abstract: CTB-MPR649-684 is a translational fusion protein consisting of the cholera toxin B subunit (CTB) and the conserved residues 649-684 of gp41 membrane proximal region (MPR). It is a candidate vaccine component aimed at early steps of the HIV-1 infection by blocking viral mucosal transmission. Bacterially produced CTB-MPR was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice and rabbits. However, the induction of high-titer MPR specific antibodies with HIV-1 transcytosis blocking ability remains a challenge as the immuno-dominance of CTB overshadows the response to MPR. X-ray crystallography was used to investigate the structure of CTB-MPR with the goal of identifying potential solutions to improve the immune response of MPR. Various CTB-MPR variants were designed using different linkers connecting the two fusion proteins. The procedures for over-expression E. coli and purification have been optimized for each of the variants of CTB-MPR. The purity and oligomeric homogeneity of the fusion protein was demonstrated by electrophoresis, size-exclusion chromatography, dynamic light scattering, and immuno-blot analysis. Crystallization conditions for macroscopic and micro/nano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro/nano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

Les études protocolaires sur les réseaux multi-sauts mobiles proposent d’améliorer la performance du réseau en liant le comportement des protocoles à sa dynamique d’évolution. Le protocole s’adapte à l’état du réseau grâce à un ensemble de métriques. Ainsi pour les protocoles de routage il s’agit de trouver des chemins tout en considérant l’état énergétique des nœuds, la bande passante des liens, la qualité de service, ou encore la dynamique de la topologie qui est liée à la qualité des liens sans fil et à la mobilité des éléments du réseau. Le travail présenté traite de l’adaptation à la mobilité dans les protocoles de routage et s’intéresse aux métriques de mobilité. Dans une première étape nous analysons le processus d’adaptation en en dégageant les caractéristiques, les contraintes et l’intérêt, et étudions qualitativement les métriques de mobilité. Nous examinons différents travaux de recherche ayant pour objet la mobilité et ses métriques dont nous proposons une taxinomie originale. Nous mettons en évidence l’intérêt des métriques obtenues par mesure locale et de voisinage. Dans une deuxième étape, nous évaluons par simulation les métriques. L’objectif est de déterminer une "meilleure" métrique de mobilité, c'est-à-dire celle qui apportera le plus de gain de performance à une adaptation protocolaire. Le postulat est que plus la métrique influe sur les performances d'un protocole, plus il est intéressant que le protocole adapte son comportement en fonction de sa valeur. L’analyse de corrélation entre performances et métriques, sur plusieurs protocoles de routage, ne permet pas de mettre en avant une meilleure métrique qui soit indépendante du contexte, à savoir de la densité ou du modèle de mobilité. Nous montrons néanmoins l’intérêt de la métrique durée de liaison. Finalement, nous développons deux applications à partir des métriques sélectionnées par l’analyse. Ce sont la densité, exprimée par le nombre de voisins, ainsi que la mobilité, exprimée par la durée de liaison pour la première application, et par le nombre d’erreurs de route pour la seconde application. La première application met en place un choix adaptatif des éléments MPR (Multi-Point Relay) dans le protocole de routage Optimized Link State Routing (OLSR), la deuxième développe une élection adaptative des chefs de clusters et un changement de mode adaptatif (avec ou sans structure) pour le protocole Cluster Source Routing (CSR). Pour les deux applications plusieurs stratégies d’adaptations sont considérées ; le meilleur résultat est obtenu dans les deux cas par la stratégie d'adaptation à la mobilité en plus de la densité. Ce travail se prolonge par l’étude de validation des résultats en rapport au modèle de mobilité. Nous en déduisons une méthode pour appliquer, selon les conditions de mobilité et de densité, des adaptations / Manquant

No description available.

On the battlefield, having accurate positions of allied troops is crucial for making effective decisions. However, obtaining this information can be challenging, particularly outside of one's own battalion network. This research focuses on developing and evaluating methods for disseminating digital geographic position data between different interconnected radio networks in a military context. The radio networks in question are ad hoc networks that use a Wide Band Waveform (WBWF) as their communication waveform. The objective is to transfer position data between networks without overloading them. The networks have a limited bandwidth and it's shared among all nodes belonging to the network. A node is a unit with a radio at hand that can communicate with other similar configured radios. To allow networks to communicate with each other, some nodes, called gateway nodes, are geared with a second radio. The second radio is used to communicate with other nearby battalions. Protocols and algorithms can use these gateway nodes to transfer data between different networks. The research evaluates two methods, one is based on Simplified Multicast Forwarding (SMF) combined with Optimized Link State Routing (OLSR) and the other is a self-developed algorithm called Gateway Node Election (GNE) algorithm. The GNE algorithm gives control to the gateway nodes, allowing for more selective transmission of position data between different networks. To evaluate the performance of methods this thesis uses a simulation tool called Aquarius to measure the performance of the methods in various scenarios, including bandwidth usage and message age. The result shows that the GNE algorithm, compared to SMF, uses less network bandwidth and it's a more robust solution since it gives redundancy by transmitting packets again if they weren't received. The findings of this research will contribute to improving the dissemination of critical information among different radio networks in a military context.

