No description available.

No description available.

No abstract available. / Thesis (LL.M.)-University of Natal, Durban, 1983.

STAT1 (Signaltransduktor und Aktivator der Transkription 1) gehört zu einer evolutionär hochkonservierten Proteinfamilie, die eine essentielle Rolle bei der Weiterleitung von Zytokinsignalen spielt. Das STAT1-Protein ist bei zahlreichen biologischen Prozessen, wie etwa der Zellproliferation oder der Steuerung der Immunantwort, von Bedeutung und gehört zu einer der am besten untersuchten humanen Trans-kriptionsfaktoren. Bekannt ist, dass die über STAT1 vermittelte Signaltransduktion durch eine Rezeptor-assoziierte Phosphorylierung eines kritischen Tyrosinrestes im carboxyterminalen Molekülbereich von STAT1 initiiert wird. Nach der Ausbildung von transkriptionell aktiven Dimeren translozieren diese in den Zellkern und binden auf den Promotoren von Interferon-gesteuerten Zielgenen. In dieser Arbeit wurden Punktmutanten in der Linker-Domäne des STAT1-Moleküls generiert, um die Bedeutung der reziproken SH2-Phosphotyrosin-Interaktion bei der Bildung von tyrosinphosphorylierten Dimeren in funktioneller Hinsicht studieren zu können. Die Substitution eines kritischen Lysinrestes in Position 550 im STAT1-Protein zu Alanin oder Glutamat war mit einer im Vergleich zum Wildtyp-Molekül verminderten Tyrosinphosphorylierung und einer verkürzten nukleären Akkumulationsphase der mutierten Proteine nach Stimulation von transient exprimierenden Zellen mit Interferon-gamma (IFN-gamma) verbunden. Bei normaler Dissoziationskinetik von STAT1-spezifischen DNA-Sequenzen fanden sich für die K550-Mutanten erhöhte In-vitro- und In-vivo-Dephosphosphorylierungsraten und identifizierten diese damit als gegenüber dem Wildtyp-Protein bevorzugte Substrate der inaktivierenden TC45-Phosphtase. Die mutationsbedingte Destabilisierung in der Architektur der Linker-Domäne resultierte in einer supprimierten Aktivierung von IFN-gamma-gesteuerten Zielgenen, wobei die Signalamplitude von der Ladung der eingeführten Seitengruppe bestimmt wurde. Zusammenfassend zeigen diese Beobachtungen, dass der Linker-Domäne eine wichtige Bedeutung bei der Regulation der Tyrosindephosphorylierung zukommt und unterstreichen überdies deren Rolle bei dem vermuteten Austausch zwischen einer parallelen und antiparallelen Dimerkonformation, die für eine optimale Genexpression essentiell ist.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

We give a detailed proof for two discrete analogues of Courant's Nodal Domain Theorem. (author's abstract) / Series: Preprint Series / Department of Applied Statistics and Data Processing

Protein interactions are often mediated through binding of protein interaction domains to short peptide motifs. However, how the specificity of interactions is regulated within domain families is not well understood. In this thesis, I investigated this issue through the study of two cross-reactive SH3 domains from the yeast adaptor proteins Nbp2p and Bem1p. Despite low sequence similarity and different cellular roles, the Nbp2p SH3 and Bem1p SH3b domains recognize targets containing the same consensus sequences, raising the question of how the specificity of these domains is controlled. The Bem1p SH3b domain is also unusual in containing a 40 amino acid long C-terminal extension required for folding and interaction with the Cdc42p GTPase, which I also investigated in this thesis. I demonstrated that although Nbp2p SH3 and Bem1p SH3b domains recognize the same consensus sequence, each domain possesses its own unique specificity within the context of eight yeast peptides. Through structural and mutagenesis studies, I demonstrated that the Nbp2p SH3 and Bem1p SH3b domains employ different mechanism for binding, explaining how each domain can still possess its own unique specificity, while recognizing the same consensus sequence. I found that the specificity of Bem1 SH3b domain is largely regulated through inhibitory interactions, where a conserved residue in the binding interface serves to decrease binding to non-specific targets. Most significantly I observed that reducing the specificity of Bem1 SH3b domain, without reducing its binding affinity decreases its ability to function in vivo. My structural and mutagenesis studies with the Bem1p SH3b domain also demonstrated that its C-terminal extension is involved in peptide binding surface and creates a separate surface required for interaction with Cdc42p. I also showed that the Bem1p SH3b domain is capable of simultaneously interacting with peptide targets and Cdc42p, suggesting a role for this domain in Cdc42p mediated activation. Overall, my findings demonstrated the ability of SH3 domains to perform complex functions and bind their targets with finely tuned specificity. Most importantly, I demonstrated the significance of negative interactions for specificity determination and the importance of precise specificity for in vivo function.

