The nitrogen-containing bisphosphonates (NBPs) are the most widely used treatment for diseases involving excessive osteoclastic bone resorption, such as osteoporosis. The clinical efficacy of NBPs is due in large part to their affinity for bone mineral, but it has been suggested that lowering affinity may have benefits due to altered distribution and duration of action possibly allowing direct anti-tumour effects. In addition, the phosphonocarboxylate (PC) analogues inhibit prenylation more selectively through a different enzyme target, Rab geranylgeranyl transferase (RGGT), which may offer additional benefits by reducing side-effects associated with farnesyl diphosphate synthase (FPPS) inhibition. Using fluorescent analogues of PCs and NBPs demonstrated that mineral affinity not only affects initial bone-binding, but also influences desorption, reattachment and penetration at the bone surface, suggesting that lower affinity compounds have lower retention and increased access to other cell types, such as tumour cells. The work presented aimed to investigate the potential of low affinity analogues by characterising their intracellular potency for inhibiting their target enzymes. The results showed that modification to the phosphonate groups to produce phosphonoalkylphosphinate analogues reduced potency for inhibiting FPPS. By contrast, removal of one of the phosphonate groups to give a monophosphonate changed the target enzyme to RGGT. Modifications to the R1 side-chain (substituting with hydrogen or a halogen) of both NBPs and PCs were studied and showed contrasting results, modifications to the R1 side-chain of NBPs affect their ability to inhibit FPPS whereas the same modification to PCs is insignificant for inhibiting RGGT. This showed the distinction between the structural requirements for inhibition of RGGT and FPPS and furthers the understanding of the structure-activity relationships of both NBPs and PCs which could guide future drug design. Within this thesis the most potent inhibitor of RGGT to date, 3-IPEHPC, was characterised which in addition to having therapeutic potential may be used as tool to investigate the importance of Rab prenylation for cellular function.

Orientador: Carlos Rossa Junior / Banca: Karina Gonzales Silverio Ruiz / Banca: Marcia Pinto Alves Mayer / Banca: Alexandra Ivo de Medeiros / Banca: Luis Carlos Spolidorio / Resumo: As interações microrganismo-hospedeiro se iniciam pela detecção de padrões moleculares associados a microrganismos (MAMPs) por receptores semelhantes à Toll (TLR) e por proteínas com domínio de ligação à nucleotídeos e oligomerização (Nod) na resposta imune inata. No entanto, como a cavidade bucal saudável é continuamente colonizada por microrganismos não patogênicos que também apresentam MAMPs, deve haver um mecanismo endógeno de regulação negativa da resposta do hospedeiro para evitar uma resposta exagerada e desnecessária com consequências negativas ao hospedeiro. Os mecanismos associados à distinção de microrganismos comensais e patogênicos na mucosa bucal são ainda pouco compreendidos. As proteínas Nod foram inicialmente descritas como 'TLRs intracelulares' capazes de reconhecer MAMPs no citosol; no entanto, estudos in vitro indicam que Nod têm papel relevante na regulação da expressão de RANKL e OPG induzidas por antígenos microbianos, bem como na modulação da atividade de vias de sinalização intracelular associadas à expressão de citocinas diretamente relacionadas à regulação do turnover do tecido ósseo. Devido à escassez de informações sobre o papel das proteínas Nod na modulação das interações microrganismo-hospedeiro na mucosa oral e com base nestas informações, nossa hipótese é que as proteínas Nod tem um papel relevante na modulação da reação inflamatória e suas consequências, incluindo a reabsorção do osso alveolar. Para testar esta hipótese, os objetivos específicos propostos foram: avaliar em camundongos knockout para Nod1, Nod2 ou Rip2, através de microtomografia computadorizada e avaliações histológicas descritivas, estereométricas e imunohistoquímicas (TRAP), o papel das proteínas Nod na inflamação e reabsorção óssea associadas à doença periodontal experimental induzida por bactérias inativadas por calor... / Abstract: Recognition of pathogenic bacteria by the host is initially mediated by the innate immune response through detection of microbe-associated molecular pattern (MAMPs) by Toll-like receptors (TLR) and Nucleotide-oligomerization domain (Nod) proteins. Since the oral cavity, as well as other mucosal surfaces, is continuously colonized with non-pathogenic bacteria that also present MAMPs, there has to be an endogenous negative regulatory mechanism in place to prevent an overt host response with deleterious consequences. Specifically in the oral mucosa, it is not clear how the immune system is able to quickly distinguish between commensal and pathogenic bacteria and tailor the host response. Nod proteins were initially described as 'intracellular TLRs' that recognize MAMPs associated with bacteria invading the cytosol; however these proteins have been shown to modulate the activation of various signaling pathways involved in the expression of inflammatory genes, including p38 MAPK and NF-κB in concert with TLR stimulation. There is paucity of information on the in vivo role of Nod proteins in the modulation of host-microbe interactions in the oral mucosa. Based on this information, our hypothesis is that Nod proteins play an important role in the modulation of the inflammatory reaction associated with periodontal diseases and its consequences, including alveolar bone resorption. To test this hypothesis, we propose the following specific aims: Assess the role of Nod proteins in the inflammation and bone resorption in experimentally-induced periodontal disease Describe the influence of Nod proteins on the cytokine and signaling networks associated with periodontal disease. / Doutor

