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Human Papillomaviruses (HPVs) are circular double-stranded DNA (dsDNA) viruses that infect human cutaneous and mucosal tissue. Most HPV infections are benign or cause only minor pathologies. However, infection with one of the ~15 high risk types of HPV is associated with a variety of head/neck and anogenital cancers. All told, HPV infection is thought to cause ~5% of all human cancers and cause ~275,000 deaths per year. Despite causing immense morbidity and mortality, many aspects of how HPV virions successfully establish infection in host cells remain poorly characterized. Infection begins with HPV virions binding the cell surface, where they are modified by the host protease furin. The HPV virions are then endocytosed by association with an unknown entry receptor(s). After endocytosis the HPV minor capsid protein L2 acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of these critical processes remain poorly characterized. In this work we investigate the viral and host factors involved in the penetration of host membranes by the HPV L2/vDNA complex. First, we elucidated many of the viral and host factors necessary for furin cleavage of L2. We also demonstrate that furin cleavage mediates the homo-oligomerization and membrane insertion of L2. Finally we demonstrate that complete translocation across the limiting membrane is dependent on host cell entry into mitosis. Overall this work provides novel insight into the molecular mechanisms used by HPV virions to breach host membranes and establish infection.

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein existing in both soluble monomeric conformation as well as integral-membrane form. The role of the domain interface in the conversion between these species was investigated. Bioinformatics-based analysis was undertaken to compare and contrast the domain interfaces of dimeric GSTs with their monomeric homologues CLIC1 and CLIC4. The mutants CLIC1-M32A and CLIC1-E81M were used as experimental case studies on the role of domain-domain interactions in the stability and folding of CLIC family proteins. A consensus interface was revealed with the prominent interaction being a conserved inter-domain lock-and-key type motif previously studied in class Alpha GSTs (Wallace et al., 2000). A number of domain-interface interactions were found to be unique to the CLIC family and as such thought to play a role in the conversion of these proteins from their soluble form to an integral membrane form. Overall the domain interfaces of monomeric CLIC1 and CLIC4 did not differ significantly from the domain interfaces of dimeric GSTs. The removal of the unique CLIC family salt-bridges between Arg29 and Glu81 and the cavity forming domain interface mutation Met32Ala did not induce significant changes in the conformational flexibility of the native state. The true role of the Arg29-Glu81 salt-bridges was masked by the introduction of stabilizing hydrophobic contacts. Removal of the inter-domain lock-and-key interaction destabilized CLIC1 significantly with concomitant loss in cooperative folding that resulted in the stabilization of a molten globule-like species. This intermediate state was less stable and less structured than the equilibrium intermediate of wtCLIC1 at pH 5.5. However the bulk of the structures found to unfold during intermediate-species formation was the same in mutant and wild-type proteins. It was concluded that formation of the membrane-competent form of CLIC1 involves re-structuring of the N-terminal thioredoxin domain that takes place after destabilization of the salt bridges connecting h1 and h3 and uncoupling of the inter-domain lock-and-key motif.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. Dysfunction in membrane insertion has been implicated in several pathologies including apoptosis, cancer and homeostatic imbalance. The transmembrane domain (TMD) is implicated in membrane penetration and pore formation and is therefore a key target for understanding amphitropism in CLIC1. The mechanism by which the TMD binds, inserts and oligomerises in membranes to form a functional chloride channel is unknown. Here the secondary, tertiary and quaternary structural changes of the CLIC1 TMD and several TMD mutants are reported in an attempt to elucidate the membrane insertion mechanism. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol (TFE), SDS micelles and POPC liposomes using far-UV CD, fluorescence and UV absorbance spectroscopy. The results suggest a fourstep mechanism whereby the TMD, which is unfolded in buffer, refolds into a helix which partitions onto the membrane, followed by insertion and dimerisation to form a membranecompetent protopore complex. These helices associate via a Lys37-mediated cation-π interaction to form weakly active dimers. The complex is then tethered to the membrane by a cationic motif acting as an electrostatic plug. Thus, electrostatic interactions provide both a strong thermodynamic driving force for helix-helix association as well as structural integrity within the membrane. This represents an important step towards understanding how amphitropism occurs in CLIC1 and offers a unique insight into how CLIC1 and other proteins defy the ‘one-sequence one-fold’ hypothesis.

Student Number : 9605361W - PhD thesis - School of Pathology - Faculty of Science / Malaria is one of the most debilitating parasitic infections to have afflicted humanity and remains an expanding health risk for many countries. This is attributed largely to the complexity of the parasite’s life cycle and refined ability to evade host immunity. During development within the erythrocyte, Plasmodium falciparum induces a wide array of changes to the ultrastructure, function and antigenic properties of the host membrane. Numerous proteins encoded by the parasite associate with the erythrocyte skeleton and appear to be essential for P. falciparum survival. The elucidation of new protein-protein interactions has therefore formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of phage display technology was used in this research. P. falciparum phage display libraries were created and biopanned against human erythrocyte skeletal protein 4.1 (4.1R). DNA sequencing and bioinformatic investigations uncovered a number of parasite proteins with binding specificity toward 4.1R. They included five hypothetical proteins, two invasion proteins, namely erythrocyte binding antigen-175 (EBA-175) and EBA-181, two predicted protein kinases and a putative aminopeptidase. A common binding motif displaying homology to muscle myosin and neurofilament sequences was also identified in four of the ten proteins. The interaction between EBA-181 and 4.1R was characterised further by mapping the domain in 4.1R responsible for binding to the parasite protein. Recombinant proteins were used in blot-overlay and pull-down experiments, which revealed specific interaction between the highly conserved 10kDa domain and the 4.1R binding region in EBA-181. Binding was concentration dependent, as well as saturable and was abolished by heat denaturation of 4.1R. Functions of the 4.1R-specific parasite proteins remain to be determined, however, they are potentially involved in parasite growth and survival during intra-erythrocytic development. Furthermore, these proteins may also participate in the entry and/or exit of parasites from the human erythrocyte. The interaction of EBA-181 with the 10kDa domain of 4.1R provides new insight into the molecular mechanisms utilised by P. falciparum during erythrocyte entry. It also highlights the multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite’s life cycle.

