In light of the world‟s growing demand for energy that is environmentally friendly and sustainable, energy sources such as hydrogen have been considered potential contenders. Hydrogen, which can be used for energy storage, can be produced efficiently by the membrane based Hybrid Sulfur (HyS) thermo-chemical process consisting of a decomposition and an electrolysis step. During the HyS electrolysis step, SO2 and H2O are converted to H2 and H2SO4, which implies that the proton exchange membranes (PEMs) to be used for this process should have a high proton conductivity, limited SO2 cross-over and good H2SO4 stability. In order to find alternatives to the costly and high-temperature unstable Nafion®, the aim of this study was to evaluate the H2SO4 stability of various novel membranes. To structure the study, the novel PEM materials were grouped according to the PBI-type base component within the blend membranes, resulting in three groups comprising non-PBI based membranes, PBIOO based membranes and F6-PBI based membranes. Nafion®212 was included as reference PEM. By repeating the H2SO4 treatment with three different Nafion®212 samples, the obtained Nafion® data was also used to determine the experimental and analytical error margins for the study. The stability of all membranes was determined by submerging the membrane samples in 80 wt% H2SO4 at 80 °C for 120 hours. To determine the influence of the acid on the membranes, all samples were characterised before and after the H2SO4 treatment and compared in terms of their acid stability. Physical characterisation of the PEMs included the evaluation of weight and thickness changes, while IEC, SEM-EDX, FTIR and TGA were used to elucidate possible chemical changes due to the H2SO4 treatment. According to the Nafion®212 data, which had been obtained in triplicate for each of the analytical techniques, the experimental error of both the analytical and H2SO4 treatment remained below 10 %, except for the SEM-EDX sulfur-content where significantly larger errors were observed. In spite of the high error margins of the SEM-EDX data (S-content), its results, combined with the results from the other analytical techniques, resulted in a better understanding (both physical and chemical) of the effect the H2SO4 had on the membrane. This further facilitated the evaluation and comparison of the various blended PEM materials in terms of their H2SO4 stability, and the subsequent relation obtained between the observed stability and the chemical constitution and cross-linking of the membranes. After the 80 wt% H2SO4 treatment, significant weight losses were reported for the non-PBI based and PBIOO based membrane groups in comparison with the minimal changes noted for the F6-PBI based group and Nafion®212. Furthermore, significant thickness changes were reported for most of the PBIOO based membranes. The small weight and thickness changes observed for the F6-PBI confirmed the improved stability of this group of membranes in an H2SO4 environment, most likely due to the protective role of the partially fluorinated basic polymer and the known strength of the C-F bonds present. The results showed a clear correlation between the H2SO4 stability and the specific polymers present in the PEM blends investigated. Specific effects found included sulfonation, salt formation, hydrolysis and the accompanied dissolution of membrane fragments. Significant physical changes, for example ascribed to sulfonation of the concerned polymers, were supported by increased IEC measurements and peak intensities of the FTIR spectra, corresponding to the additional –SO3H groups present, while a variation in TGA signals served to further support the altered membrane composition and structure due to the H2SO4 treatment. In the case of dissolution, the corresponding chemical changes (analytical techniques) were supported by the decreased peak intensities of FTIR spectra, IEC measurements and TGA signals associated with degradation of the polymer backbone. It was shown that the stability of the blended membranes depended on the composition (blend components) of the membrane and the effective cross-linking (interaction) between the blend components. For all three groups examined, it became apparent that blend components sFS and sPSU were, for example, more stable than sPEEK and that ionical cross-linking seemed more effective than covalent cross-linking of blend components. When considering all membranes tested, the non-PBI based blend membranes consisting of (s)PSU and PFS copolymers in the presence of fluorinated cross-linkers and the PBIOO-sPSU blended membranes including most of the F6-PBI based membranes showed sufficient stability to be recommended for SO2 electrolysis. / MSc (Chemistry), North-West University, Potchefstroom Campus, 2014