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / The bisphosphonate tiludronic acid (TIL) presents anti-resorptive and anti-inflammatory properties and it has not been evaluated in the association periodontitis-diabetes mellitus (DM) to date. The purpose of this study was to evaluate the effects of local administration of TIL on experimental periodontitis (EP) in rats with streptozotocin (STZ)-induced DM. On day 1, thirty two rats received STZ injection. The animals were divided into groups (n=8): DM/C (Control), DM/EP, DM/EP/TIL1 and DM/EP/TIL3. In groups EP, a ligature was placed around the cervical area of mandibular first molars at day 8. In groups DM/EP/TIL1 and DM/EP/TIL3, TIL solutions of 1 and 3 mg/kg body weight, respectively, were injected into the buccal gingival margin of mandibular first molars every other day. Animals were euthanized at day 18. Histomorphometric analyses were performed. Data were statistically analyzed (p<0.05). Group DM/EP/TIL3 presented reduced alveolar bone loss and attachment loss when compared with group DM/EP (p<0.05). Within the limits of this study, it can be concluded that i) the local administration of TIL solutions presented a protective effect on tissue destruction in EP in diabetic rats and ii) the dosage of TIL may influence its effects. / O bisfosfonato Ãcido tiludrÃnico (TIL) apresenta propriedades antirreabsortivas e anti-inflamatÃrias e ainda nÃo foi estudado na associaÃÃo periodontite-diabetes mellitus (DM). O objetivo deste estudo foi avaliar os efeitos da administraÃÃo local do TIL na periodontite experimental (PE) em ratos com DM induzido por streptozotocina (STZ). No 1 dia, trinta e dois ratos receberam injeÃÃo de STZ. Os animais foram divididos nos grupos (n = 8): DM/C (Controle), DM/PE, DM/PE/TIL1 e DM/PE/TIL3. Nos grupos PE, uma ligadura foi colocada na Ãrea cervical dos primeiros molares inferiores no 8 dia. Nos grupos DM/PE/TIL1 e DM/PE/TIL3, soluÃÃes de TIL (1 e 3 mg/kg de peso corporal, respectivamente) foram injetadas na margem gengival vestibular dos primeiros molares inferiores em dias alternados. Os animais foram submetidos à eutanÃsia no 18 dia. AnÃlises histomorfomÃtricas foram realizadas. Os dados foram estatisticamente analisados (p<0,05). O grupo DM/PE/TIL3 apresentou perda Ãssea alveolar e perda de inserÃÃo reduzidas quando comparado com o grupo DM/PE (p<0,05). Dentro dos limites deste estudo, pode-se concluir que i) a administraÃÃo local de soluÃÃes de TIL apresentou um efeito protetor na destruiÃÃo tecidual na PE em ratos diabÃticos e ii) a dosagem de TIL pode influenciar seus efeitos.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 44-57).

Thesis (M.S.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 68-76).

No description available.

As implant survival extends into the second and third decades focal osteolysis around cemented femoral components in total hip replacement is emerging as an important failure mechanism. Whilst the problem of focal osteolysis is well recognised, there are many aspects of its development which are poorly understood. The broad aim of this thesis is to try to provide some insights into how, why and where focal osteolysis develops around the cemented femoral component. There are broadly two sections to this thesis, chapters 2-5 present clinical and geometrical studies and chapters 6-10 a series of experimental studies. The aim of the first section was to establish what is observed in clinical practice, the aim of the second to try to explain these findings. A mid-term clinical study showed that focal osteolysis is more common with rough than polished stems that differed in no aspect other than their surface finish. Further studies established that focal osteolysis is probably always associated with defects in the cement mantle. These defects occur anteriorly at the mid-stem of the prosthesis and posteriorly at the component tip. The distribution of focal osteolysis and its strong association with cement mantle defects suggests the importance of the stemcement interface as a pathway for fluid and debris to reach the distal femur. However, at 15-25 years, osteolysis rarely develops with the polished Exeter stem even in the presence of confirmed defects in the cement mantle, suggesting that the stem seals the stem-cement interface against fluid and debris. In an attempt to explain the clinical findings a series of bench top experiments were undertaken. These studies showed that the behaviour of fluid and dye at the stemcement interface was significantly influenced by component surface finish. Bonded and debonded stem-cement interfaces of rough stems provided an incomplete barrier to fluid movement along this interface. In contrast, polished stems both bonded and debonded were able to provide a seal at the stem-cement interface. The seal at this interface was improved with component subsidence in the presence of rotational stability. It is believed that this thesis provides a rationale explanation for why focal osteolysis rarely develops around the Exeter stem in clinical practice. It also explains how, where, and why osteolysis develops around certain designs of cemented femoral components used in total hip replacement.

