No description available.

No description available.

No description available.

No description available.

No description available.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science (Medicine) in Human Genetics / Maintaining suitable bone health is emerging as a serious point of concern worldwide, as the prevalence of skeletal disorders threatens to reach unmanageable proportions. Despite unfavourable environmental factors, black South Africans demonstrate elevated bone mass, especially at the femoral neck, when compared to whites. Genetic factors are thought to mediate this effect, which may have clinical or therapeutic value. Using a candidate gene approach, this study investigated associations of six candidate genes (ESR1, TNFRSF11A, TNFRSF11B, TNFSF11, SOST and SPP1) with bone mineral content amongst pre-pubertal black South African children that formed part of the longitudinal Birth to Twenty cohort. The GoldenGate genotyping assay with VeraCode microbeads was used to genotype 151 black children at 366 polymorphic loci, including 112 previously associated and 254 tagging SNPs. A linear regression approach was implemented to highlight significant associations whilst adjusting for height, weight, sex and bone area. Twenty seven markers (8 previously associated and 19 tag SNPs; P <0.05) were found to link to either femoral neck (18) or lumbar spine (9) BMC. These signals derived from three genes, namely ESR1 (17), TNFRSF11B (9) and SPP1 (1). One marker (rs2485209) maintained its association with the femoral neck after correction for multiple testing (P = 0.038). These results fully support the existence of a strong genetic effect acting at the femoral neck in African ancestry individuals. Tagging SNP signals suggest the presence of a number of population specific variants that require further investigation. Combined, these markers may help to account for increased bone mass amongst black South Africans, when adjusted for covariates.

A number of growth factors and cytokines involved in the local regulation of bone remodelling are either synthesised by osteoblasts or have osteoblasts as their target. These include the RANK-L/OPG system, the gp130 cytokine family, including IL-6, and insulin like growth factors. In addition, aberrant cytokine signalling is strongly linked with pathological states characterised by increased bone resorption, including osteoporosis and renal osteodystrophy. The range of action and potency of these osteotropic cytokines requires that their actions are tightly regulated. Amongst such potential control mechanisms are the suppressors of cytokine signalling (SOCS), the presence and role of which in bone has not been studied in detail. The aim of this thesis was (i) to examine the direct effect of uraemia on cytokine release in human osteoblastic cells; (ii) to determine if the regulatory SOCS genes are expressed in these cells and, if so, (iii) to characterise their functional significance. In initial studies, osteoblastic cells were cultured in media containing sera from either healthy volunteers or haemodialysis treated chronic kidney disease patients. Concentrations of OPG and IL-6 were then measured in harvested supernatants. Additionally, individual serum samples collected prior to, and during, a haemodialysis (HD) session were assayed for IL-6, IL-1β and soluble IL-6 receptor (sIL-6R). HD patients had significantly higher concentrations of IL-6 than normal subjects, but there were no significant differences in either IL-1β or sIL-6R. These concentrations did not change significantly during HD. There were no differences in OPG production by osteoblastic cells after exposure to either normal or uraemic serum. Incubation with untreated sera from normal subjects increased IL-6 production by ~6-fold above control, whereas sera from uraemic subjects increased it only ~2-3-fold. HD did not restore the capacity of uraemic serum to augment IL-6 release to the same degree as normal serum. Further work examined a variety of osteotropic stimuli for their ability to induce SOCS1- 3 and CIS expression in human osteoblastic cells. The utility of both conventional RTPCR and fluorescence-based kinetic real time PCR for this purpose are compared. These SOCS were found to be expressed constitutively and could be induced to a variable degree by relevant growth factors. In general, the temporal pattern of SOCS expression was consistent with a negative feedback function. Potential functionality was explored following transfection with SOCS1 and SOCS3 plasmid DNA. Significantly enhanced IL-6 secretion was found in both the basal and stimulated state, whilst OPG production was enhanced only in the latter. Function was also studied in the context of osteoblastic apoptosis, the regulation of which is highly relevant to skeletal disease. Initial experiments developed a framework for subsequent studies: serum starvation for 24h produced reproducible cell death that could be attenuated in a dose dependent manner by IGF-I. SOCS1 and SOCS3 overexpression had limited influence on osteoblast survival, whereas gene knock down experiments using siRNA indicated that IL-1β-induced cell death is mediated differentially, depending on the type of cell death involved. SOCS1 and SOCS3 are involved in the apoptotic cascade, while IL-1β-induced necrosis appears to be independent of SOCS3. Collectively these studies demonstrate that the augmentation of IL-6 production by osteoblastic cells after exposure to normal serum is greater than after uraemic serum. HD does not correct this disparity; perhaps indicating a non-dialysable inhibitor of IL-6 release is involved in the dysregulated bone turnover of uraemic patients. Further work establishes the constitutive presence of the SOCS family in human osteoblastic cells, as well as their transient inducibility by key osteotropic stimuli. Several novel aspects of SOCS function, including influence on IL-6 and OPG production and involvement within apoptotic pathways are demonstrated.

Thesis (M.D.)--Boston University

Thesis (M.S.)--University of Michigan, 1948.

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.