Ph.D, Faculty of Science, University of the Witwatersrand, 2011 / Chloride intracellular channel proteins (CLICs) are able to exist in a soluble or membrane-bound state. The mechanism by which the transition between the two states takes place is yet to be elucidated. It is proposed that structural rearrangements of the N-terminal domain take place when CLICs encounter the lower pH environment of the membrane surface (pH 5.5). This prompts the CLICs to form a soluble membrane-ready state prior to pore formation and membrane transversion. Since the insertion of CLIC1 into membranes occurs at low pH, perhaps protonation and electrostatic effects of key conserved residues at the domain interface situated within the transmembrane region bring about the structural changes necessary for this transition. Structural and sequence alignments revealed that a conserved salt-bridge interaction between conserved residues on helices 1 and 3 of the N-terminal domain is present at the domain interface of CLICs. Therefore, this interaction was proposed to play an important role in maintaining the structural integrity and conformational stability of the N-terminal domain. This hypothesis was tested by mutating conserved CLIC1 residues Arg29 and salt-bridge partner Glu81 to methionine, thus removing the salt-bridge interaction. The conformational stabilities of each mutant at pH 7 (cytosol) and pH 5.5 (membrane surface) in the absence of membranes was then measured and compared to that of the wild type protein. The mutations did not impact upon the structural integrity of the protein. However, removal of the salt-bridge and hydrogen bonding interactions caused a loss in the cooperativity of unfolding from the native to unfolded state that resulted in the formation of an intermediate species. The intermediate species are less stable than the intermediate species of wild type CLIC1 at pH 5.5. Nevertheless, the properties (secondary and tertiary structure, ANS binding and cooperative unfolding (N ↔ U)) of the intermediate species are the same for all mutants and wild type protein. It can be concluded that the salt-bridge and more importantly hydrogen bonding interactions between helices 1 and 3 stabilise the Nterminal domain of CLIC1. It can be hypothesised that in the absence of membranes under acidic conditions, such as those at the surface of the membrane, protonation of acidic amino acid residues at the domain interface cause destabilisation of the Nterminal domain. This causes a reduction in the activation energy barrier for the conversion of soluble CLIC1 to its membrane-insertion conformation.

Yu, Chi Shing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 115-125). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

The conventional methods for diagnosing pathological conditions of the tympanic membrane (TM) and other abnormalities require measuring its motion to an acoustic excitation for its use in a clinical environment. To obtain comprehensive quantitative diagnostic information from the motion of the entire surface of the TM, it is necessary to devise an integrated system capable of accurately recording the motion and induce an acoustic stimulus. To accomplish this goal, a sound presentation system (SPS) capable of impinging acoustic stimulus in the frequency range of 20Hz to 8 kHz at known amplitudes is synthesized in this thesis. This system is then integrated with optoelectronic digital holographic system (OEDHO) which utilizes laser interferometry to record and reconstruct phase shifted images with the help of a digital camera. The OEDHO is capable of accurately recording nanometer scale motion of the TM. The preliminary design of the SPS depends on the physical dimensions of the human ear, such as the diameter of the TM (6-9mm), depth of the ear canal (about 30mm), and also dimensions of the OEDHO system such as: diameter of tip of the otoscope head for optical access (8mm), and possible locations for integration with the OEDHO. The characteristics of the system are based on the intensity of the acoustic stimulus necessary to vibrate the TM (90-110dB SPL), and method of impinging the stimulus. To accomplish this goal, the nature of sound wave propagation through a circular pipe with known dimensions is analyzed analytically, experimentally, and by using finite element analysis (FEA). The pipe is further investigated for optimum parameters using FEA by introducing changes in the diameter (3.8mm, 6mm, 10mm), length of the pipe (30mm, 60mm, 90mm), radius of the curvature (50mm, 75mm, 100mm), and strength of the sound power source (0.2W, 0.4W, 0.6W). The comparative results provide guidelines for the design of the first version of the SPS (SPS_V1). The SPS_V1 consists of a symmetric design to impinge the acoustic stimulus towards the TM and a microphone to measure the sound pressure at the TM. The system is capable of housing a range of speakers from 2mm to 15mm in diameter. The SPS_V1 can directly interface with the standard medical speculums used for human ear testing. Also, the system is capable of interfacing with all available versions of the OEDHO. The SPS_V1 is currently being evaluated in a medical-research environment to address basic otological questions regarding TM function. The performance characterization of the system inside an artificial ear canal with two different speaker configurations is herein shown, and the potential improvements and utilization are discussed

"This project investigates the effect of membrane thickness on the performance and durability of a Direct Methanol Fuel Cell (DMFC) using a commercially available Celtec®P-1000 PBI-based membrane electrode assembly (MEA). The PBI-based membranes tested were the 100µm, the standard thickness, 200µm and 250µm thick. With various methanol feed concentrations and cathode feeds, oxygen and air, the PBI-based MEAs were operated between 160 and 180°C with vaporized methanol feed. Results showed that the DMFC performance increased with temperature and with PBI membrane thickness. The optimal concentration for the 100µm membrane was at 5M while the best performance with the 200µm membrane was obtained at 3M. The 250µm membrane looked like it could have had better performance than the 200µm, but unfortunately experimental issues didn’t allow completion of these results."