The concept of 3D proteomics is a technique that couples chemical cross-linking with mass spectrometry and has emerged as a tool to study protein conformations and protein-protein interactions. In this thesis I present my work on improving the analytical workflow and developing applications for 3D proteomics in the structural analysis of proteins and protein complexes through four major tasks. I. As part of the technical development of an analytical workflow for 3D proteomics, a cross-linked peptide library was created by cross-linking a mixture of synthetic peptides. Analysis of this library generated a large dataset of cross-linked peptides. Characterizing the general features of cross-linked peptides using this dataset allowed me to optimize the settings for mass spectrometric analysis and to establish a charge based enrichment strategy for cross-linked peptides. In addition to this, 1185 manually validated high resolution fragmentation spectra gave an insight into general fragmentation behaviours of cross-linked peptides and facilitated the development of a cross-linked peptide search algorithm. II. The advanced 3D proteomics workflow was applied to study the architecture of the 670 kDa 15-subunit Pol II-TFIIF complex. This work established 3D proteomics as a structure analysis tool for large multi-protein complexes. The methodology was validated by comparing 3D proteomics analysis results and the X-ray crystallographic data on the 12-subunit Pol II core complex. Cross-links observed from the Pol II–TFIIF complex revealed interactions between the Pol II and TFIIF at the peptide level, which also reflected the dynamic nature of Pol II-TFIIF structure and implied possible Pol II conformational changes induced by TFIIF binding. III. Conformational changes of flexible protein molecules are often associated with specific functions of proteins or protein complexes. To quantitatively measure the differences between protein conformations, I developed a quantitative 3D proteomics strategy which combines isotope labelling and cross-linking with mass spectrometry and database searching. I applied this approach to detect in solution the conformational differences between complement component C3 and its active form C3b in solution. The quantitative cross-link data confirmed the previous observation made by X-ray crystallography. Moreover, this analysis detected the spontaneous hydrolysis of C3 in both C3 and C3b samples. The architecture of hydrolyzed C3-C3(H2O) was proposed based on the quantified cross-links and crystal structure of C3 and C3b, which revealed that C3(H2O) adopted the functional domain arrangement of C3b. This work demonstrated that quantitative 3D proteomics is a valuable tool for conformational analysis of proteins and protein complexes. IV. Encouraged by the achievements in the above applications with relatively large amounts of highly purified material, I explored the application of 3D proteomics on affinity purified tagged endogenous protein complexes. Using an on-beads process which connected cross-linking and an affinity purification step directly, provided increased sensitivity through minimized sample handling. A charge-based enrichment step was carried out to improve the detection of cross-linked peptides. The occurrence of cross-links between complexes was monitored by a SILAC based control. Cross-links observed from low micro-gram amounts of single-step purified endogenous protein complexes provided insights into the structural organization of the S. cerevisiae Mad1-Mad2 complex and revealed a conserved coiled-coil interruption in the S. cerevisiae Ndc80 complex. With this endeavour I have demonstrated that 3D proteomics has become a valuable tool for studying structure of proteins and protein complexes.

No description available.

No description available.

With widespread use of the Internet, data is often shared between organizations in B2B health care networks. Integrating data across all sources in a health care network would be useful to public health surveillance and provide a complete view of how the overall network is performing. Because of the lack of standardization for a common data model across organizations, matching identities between different locations in order to link and aggregate records is difficult. Moreover, privacy legislation controls the use of personal information, and health care data is very sensitive in nature so the protection of data privacy and prevention of personal health information leaks is more important than ever. Throughout the process of integrating data sets from different organizations, consent (explicitly or implicitly) and/or permission to use must be in place, data sets must be de-identified, and identity must be protected. Furthermore, one must ensure that combining data sets from different data sources into a single consolidated data set does not create data that may be potentially re-identified even when only summary data records are created. In this thesis, we propose new privacy preserving data integration protocols for public health surveillance, identify a set of privacy preserving data integration patterns, and propose a supporting framework that combines a methodology and architecture with which to implement these protocols in practice. Our work is validated with two real world case studies that were developed in partnership with two different public health surveillance organizations.

A crucial step in the biosynthesis of membrane proteins is their incorporation into the hydrophobic environment of the lipid bilayer. In eukaryotic cells this event occurs largely in concert with translation on ribosomes bound to the membrane of the endoplasmic reticulum (ER) at a site termed the ER translocon. This dynamic proteinaceous complex forms an aqueous conduit across the ER membrane and is laterally gated to allow transmembrane (TM) segments to partition into the lipid phase. In the case of polytopic membrane proteins, the coordinated release of multiple TM segments by the ER translocon is a poorly defined process and appears to be highly substratespecific. In this study, the ion channel subunit P2X2 was used as a novel model to examine themolecular details of membrane protein integration at the ER translocon. A primarily in vitro approach was taken using stable biosynthetic intermediates to simulate each stage of the membrane translocation and integration of P2X2. Chemical and photoreactive site-specific cross-linking analyses were then conducted to determine the molecular environment of the P2X2 TM segments throughout biosynthesis. Remarkably, both TM1 and TM2 of P2X2 were found to remain directly adjacent to the ER translocon throughout P2X2 biosynthesis and were only dislocated into the lipid phase by artificial termination of translation and disruption of the ribosome-translocon interaction. Retention of P2X2 TM1 at the ER translocon is maintained despite the synthesis of over 300 amino acid residues separating it from the ribosome peptidyl transferase centre. Premature dislocation of TM1 from the ER translocon site resulted in a pronounced aggregation of TM1 fragments both in vitro and in vivo. This is in stark contrast to previous passive-partitioning models of membrane integration and suggests that the ER translocon regulates the integration of polytopic membrane proteins in order to accommodate the specific requirementsof the substrate protein itself. The detailed characterisation of P2X2 biosynthesis was then exploited in order to examine the effect of a novel small inhibitor of ER translocon function. Eeyarestatin 1 (ESI) was found to cause a substantial inhibition of protein secretion in vivo and dramatically reduced the ER translocation of three distinct classes of substrate, including P2X2, in vitro. Using both a cross-linking analysis and a protease-protection assay for a specialised translocation reaction, ESI was shown to prevent the transfer of the nascent polypeptide chain from the membrane delivery machinery to the ER translocon complex. Further evidence that ESI targets the Sec61 complex is presented and a model for ESI-mediated inhibition of ER translocation is suggested. Taken together these data establish ESI as a novel small molecule inhibitor that selectively inhibits protein translocation both in vitroand in vivo.

Context. Software defect prediction aims to reduce the large costs involved with faults in a software system. A wide range of traditional software metrics have been evaluated as potential defect indicators. These traditional metrics are derived from the source code or from the software development process. Studies have shown that no metric clearly out performs another and identifying defect-prone code using traditional metrics has reached a performance ceiling. Less traditional metrics have been studied, with these metrics being derived from the natural language of the source code. These newer, less traditional and finer grained metrics have shown promise within defect prediction. Aims. The aim of this dissertation is to study the relationship between short Java constructs and the faultiness of source code. To study this relationship this dissertation introduces the concept of a Java sequence and Java code snippet. Sequences are created by using the Java abstract syntax tree. The ordering of the nodes within the abstract syntax tree creates the sequences, while small sub sequences of this sequence are the code snippets. The dissertation tries to find a relationship between the code snippets and faulty and non-faulty code. This dissertation also looks at the evolution of the code snippets as a system matures, to discover whether code snippets significantly associated with faulty code change over time. Methods. To achieve the aims of the dissertation, two main techniques have been developed; finding defective code and extracting Java sequences and code snippets. Finding defective code has been split into two areas - finding the defect fix and defect insertion points. To find the defect fix points an implementation of the bug-linking algorithm has been developed, called S + e . Two algorithms were developed to extract the sequences and the code snippets. The code snippets are analysed using the binomial test to find which ones are significantly associated with faulty and non-faulty code. These techniques have been performed on five different Java datasets; ArgoUML, AspectJ and three releases of Eclipse.JDT.core Results. There are significant associations between some code snippets and faulty code. Frequently occurring fault-prone code snippets include those associated with identifiers, method calls and variables. There are some code snippets significantly associated with faults that are always in faulty code. There are 201 code snippets that are snippets significantly associated with faults across all five of the systems. The technique is unable to find any significant associations between code snippets and non-faulty code. The relationship between code snippets and faults seems to change as the system evolves with more snippets becoming fault-prone as Eclipse.JDT.core evolved over the three releases analysed. Conclusions. This dissertation has introduced the concept of code snippets into software engineering and defect prediction. The use of code snippets offers a promising approach to identifying potentially defective code. Unlike previous approaches, code snippets are based on a comprehensive analysis of low level code features and potentially allow the full set of code defects to be identified. Initial research into the relationship between code snippets and faults has shown that some code constructs or features are significantly related to software faults. The significant associations between code snippets and faults has provided additional empirical evidence to some already researched bad constructs within defect prediction. The code snippets have shown that some constructs significantly associated with faults are located in all five systems, and although this set is small finding any defect indicators that transfer successfully from one system to another is rare.

