Lipid-protein and protein-protein interactions in the mechanisms of photosynthetic reaction centre and the Na+,K+-ATPase

Lipid-protein and protein-protein interactions are likely to play important roles in the function and regulation of charge-transporting membrane proteins. This thesis focuses on two different membrane proteins, the photosynthetic reaction centre (RC) from purple bacteria and the Na+,K+-ATPase. The influence of the lipid surroundings and cholesterol derivatives on the kinetics of electron transfer of the RC were investigated by reconstituting the protein in phosphatidylcholine vesicles containing cholesterol and derivatives known to modulate the membrane dipole potential. The experiments performed on the Na+,K+-ATPase were designed to contribute to a better understanding of the role that oligomeric protein-protein interactions have in the enzyme’s mechanism. Our results show that the cholesterol derivatives significantly modify the electron transfer kinetics within the RCs and their multiphasic behavior. These effects seem to be associated with the extent of the dipole potential change experienced by the RC within the phospholipid membrane. Indeed, the largest effects on the rates are observed when 6-ketocholestanol and cholesterol are present, consistent by with their previously demonstrated significant increase of the dipole potential. We interpret this data as indicating an increased free energy barrier for protons to enter the protein. The consequences of the increased dipole potential seem to be experienced across the entire protein, since the rates of the P+QA- charge recombination in the presence of AQ- acting as QA are also modified by the same effectors. Also interesting is the effect of the dipole potential on the two conformational states of the RCs (previously reported) as revealed by the biphasic decays of the electron transfer kinetics. In particular, we report for the first time a biphasicity of the P+QA- charge recombination in the WT RCs. This non exponential behaviour, absent in the phospholipid membrane or isolated RCs, is induced by the presence of the cholesterol derivatives, suggesting that the equilibration time between the two RC conformations is slowed down significantly by these molecules. According to this work, the dipole potential seems to be an important parameter that has to be taken into account for a fine understanding of the charge transfer function of the RCs. Reported literature values of the dissociation constant, Kd, of ATP with the E1 conformation of the Na+,K+-ATPase based on equilibrium titrations and kinetic methods disagree. Using isothermal titration calorimetry (ITC) and simulations of the expected equilibrium behaviour for different binding models, this thesis presents an explanation for this apparent discrepancy based on protein-protein interactions. Because of the importance of Mg2+ in ATP hydrolysis, kinetic studies of Mg2+ binding to the protein were also carried out. These studies showed that ATP alone is responsible for Mg2+ complexation, with no significant contribution from the enzyme environment

[Loading and strength of single- and multi-unit fixed dental prostheses. 1. Retention and resistance]

Item does not contain fulltextThe degree to which single- and multi-unit fixed dental prostheses are able to withstand loading forces is dependent, among other things, on the quality of their retention and resistance. The quality of the retention and resistance of the configuration of an abutment tooth prepared for a metal and metal-ceramic single-unit fixed dental prosthesis is determined by the configuration's convergence angle, the height, the volume, the interocclusal space, the cervical outline design, the additional preparations, the quality of the (build-up) restoration, and the surface roughness. A silicate ceramic single-unit fixed dental prosthesis is attached through adhesion using a composite cement, but the retention and resistance of an oxide ceramic single-unit fixed dental prosthesis is dependent on the abutment tooth configuration. Most types of multi-unit fixed dental prosthesis have the following additional retention and resistance determining factors: the position in the occlusal system, the number of abutment teeth and their mutual configurations, and the length of (cantilever) pontics. A resin-bonded fixed partial denture's retention and resistance are determined by its bonding as well as its enamel surface coverage and its resistance preparations

The CHEK2*1100delC variant acts as a breast cancer risk modifier in non-BRCA1/BRCA2 multiple-case families.

Item does not contain fulltextThe frame-shifting mutation 1100delC in the cell-cycle-checkpoint kinase 2 gene (CHEK2) has been reported to be associated with familial breast cancer in families in which mutations in BRCA1 and BRCA2 were excluded. To investigate the role of this variant as a candidate breast cancer susceptibility allele, we determined its prevalence in 237 breast cancer patients and 331 healthy relatives derived from 71 non-BRCA1/BRCA2 multiple-case early onset breast cancer families. Twenty-seven patients (11.4%) were carrying the CHEK2*1100delC variant. At least one carrier was found in 15 of the 71 families (21.1%). There was no evidence of cosegregation between the variant and breast cancer, but carrier patients developed breast cancer earlier than did noncarriers. We studied CHEK2 protein expression in 111, and loss of heterozygosity at CHEK2 in 88 breast tumors from these patients. Twelve of 15 tumors from carriers showed absent protein expression as opposed to 3 of 76 tumors from noncarriers (P < 0.001). CHEK2 loss of heterozygosity was associated with absence of protein expression but not with 1100delC carrier status. Thus, selecting for breast cancer cases with a strong familial background not accounted for by BRCA1 or BRCA2 strongly enriches for carriers of CHEK2*1100delC. Our results support a model in which CHEK2*1100delC interacts with an as yet unknown gene (or genes) to increase breast cancer risk

RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gels systems.

Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in combination with RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3, EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however, are more or less restricted to HSCR associated with specific phenotypes. We have developed a novel comprehensive mutation detection system to analyse all but three amplicons of the RET and GDNF genes, based on denaturing gradient gel electrophoresis. We make use of two urea-formamide gradients on top of each other, allowing mutation detection over a broad range of melting temperatures. For the three remaining (GC-rich) PCR fragments we use a combination of DGGE and constant denaturing gel electrophoresis (CDGE). These two dual gel systems substantially facilitate mutation scanning of RET and GDNF, and may also serve as a model to develop mutation detection systems for other disease genes. In a screening of 95 HSCR patients, RET mutations were found in nine out of 17 familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a sporadic cas