Inflammatory bone diseases like rheumatoid arthritis and periodontal disease lead to increased bone loss in the inflamed areas. The multinucleated bone resorbing cells, the osteoclasts, present in these diseases are larger than normal, and these larger osteoclasts (10+ nuclei) resorb more bone and more often than smaller osteoclasts (2-5 nuclei). Thus, the focus of this thesis was to determine if there are differences in mechanisms of osteoclast activation between large and small osteoclasts. Experiments using authentic rabbit osteoclasts and RAW 264.7-derived osteoclasts revealed differences in the expression of a number of activating factors; with large osteoclasts expressing higher levels of activating receptors (RANK, IL-1RI, TNFR1 and integrins αv and β3), as well as enzymes involved in cellular resorption, while small osteoclasts expressed higher levels of an alleged fusion receptor and the inhibitory receptor, IL-1RII. Further studies revealed that large osteoclasts more readily responded to stimulation by IL-1 compared to small osteoclasts and at lower concentrations suggesting this is a result of their higher expression of activating receptors. Differences in responses to the IL-1 isoforms, IL-1α and IL-1β, were also seen in large osteoclasts: IL-1α generated more large osteoclasts over the course of differentiation, while IL-1β induced changes in cell morphology and in the induction of integrin β3 phosphorylation. These observations suggested that differences in osteoclast responses are induced by IL-1α and IL-1β and it led to the hypothesis that there are differences in signaling between large and small osteoclasts. To elucidate differences in signaling mechanisms a signaling pathway microarray was used which revealed higher expression of Vegfa in large compared to small osteoclasts. Osteoclast differentiation with RANKL increased Vegfa gene expression in a time-dependent manner and VEGF-A secretion was elevated in populations enriched for large osteoclasts. Furthermore, mechanistic studies with inhibitors of transcription factors involved in differentiation revealed that RANKL-mediated Vegfa expression in large osteoclasts was regulated by the NF-κB pathway via induction of Hif1α. These results support the hypothesis that signaling differences exist between large and small osteoclasts and implicates VEGF-A in osteoclast hyperactivity in inflammatory conditions.

This study used vital bone markers to investigate mineralized tissue adaptation in the periodontium of rats after a hypothermic insult to their maxillary first molars. This hypothermic insult has been shown in previous studies to induce aseptic root resorption with variable effects on ankylosis. A secondary objective was to assess the pulpal changes that occurred concurrent with the changes in the periodontium. Four groups of 7, eight-week old male Sprague Dawley rats were assigned to be euthanased at the day 7, 14, 21 and 28 observation periods. At day 0, 4 groups of 6 rats were subject to a single 20 minute application of dry ice on their maxillary right first molar. The remaining 1 rat within each group did not receive the dry ice. All rats were given 2 sequential bone labels, calcein 5mg/kg and alizarin red 30mg/kg, administered intraperitoneally 8 days apart. The timing of the labels was such that all rats were euthanased 2 days after the last label. The rat maxillae were fixed in ethanol and embedded undecalcified in methylmethacrylate. Ten micrometre coronal sections were obtained through the furcation of the first molars with three of each group of ten consecutive sections being unstained, stained with von Kossa/ hematoxylin and eosin counterstain, or decalcified and stained with hematoxylin and eosin, respectively. Unstained sections were viewed under fluorescence, while transmitted light microscopy was used for the other sections. Mineral apposition rates along the bone, root and pulpal surfaces as well as periodontal ligament width were measured using histomorphometry. Semiquantitative measurements of the resorptive surfaces within the periodontium were also noted. Multivariate and negative binomial regression statistical analyses were used to identify influencing variables. A focal pattern of ankylosis was observed at days 14 and 21 in 3 rats and was not seen at day 28. In both the treated and control teeth, appositional activity was greatest along bone and least along the root surface. Mineral tissue apposition rates along the bone and root surface displayed an initial spike during day 14 but declined to levels of the control teeth by day 28. A longer time lag was observed with appositional activity in the pulp which also displayed a declining trend towards the control teeth values by day 28. Resorption levels along the root surface continued to remain significantly (p<0.0001) elevated. The significantly (p<0.0001) wider periodontal ligament width in the treated molars showed a declining trend towards that of the control teeth by day 28. There was a temporary disturbance of mineralized tissue adaptation on the bone and root surfaces with a recovery of cellular vitality within the periodontium and pulp and a trend towards homeostasis of the periodontal ligament width. The null hypotheses that a single prolonged thermal insult on a rat has no effect on mineralized tissue adaptation within the periodontium and pulp chamber and that the periodontal ligament width within the interradicular region does not change in response to thermal trauma induced by the present study were rejected. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345386 / Thesis (D.Clin.Dent) - University of Adelaide, School of Dentistry, 2008

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.