sich durch eine erhöhte Tumor-Inzidenz des neuralen Gewebes in der Haut (Neurofibroma). Neben diesen häufigeren klinischen Manifestationen haben rund 50% der NF1-Patienten Skelett-Anomalien. Häufiger sind Röhrenknochen betroffen, die klinischen Symptome reichen von Tibia-Krümmung über Spontanfrakturen bis hin zu Nonunions. Diese Studie analysiert den Heilungsverlauf von Femurfrakturen in Nf1Prx1- Mäusen. Der Frakturkallus von Mäusen wurde an den Tagen 7, 10, 14 und 21 durch µCT, Histologie und molekulare Analysen evaluiert. µCT und histologische Analysen haben eine beeinträchtigte Knochenheilung in Nf1Prx1-Mäusen gezeigt. Eine erhöhte periostale Knochenbildung in den frühen Stadien der Heilung war zu beobachten, sowie eine reduzierte, aber anhaltende Knorpelbildung und Bindegewebs-Akkumulation innerhalb der Fraktur. Wir konnten zeigen, dass der normalen Heilungsprozess durch dieses Bindegewebe behindert wird, welches durch alpha smooth muscle actin-positive Myofibroblasten gebildet wird, die ihrerseits aus einer bisher noch nicht identifizierten Muskelfaszie abgeleitet sind. Dieser Zusammenhang wird durch eine Microarray-Analyse der Kallus-Gewebe bestätigt, die ergab, dass durch den Knock-Out Gene reguliert wurden, die in Physiologie, Proliferation und Differenzierung von Muskelzellen involviert sind. Darüber hinaus waren extrazelluläre-Matrix-Gene in den Mutanten hoch regeuliert. Zusammenfassend konnten wir zeigen, dass eine Ähnlichkeit des Heilungsverlauf zwischen dem Nf1Prx1-Mausmodell und NF1-Patienten besteht. Folglich kann an diesem Mausmodell untersucht werden, durch welche Mechanismen die Mutationen im NF1 zu Knochenheilungsstörungen führen. Außerdem konnte in einer Pilotstudie der Effekt des Neurofibromin-Mangels auf die Knochenheilung durch Behandlung mit MEK-Inhibitoren in vitro und in vivo weitestgehend behoben werden
Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 patients – around 50% – have abnormalities of the skeleton. Long bones are often affected, and the clinical signs range from tibial bowing to spontaneous fractures and even non-unions. Moreover, NF1 mice models could provide the understanding of the cell types involved in the resulting non-union and their behavior. This study analyzed the healing progress of femur fractures in a model of NF1 long bone dysplasia. Fracture callus was assessed at days 7, 10, 14, and 21 by µCT, histology, biomechanics, and molecular analyses. Bone healing was impaired in Nf1Prx1 mice femoral fracture. Results revealed increased periosteal bone deposition at the early stages of healing, decreased but persistent cartilage formation concomitant with fibrous tissue accumulation within the fracture site, decreased torsional stiffness, decreased bone mineral density, and increased fibrous tissue infiltration in the callus of mutant mice. This fibrous tissue accumulation hindered bone fracture healing, and was deposited by alpha smooth muscle actin-positive myofibroblasts, which were derived from a yet unidentified muscle fascia. This is further supported by the microarray analysis of callus tissues showing that genes crucial to muscle cells physiology, proliferation and differentiation were affected. In addition, extracellular matrix related genes were up-regulated in the mutants. In summary, this study shows a resemblance in the healing progression to the Nf1Prx1 mice model and NF1 patients, thereby, confirming the suitability of this mice model to explore the mechanism by which mutations in NF1 lead to non-unions. Moreover, in vitro and in vivo pilot assessments of MEK inhibitor treatment demonstrated a potential remedy for the lack of neurofibromin in bone healing.