A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, June 2017 / Introduction: Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease of unknown aetiology. There is growing evidence environmental factor(s) trigger the disease in the genetically susceptible host. Fragment crystallisable receptor (FCR) genes encode receptors that recognise the fragment crystallisable (Fc) portion of immunoglobulins (IgG) play an important role in the removal of antigen-antibody complexes from the circulation. Genes that code for these receptors have shown to be associated with susceptibility to SLE in various populations. The aim of the present study is to determine the role of single nucleotide polymorphisms (SNPs), allotypes and copy number variations of FC Gamma receptor genes IIIA and IIIB in susceptibility for the Black South Africans with SLE. Methods: DNA from 162 Black South African SLE patients and 155 matched controls were investigated using Taqman assays to determine SNP genotyping differences (FCGRIIIA) and copy number variation (CNV) number (FCGRIIIB). A PCR was optimised in order to determine the allotype differences (FCGRIIIB) via agarose gel electrophoresis. Statistical analyses were then performed on the data to see if the results displayed significance in susceptibility to SLE. Results: The minor allele of the allotypes (FCGRIIIB) and the rs396991 SNP (FCGRIIIA) were not statistically significant in conferring susceptibility to SLE in cases or controls. The rs10127393 SNP (FCGRIIIA) was shown to be monomorphic within both cases and controls for the T allele and is not associated with SLE. The cumulative percentage of copy numbers (FCGRIIIB) ≤2 copies were 0.6% larger in cases than seen in controls. Although this was not significant, this was what has been previously suggested in the literature. Almost half of the cases (43.8%) had lupus nephritis (LN). Upon investigation the NA1/NA2 alleles were found to confer susceptibility to LN (p=0.018), whereas the rs396991 G allele did not (p=0.643). Conclusion: In this study the allotypes, SNPs and CNV investigated were not found to confer susceptibility to SLE. However, subtle trends suggest that further studies are required with larger sample sizes to acquire more data. Almost half of the cases were diagnosed with LN and the NA2 allele was shown to be a risk factor in developing LN. / MT2017

Molecular dynamics (MD) simulations were used to investigate the structural basis for the functions of three proteins: Fc(gamma) receptor III (CD16), von Willebrand factor (VWF), and integrin. CD16, a heavily glycosylated protein expressed on human immune cells, plays a crucial role in immune defense by linking antibody-antigen complexes with cellular effector functions. Glycosylation of CD16 decreases its affinity for IgG. MD simulations were run for CD16-IgG Fc complexes with or without an N-glycan on CD16. The two simulated complexes show different conformations. Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) approach was used to calculate the binding free energy of the CD16-IgG Fc complexes. The calculated binding free energy helped to identify critical residues. VWF, a multimeric multidomain glycoprotein, initiates platelet adhesion at the sites of vascular injury. A specific VWF metalloprotease, A Disintegrin And Metalloprotease with ThromboSpondin motifs member 13 (ADAMTS-13), cleaves the Tyr1605-Met1606 bond in the VWF A2 domain to generate the full spectrum of plasma VWF species. Shear stress or denaturants assist VWF cleavage by ADAMTS-13 due to the unfolding of A2. MD was used to simulate the unfolding processes of A2 under force or high temperature. The beta-strands of A2 were pulled out sequentially by force, during which the cleavage site changed in steps from the fully buried state to the fully exposed state. Thermal unfolding follows a very different pathway. Integrins are adhesion molecules mediating cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Experiments suggest that integrins can undergo a large-scale change from a bent to an extended conformation, associating with a transition from low to high affinity states, i.e., integrin activation. Steered MD was utilized to simulate the bent-to-extended conformational transition in time of aVb3 integrin. The integrin was observed to change smoothly from the bent to the extended conformation. One major energy barrier was overcome, corresponding to the disruption of the interactions at Hybrid/EGF4/bTD interfaces. A partially extended conformation tends to bend back while a fully extended conformation is stabilized by the coordination of Asp457 with Ca2+ at alpha-genu. Unbending with separated legs overcomes more energy barriers.