Ce travail s’intéresse au renforcement mécanique de films lubrifiants auto-assemblés en milieu aqueux par réticulation. Deux stratégies ont été suivies. La première consiste à réticuler physiquement un film de tensioactifs cationiques par coadsorption d’un copolymère associatif anionique. La deuxième est de réticuler chimiquement un film de copolymères triblocs par la création de liaisons covalentes entre les différentes macromolécules. Nous étudions l’auto-assemblage des films par microbalance à cristal de quartz et microscopie à force atomique (AFM). Nous discutons ensuite les effets de la réticulation sur la morphologie des films (caractérisée par AFM), et sur leurs propriétés mécaniques mesurées avec l’appareil à mesure de force SFA-nanotribomètre. Cette étude a montré que dans les deux cas, la réticulation modifie les propriétés des films lubrifiants et permet une augmentation de la cohésion des films adsorbés. / In this work, we study the effect of cross-linking on boundary lubricant films in aqueous media. Two strategies have been followed. First, we investigate a physical network on a cationic surfactant bilayer by coadsorption of an anionic copolymer. Secondly a tribloc copolymer film was chemically cross-linked by creating some chemical bounds between macromolecules. We study film self-assembling by Quartz Crystal Microbalance and Atomic Force Microscopy. We discuss then on the effect of cross-linking on the morphology of the film and its mechanical properties by combining AFM and Surface Force Apparatus SFA-nanotribometer. We have shown that in both cases, lubricant films properties are modified and the cohesion of the adsorbed films is increased by cross-linking.

During production of different Hyaluronic acid (HA) products Galderma use a high concentration of NaOH in the crosslinking process of HA. HA’s degradation kinetics are widely unknown at higher pH’s and is interesting for the future of product development of HA. Static Light Scattering (SLS) was used to determine the weight average molecular weight (MW ) of samples with four different NaOH concentrations, four temperatures, three reaction times and one t0  sample. The results were evaluated in forms of Arrhenius-graphs for different NaOH concentrations, the activation energy (EA ) with respect to pH and the reaction rate for each temperature with respect to NaOH concentration. It was found that the degradation of HA was more strongly affected by temperature than by NaOH concentration and that the EA  did not show any significant signs of change between higher concentrations of NaOH.

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence.  The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over a long term at a closer physiological level. Yet, efficient gene delivery reagents are needed. In paper IV, two novel gene delivery nanoplexes were developed by post coating DNA-nanoplexes with chondroitin sulfate (CS). To ensure the stability, aldehyde-modified CS (CS-CHO) reacted with free amines of pDNA/PEI complexes. We provided first evidence that CS-CHO coated nanoplexes controlled the release from endosomes, which is essential for higher transfection efficiency.