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El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP. / National Institutes of Health / Revisión por pares

Student Number : 9903144J - MSc (Med) dissertation - Faculty of Health Sciences / Since the introduction of vaccination against hepatitis B virus (HBV) infection in South Africa, at least one case of infection despite vaccination has occurred. The purpose of this study was to determine whether this infection was the result of mutations within the region of the surface (S) gene encoding the a determinant epitopes of the hepatitis B surface antigen, which permitted viral vaccine-escape. HBV DNA was extracted from the serum and liver tissue of the patient and amplified within the complete 3 215 bp genome and S gene, respectively. Following cloning, sequencing revealed a minor population displaying unique or uncommon S gene mutations that resulted in C138R, C139R, K141R, P142L, T143A, N146D, and T148A amino acid substitutions in the clones from the serum, and C139Y and D144N in the clones from the liver. Such isolates may represent South African HBV vaccine-escape mutants that caused chronic infection in the host prior to their reversion to wild-type.

Collagen and glycosaminoglycans, such as hyaluronan and chondroitin sulfate, are the major components of bone extracellular matrix, and extracellular matrix composites are being evaluated for a wide range of clinical applications. The molecular and cellular effects of native and sulfatemodified glycosaminoglycans on osteocytes were investigated as critical regulators of bone remodeling. The effects of glycosaminoglycans on viability, necrosis, apoptosis, and regulation of gene expression were tested in two osteocyte-like cell lines, the murine MLO-Y4 and the rat UMR 106-01 cells. Glycosaminoglycans were non-toxic and incorporated by osteocytic cells. In MLO-Y4 cells, sulfation of glycosaminoglycans led to a significant inhibition of osteocyte apoptosis, 42% inhibition for highly sulfated chondroitin sulfate and 58% for highly sulfated hyaluronan, respectively. Cell proliferation was not affected. While treatment with highly sulfated chondroitin sulfate increased cell viability by 20% compared to the native chondroitin sulfate. In UMR 106- 01 cells, treatment with highly sulfated hyaluronan reduced the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio by 58% compared to the non-sulfated form, whereas highly sulfated chondroitin sulfate led to 60% reduction in the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio in comparison to the native chondroitin sulfate. The expression of SOST, the gene encoding sclerostin, was reduced by 50% and 45% by highly sulfated hyaluronan and chondroitin sulfate, respectively, compared to their native forms. The expression of BMP- 2, a marker of osteoblast differentiation, was doubled after treatment with the highly sulfated hyaluronan in comparison to its native form. In conclusion, highly sulfated glycosaminoglycans inhibit osteocyte apoptosis in vitro and promote an osteoblast-supporting gene expression profile.