In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van't Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases.

In this thesis, the effect of the sample properties on the characteristics of the hot carrier luminescence in graphene is investigated. The present work focuses on the two main issues described below. The first issue is the modification effects of near-infrared pulsed laser excitation on graphene. For excitation fluences several orders of magnitude lower than the optical damage threshold, the interaction with ultrafast laser pulses is found to cause a stable change in the properties of graphene. This photomodification also results in a decrease of the hot photoluminescence intensity. The detailed analysis shows that ultrafast photoexcitation leads to an increase in the local level of hole doping, as well as a change in the mechanical strain. The variation of doping and strain are linked with the enhanced adsorption of atmospheric oxygen caused by the distortion of the graphene surface. These findings demonstrate that ultrashort pulsed excitation can be invasive even if a relatively low laser power is used. Secondly, the variation of the hot photoluminescence intensity with the increasing charge carrier density in graphene is investigated. The electro-optical measurements performed using graphene field-effect transistors show a strong dependence of the photoluminescence intensity on the intrinsic carrier concentration. The emission intensity has a maximum value in undoped graphene and decreases with the increasing doping level. The theoretical calculations performed using a refined two-temperature model suggest that the reduction of the photoluminescence intensity is caused by an increase in the hot carrier relaxation rate. The modification of the carrier relaxation dynamics caused by photoinduced doping is probed directly using the two-pulse correlation measurements. The discovered sensitivity of the hot photoluminescence to the intrinsic carrier concentration can be utilised for spatially-resolved measurements of the Fermi level position in graphene samples, offering an advantage in resolution and speed.

Oxaliplatin is one of the three FDA-approved platinum anticancer drugs and considered a third generation drug, discovered after the first generation drug cisplatin and second generation drug carboplatin. It is known to react with proteins and DNA nucleotides in the body. Reaction with DNA occurs primarily at guanosine residues and secondarily at adenine residues for oxaliplatin and other platinum drugs. We have previously studied oxaliplatin and an analog with additional steric hindrance in the amine ligand and found that the analog had different reactivity with methionine. Now, we have prepared oxaliplatin and its three analogs Pt(Me2dach)(ox), Pt(en)(ox) and Pt(Me4en)(ox) and have reacted each platinum compound with both guanine and adenine nucleotides at pH 4 and pH 7 at different molar ratios. These reactions have been characterized by Nuclear Magnetic Resonance (NMR) spectroscopy equipment over time to observe the formation of products and compare them on the basis of their kinetics and binding affinities. NMR has shown that even under the conditions of excess platinum, the dominant products are usually those with two nucleotides coordinated to one platinum center. Reactions are faster at pH 7 than pH 4 due to deprotonation of phosphate group. Reactions of GMP with a platinum center are faster than reaction with AMP because of the chelate formed by the oxalate ligand. The extra methyl groups on the oxaliplatin analogs do not appear to slow down the reactions with nucleotides considerably. The pH generally affects the rate but does not substantially affect the product distribution.

<p>Acyl-carrier-protein (ACP) is the principal transporter of fatty acids, coordinating acyl-transfer among a vast network of diverse enzymes and biochemical processes. ACP association with protein partners is thought to be exceedingly transient. This paradigm has posed challenges for understanding the molecular basis for acyl-delivery and dissociation. During biosynthesis of the lipid A component (endotoxin) of lipopolysaccharides, ACP shuttles acyl-intermediates thioester-linked to its 4'-phosphopantetheine arm among four acyltransferases: LpxA, LpxD, LpxL, and LpxM. LpxA and LpxD are essential cytoplasmic enzymes, which not only provide an excellent model system to study ACP-based interaction, but also offer an important therapeutic target for development of novel antibiotics. The current dissertation reports the crystal structures of three forms of <italic>Escherichia coli</italic> ACP engaging LpxD, which represent stalled substrate and breakage products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4'-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release. Moreover, the crystal structure of <italic>E. coli</italic> LpxA in complex with one form of ACP (holo-ACP) is presented. The structure reveals three molecules of holo-ACP localize to the C-terminal domain of the LpxA homotrimer, and shows the functional role of this domain is two-fold: ACP recognition and nucleotide binding of UDP-GlcNAc. A comparison with the LpxD:ACP complexes uncovers that ACP utilizes different surface residues for recognition even amongst closely related acyltransferases, yet still relies on "electrostatic steering" for docking to its enzyme partner. Insights gleaned from the presented structures have provided not only a better understanding of ACP interaction with acyltransferases, but also has identified the "drugable molecular landscape" for the development of novel antibiotics against infective bacteria.</p> / Dissertation

International Telemetering Conference Proceedings / October 26-29, 1998 / Town & Country Resort Hotel and Convention Center, San Diego, California / In any satellite communication, the Doppler shift associated with the satellite’s position and velocity must be calculated in order to determine the carrier frequency. If the satellite state vector is unknown then some estimate must be formed of the Doppler-shifted carrier frequency. One elementary technique is to examine the signal spectrum and base the estimate on the dominant spectral component. If, however, the carrier is spread (as in most satellite communications) this technique may fail unless the chip rate-to-data rate ratio (processing gain) associated with the carrier is small. In this case, there may be enough spectral energy to allow peak detection against a noise background. In this paper, we present a method to estimate the frequency (without knowledge of the Doppler shift) of a spread-spectrum carrier assuming a small processing gain and binary-phase shift keying (BPSK) modulation. Our method relies on a simple, averaged discrete Fourier transform along with peak detection. We provide simulation results indicating the accuracy of this method. In addition, we will describe an all-digital hardware design based around a Motorola DSP56303 and high-speed A/D which implements this technique in real-time. The hardware design is to be used in NMSU’s implementation of NASA’s demand assignment, multiple access (DAMA) service.

Thesis (MScEng (Electrical and Electronic Engineering))--University of Stellenbosch, 2006. / A low earth orbit satellite is to provide the telecommunications link to facilitate email services to rural areas, where the infrastructure necessary for e-mail is lacking (e.g. no telephone lines). Communication time with this satellite from any particular point on the ground is less than one hour per day. It is thus of utmost importance to maximise the data throughput rate for the system. The contribution of this thesis is to improve the performance of CSMA/CA by adapting and optimising it for the above application. This improved protocol is used to regulate data flow through the system. Specific attention is given to the comparison of various random variable distributions for use as the back-off random variable. Two pieces of software are further contributed. First, a set of MATLAB scripts which are used for comparing various back-off random variable distributions and optimising each of these distributions. Secondly, an extensive (more than 2500 lines of code) OMNeT++ simulation of the improved CSMA/CA protocol, complete with MATLAB scripts for setting up multiple simulation runs and plotting the results. Both pieces of software accept the system constraints as parameters, and are thus easily adaptable for a similar system which may use the same protocol, but has different parameters. It is concluded that the set of MATLAB scripts are a fairly accurate tool for optimising throughput, as is confirmed by the OMNeT++ simulations, and that OMNeT++ has merit for simulating the given type of system and protocol.

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy