B and T cells comprise the two major arms of the adaptive immune response tasked with clearing and preventing infection; molecular recognition in these cells occurs through antibodies and T cell receptors (TCRs), respectively. Highly successful therapeutics, clinical diagnostics and laboratory tools have been engineered from fragments of these parent molecules. The binding specificity, affinity and biophysical characteristics of these fragments determine their potential applications and resulting efficacies. Thus engineering desired properties into antibody and TCR fragments is a major concern of the multi-billion dollar biopharmaceutical industry. Toward this goal, we (1) designed antibody specificity using a novel computational method, (2) engineered thermoresistant Fabs by phage-based selection and (3) modulated binding kinetics for a single-chain TCR. In the first study, de novo modeling was used to generate libraries of FLAG peptide-binding single-chain antibodies. Phage-based screening identified a dominant design, and activity was confirmed after conversion to soluble Fab format. Bioinformatics analysis revealed potential areas for design process improvement. We present the first experimental validation of this in silico design method, which can be used to guide future antibody specificity engineering efforts. In the second study, the variable heavy chain of a moderately stable EE peptide-binding Fab was subjected to random mutagenesis, and variants were selected for resistance to heat inactivation. Thermoresistant clones where biophysically characterized, and structural analysis of selected mutations suggested general mechanisms of stabilization. Framework mutations conferring thermoresistance can be grafted to other antibodies in future Fab stabilization work. In the third study, TCR fragment binding kinetics for a clonotypic antibody were modulated by varying valence during phage-based selection. Binding affinity and kinetics for representative variants depended on the display format used during selection, and all TCR fragments retained binding to native pMHC antigen. This work demonstrates a general engineering platform for tuning protein-protein interactions. Using a combination of computational design and phage-based screening, we have identified antibodies and TCR fragments with improved binding properties or biophysical characteristics. The optimized variants possess a wider range of potential applications compared to their parent molecules, and we detail engineering methods likely to be useful in the engineering of many other protein-based therapeutics. / text

Block copolymers demonstrate potential in next-generation lithography as a solution for overcoming the limitations of conventional lithographic techniques. Ideal block copolymer materials for this application can be synthesized on a commercial scale, have high [chi]-parameters promoting self-assembly into sub-20 nm pitch domains, have controllable alignment and orientation, and have high etch contrast between the domains for facilitating pattern transfer into the underlying substrate. Block copolymers that contain silicon in one domain are attractive for nanopatterning since they often fulfill at least three of these requirements. However, silicon-containing materials are notoriously difficult to orient in thin films due to the low surface energy of the silicon-containing block, which typically wets the free surface interface. In this work, the methodology behind material choice and the synthesis of new silicon-containing block copolymers by a variety of polymerization techniques will be described. Thin film self-assembly of the block copolymers with domains oriented perpendicular to the plane of the substrate is achieved using different solvent annealing and neutral surface treatments with thermal annealing conditions. Block copolymer patterns are transferred to the underlying substrate by reactive ion etching and directed self-assembly of the polymers is demonstrated using chemical contrast patterns. Interesting thermodynamics governing the self-assembly of block copolymers with solvent annealing will also be discussed. Finally, new amphiphilic block copolymers will be described that were created with lithographic applications in mind but that are most useful for biological applications in drug delivery. / text

Nonsuicidal self-injury (NSSI) is a behavior recently added to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) as a condition for further study. In this dissertation, I present findings from three studies that inform the clinical description of NSSI as well as some of the cognitive and emotional aspects of NSSI. In Study 1, I examined data from a community sample of adult women who met proposed DSM-5 criteria for NSSI or subthreshold NSSI. The findings show that any experience with NSSI is associated with significant impairment. Further, they suggest that greater self-criticism may be a key variable capable of distinguishing between people who engage in more versus less frequent NSSI. In Study 2, I examined the extent to which cognitive deficits in inhibiting emotional information may characterize people who engage in NSSI. Although NSSI participants endorsed greater difficulty with negative thoughts relative to controls, the groups demonstrated no differences on a directed forgetting task. These findings are consistent with previous research on emotional reactivity and impulsivity in NSSI that shows a similar dissociation between self-report and behavioral-based results. In Study 3, I examined how people process NSSI images. Results suggest that people who engage in NSSI view NSSI stimuli as relatively non-aversive. This is in sharp contrast to healthy controls, who consider NSSI images as highly aversive. These finding are consistent with models of NSSI that regard self-injury as providing reinforcement (positive and negative). Overall, the findings from these studies add to the clinical description of NSSI and support its diagnostic validity. Our examination of the clinical characteristics of the NSSI as described in DSM-5 highlights the importance of assessing the presence of any NSSI behavior as well as highly self-critical thoughts. Importantly, we found no evidence of memory deficits in NSSI. Of great clinical concern, however, is the extent to which engaging in NSSI appears to erode the aversive nature of NSSI stimuli. Taken together, our findings support a model in which self-criticism may reduce the initial barriers to engaging in NSSI, with the mood benefits associated with NSSI subsequently serving to maintain it. / Psychology

The conflict between HIV-1 and the host immune system plays out over a time-scale of months and years, and on a grander scale in the co-evolution of lentiviruses and the immune systems of their host species. Directed evolution of HIV-1 entry inhibitors using controlled randomization together with a display system offers a means of recapitulating one side of this conflict in vitro on an accelerated time-scale. To address limitations in existing display systems, we constructed a vector (pDQ1) integrating phage-display and mammalian-expression systems. This vector displays on phage when expressed in bacteria, and as an Fc-fusion when expressed in tissue culture, thus accelerating the iterative process of randomization, display, and characterization. We demonstrated the utility of this vector in the evolution of a CD4-mimetic peptide.

Lipoxygenases (LOX) catalyze regio- and stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxides. These hydroperoxides are further metabolized to leukotrienes and lipoxins in mammals, and are involved in asthma and inflammation. LOX of animals and plants contain iron as catalytic metal (FeLOX). Filamentous fungi use both FeLOX, and manganese containing LOX (MnLOX). The role of LOX in fungi is still not known. This thesis focuses on expression of novel MnLOX, analyses of their reaction mechanism and products by HPLC-MS/MS, protein crystallization and analysis of the first MnLOX structure.   MnLOX from G. graminis, M. salvinii, M. oryzae, F. oxysporum and C. gloeosporioides were expressed in Pichia pastoris, purified and characterized by HPLC-MS/MS. All MnLOX catalyzes suprafacial hydrogen abstraction and oxygen insertion. Replacement of one Ile to Phe in the active site of MnLOX of G. graminis could switch the mechanism from suprafacial to mainly antarafacial. MnLOX of F. oxysporum was interesting since it catalyzes oxygenation of linoleic acid to 11R- instead of the more common 11S-hydroperoxides. This feature could be attributed to a single Ser/Phe exchange in the active site.   We found that Gg-MnLOX utilizes hydrogen tunneling in the reaction mechanism, but was slightly more temperature dependent than soybean FeLOX. It is an intriguing question why some fungal LOX use manganese and not iron as catalytic metal and whether the large redox potential of Mn2+/Mn3+ (1.5 V) can be tuned close to that of Fe2+/Fe3+ (0.77 V) for redox cycling and catalysis. We present crystallization conditions for two MnLOX, and the 2.07 Å crystal structure of MnLOX from M. oryzae, solved using sulfur and manganese single anomalous dispersion (SAD). The structure reveals a similar metal coordinating sphere as FeLOX but the metal ligand Asn473 was positioned on a short loop instead of a helix and formed interactions with a conserved Gln. This feature could be essential for the use of manganese as catalytic metal in LOX. We found three Phe residues that likely facilitate the suprafacial hydrogen abstraction and oxygen insertion for MnLOX. These findings provide new insight into the unique reaction mechanism of MnLOX.

In this thesis we study three basic issues related to international black tea markets: Are black tea markets integrated? Where is the price of black tea discovered? Are there leaders and followers in black tea markets? We use two statistical techniques as engines of analysis. First, we use time series methods to capture regularities in time lags among price series. Second, we use directed acyclic graphs to discover how surprises (innovations) in prices from each market are communicated to other markets in contemporaneous time. Weekly time series data on black tea prices from seven markets around the world are studied using time series methods. The study follows two paths. We study these prices in a common currency, the US dollar. We also study prices in each country's local currency. Results from unit root tests suggest that prices from three Indian markets are not generated through random walk-like behavior. We conclude that the Indian markets are not weak form efficient. However, prices from all non-Indian markets cannot be distinguished from random walk-like behavior. These latter markets are weak form efficient. Further analysis on these latter markets is conducted to determine whether information among the markets is shared. Vector Autoregressions (VARs) on the non-Indian markets are studied using directed acyclic graphs, impulse response functions and forecast error decomposition analyses. In both local currencies and dollar-converted series, the Sri Lankan and Indonesian markets are price leaders in contemporaneous time. Kenya is an information sink. It is endogenous in current time. Malawi is an exogenous price leader in dollar terms, but it is endogenous in local currency in contemporaneous time. In the long run, Sri Lanka, Indonesia and Malawi are price leaders in US dollar terms. In local currency series, Indonesia, Kenya and Malawi are price leaders in the long run. We use Theil's U-statistic to test the forecasting ability of the VAR models. We find for most markets in either dollars or on local currencies that a random walk forecast outperforms the VAR generated forecasts. This last result suggests the non-Indian markets are both weak form and semi-strong form efficient.

No description available.