Protein interactions are often mediated through binding of protein interaction domains to short peptide motifs. However, how the specificity of interactions is regulated within domain families is not well understood. In this thesis, I investigated this issue through the study of two cross-reactive SH3 domains from the yeast adaptor proteins Nbp2p and Bem1p. Despite low sequence similarity and different cellular roles, the Nbp2p SH3 and Bem1p SH3b domains recognize targets containing the same consensus sequences, raising the question of how the specificity of these domains is controlled. The Bem1p SH3b domain is also unusual in containing a 40 amino acid long C-terminal extension required for folding and interaction with the Cdc42p GTPase, which I also investigated in this thesis. I demonstrated that although Nbp2p SH3 and Bem1p SH3b domains recognize the same consensus sequence, each domain possesses its own unique specificity within the context of eight yeast peptides. Through structural and mutagenesis studies, I demonstrated that the Nbp2p SH3 and Bem1p SH3b domains employ different mechanism for binding, explaining how each domain can still possess its own unique specificity, while recognizing the same consensus sequence. I found that the specificity of Bem1 SH3b domain is largely regulated through inhibitory interactions, where a conserved residue in the binding interface serves to decrease binding to non-specific targets. Most significantly I observed that reducing the specificity of Bem1 SH3b domain, without reducing its binding affinity decreases its ability to function in vivo. My structural and mutagenesis studies with the Bem1p SH3b domain also demonstrated that its C-terminal extension is involved in peptide binding surface and creates a separate surface required for interaction with Cdc42p. I also showed that the Bem1p SH3b domain is capable of simultaneously interacting with peptide targets and Cdc42p, suggesting a role for this domain in Cdc42p mediated activation. Overall, my findings demonstrated the ability of SH3 domains to perform complex functions and bind their targets with finely tuned specificity. Most importantly, I demonstrated the significance of negative interactions for specificity determination and the importance of precise specificity for in vivo function.

The speed of fuzzy controllers implemented on dedicated hardware is adequate for control of any physical process, but too slow for todays high-complexity data networks. Defuzzification has been the bottleneck for fast implementations due to the large number of computationally expensive multiplication and division operations. In this thesis, we propose a high-speed fuzzy inferential system based on log-domain arithmetic, which only requires addition and subtraction operations. The system is implemented on a Xilinx Virtex-II FPGA with a processing speed of 67.6 MFLIPS having a maximum combinational path delay of 4.2 ns. It is a clear speedup compared to the reported fastest 50 MFLIPS implementation. A pipelined version of the controller is also implemented, which achieves a speed of 248.7 MFLIPS. Although a small approximation error is introduced, software simulation and hardware implementation on FPGA confirm high similarity of the outputs for control surfaces and a number of second-order plants. / Software Engineering and Intelligent Systems

We study current-induced domain wall dynamics in a thin ferromagnetic nanowire. We derive the effective equations of domain wall motion, which depend on the wire geometry and material parameters. We describe the procedure to determine these parameters by all-electric measurements of the time-dependent voltage induced by the domain wall motion. We provide an analytical expression for the time variation of this voltage. Furthermore, we show that the measurement of the proposed effects is within reach with current experimental techniques. We also show that a certain resonant time-dependent current moving a domain wall can significantly reduce the Joule heating in the wire, and thus it can lead to a novel proposal for the most energy efficient memory devices. We discuss how Gilbert damping, non-adiabatic spin transfer torque, and the presence of Dzyaloshinskii-Moriya interaction can effect this power optimization. Furthermore, we propose a new nanodot magnetic device. We derive a specific time-dependent current that is needed to switch the magnetization of the nanodot the most efficiently.