Antibodies are proteins capable of specific interactions to a wide range of molecules. These interactions are facilitated by the complementary determining regions (CDR). Carcinomas are the most common of human cancers and they release significant amount of cytokeratins (CK) in the necrotic areas of the tumors. The CKs stay in the tumor, since they have low solubility. The antibody studied in this thesis, the anti-CK 8 antibody TS1, has shown to be effective in tumor targeting and is proposed to be useful in therapy. Single-chain antibodies (scFv) are recombinant antibodies which are much smaller than the intact IgG. This is an advantage when used in tumor therapy, since they can penetrate the tumors more easily than the larger IgG. Moreover, they are expressed by one single gene which make them easy to modify, for example by site-directed mutagenesis. The anti-idiotypic antibody αTS1 can be used to clear the TS1 form the circulation and thereby clear the body from non-tumor bound TS1 in therapy. To be able to modify the binding of an antibody to its antigen and or anti-idiotype, these interactions must be studied. In this study this is accomplished by chemical modifications of the IgGs TS1 and αTS1 and the antigen CK 8. Guided by these results, amino acid residues were mutated by using site-directed mutagenesis in the TS1-218 scFv and the effects were studied. From mutational study results, the functional epitope could be mapped and it was found that there are mainly tyrosines, but also charged residues, serine and a tryptophan that are important for both interactions. The binding of TS1-218 to both αTS1 and CK 8 could be improved by changing the negatively charged side-chains by mutations to their corresponding amide or alanine. Both the IgG and scFv versions of TS1 were administered in vivo. The IgG αTS1 was used to clear the TS1 from the circulation by forming immune complexes. The immune complexes, consisting of four or more antibodies, were mainly metabolized by the liver. The scFv TS1-218 could localize to the tumor in a tumor xenograft mouse model, although a higher uptake would be desired in a therapeutic strategy. The scFv was cleared rapidly by the kidneys, but the clearance could be slowed by pre-formed immune complexes with anti-TS1 scFv in vitro, prior to administration in vivo.

Modern superscalar pipelines have tremendous capacity to consume the instruction stream. This has been possible owing to improvements in process technology, technology scaling and microarchitectural design improvements that allow programs to speculate past control and data dependencies in the superscalar architecture. However, the speed of the memory subsystem lags behind due to physical constraints in bringing in huge amounts of data to the processor core. Cache hierarchies have subdued the impact of this speed gap; however, there is much that can be still done in improving microarchitecture. Data prefetching techniques bring in memory content significantly before the instruction stream actually witnesses demand misses. However, a majority of the techniques proposed so far depend upon an initial demand miss that initiates a stream of previously identified prefetches. In this thesis, we propose a novel prefetching algorithm, which leverages branch prediction to facilitate deep memory system speculation. The branch predictor directed lookahead mechanism builds a speculative control flow path for the instruction stream about to be fetched by the main superscalar pipeline. Prefetches are generated along this speculative path from a condensed representation of the memory instructions, leveraging register index based correlation. The technique integrates eloquently with the main pipeline's branch predictor to filter out prefetches along invalid speculative paths. Impact of the prefetching scheme is analyzed using out- of-order model of the Gem5 cycle accurate simulator. Evaluation shows that on a set of 13 memory intensive SPEC CPU2006 benchmarks, our prefetching technique improves performance by an average of 5.6% over the baseline out-of-order processor.

Innovation is a necessity, not a nicety - but many companies still think of innovation as being important rather than urgent. For innovation to be successful, it requires different behaviours and new ways of thinking. It is fundamentally a human activity; hence the people that make innovation a reality are the inner workings of this process. In this study, a measurement tool has been designed to assess the different types of personalities that exist in new product development teams. A model has also been proposed. This model classifies the different personalities according to their dominant traits. It was derived from a tool that is well known within many innovation driven organisations: the model for assessing brand personalities that is very similar to the Heylen model. Using this model, a new model is proposed for the assessment of individual personalities. The individual personality types were established and the overall team structure was examined to determine if diverse personality innovation teams have any correlation with the perceived output of innovation. This study served the purpose of determining if this relationship exists as well as introducing a new model for the classification of different personality types. Three companies within the Durban region were selected and upon investigation it was learned that there does exist a relationship between diverse personality innovation teams and the perceived output of the process. It was learned that diversity does contribute to the measured innovation output. There were four different personality types classified. It was established that too many of one or more type of personality (e.g. originators or effectors) or the lack of other types (in this case motivators) in a new product development team hinders the optimal output of the process i.e. it effectively delays innovation and a valuable market offering since the abundant personality types dominate with their respective functions and inhibit other critical functions for the innovation journey to run smoothly. Effective innovation is about each personality type adding his/her contribution to the process. In this study it was established that not many motivators were identified in teams and an increase in originators and effectors correlated with a decrease in perceived innovation output. Each team member exists in a team at the opportunity cost of another, and it is essential that the right mix of personalities be present for effective innovation. / Thesis (MBA)-University of Natal, 2003.