No description available.

No description available.

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi. / The proper sorting and trafficking of acid hydrolases plays a critical role in the normal function of cells. Over 50 known human diseases are caused by mutations of lysosomal enzymes, of proteins that regulate key processes of transport to the lysosome or of enzymes that perform posttranslational modifications which are important for the function of the lysosome. The main objective of this thesis is to identify proteins and mechanisms that allow the cell to regulate the transport of enzymes toward the lysosome. We formulated the hypothesis that proteins mutated in lysosomal diseases and that have no known functions could play a role in transport toward the lysosome. Neuronal ceroid-lipofuscinoses form a family of lysosomal storage disorders that are very rare but are also the most frequent infantile neurodegenerative diseases. The work presented in this thesis identified ceroid-lipofuscinosis neuronal-5 (CLN5), which is located at the late-endosomal/lysosomal compartment as a necessary element for the recruitment and activation of Rab7. Rab7 is an important GTPase that controls traffic from the late-endosome to the trans-Golgi network. Rab7 has been implicated in the recruitment of the retromer complex, which regulates retrograde transport of the lysosomal sorting receptor such as sortilin and the mannose-6-phosphate receptor. In the cells where CLN5 is depleted, the lysosomal sorting receptors are less recycled and degraded more rapidly. Using photolabelling assays we were also able to show that Rab7 is less activated in the absence of CLN5. To perform their function, Rab proteins have to be recruited to membranes and activated by the exchange of a GDP nucleotide for GTP. The recruitment of Rabs to membranes necessitates a lipidic posttranslational modification to raise the affinity. Using yeast as a model we demonstrated that the Rab7 homolog, Ypt7 is palmitoylated. We have also showed that the yeast palmitoyltransferase Swif1 is required for Ypt7 membrane recruitment. We have also observed that retromer subunits in yeast are less recruited when palmitoyltranferases are depleted. In mammals we have shown that Rab7 is also palmitoylated and that this palmitoylation may be done by palmitoyltransferases DHHC1 and DHHC8. The palmitoylation of Rab7 occurs on the C-terminal cysteines that are required for membrane recruitment and were previously only shown to be prenylated. By using Click chemistry we have discovered that when Rab7 prenylation is blocked the level of palmitoylation is augmented. To characterize the interaction of Rab7 and CLN5 we performed experiments to definitively establish the topology of this latter protein. Our results show that CLN5 is a heavily glycosylated protein that is initially translated as a type II transmembrane protein and subsequently cleaved by a member of the signal-peptide peptidase (SPP) family. This protein can then possibly interact with another member of the CLN family, CLN3 that is predicted to be palmitoylated to recruit and activate Rab7. Our studies establish for the first time that CLN5 is required for the recruitment and activation of Rab7 and may cooperate with the possibly palmitoylated protein CLN3 to sequester Rab7 in specific membrane domains with sorting receptors to allow their recycling toward the trans-Golgi network.

Please read the abstract in the section 00front of this document / Dissertation (MA(Human Movement Science))--University of Pretoria, 2006. / Biokinetics, Sport and Leisure Sciences / unrestricted

This study identifies, tests and modifies factors for effective corporate online communication using a marketing public relations (MPR) perspective. An MPR perspective entails an integrated cross-disciplinary approach with a strong product and/or service focus. The need for the study is underscored by the fact that there is undoubtedly a lack of a theoretical framework in which to practise corporate online communication in the context of selling the organisation's products or services. In order to test the identified factors for effective corporate online communication, namely credibility, trust and long-term relationships, this study uses Q methodology as a research method and applies Q sorting as a means of data collection. Participants are asked to sort statements about corporate online communication in their preferred order of importance on a large board in the presence of the researcher. This is known as the Q sorting process. The identified factors are tested among 20 communicators and 20 receivers of corporate online communication. The participants' sortings of statements are compared by means of Q factor analysis and then analysed. The results of the study indicate that only one factor is perceived as an absolute significant factor for effective corporate online communication and that two of the factors necessitate modification. Consequently, based on the results, four factors are identified for effective corporate online communication, using an MPR perspective. These four factors are derived from the perspectives of both the communicators and receivers of corporate online communication and are included in a proposed theoretical framework of corporate online communication using an MPR perspective. / Communication / D. Litt et Phil.

Asynchronous multiplexing schemes are efficient than synchronous schemes, in a bursty traffic environment of multiple access local area network (LAN), as fixed bandwidth is not allocated among the users and there is no access delay. Fiber- Optic Code-Division Multiple Access (FO-CDMA) is one such asynchronous multiplexing scheme suitable for high speed LAN networks. While FO-CDMA offers potential benefits it also faces challenges in three diverse areas which are 1) coding algorithms and schemes 2) advanced encoding and decoding hardware and 3) network architecture. In this thesis, as a solution to the first challenge, we propose the design and construction of a new family of codes, wavelength/time multiple-pulses-per-row (W/T MPR) codes. These codes have good cardinality, spectral efficiency and minimal cross-correlation values. Performance analysis of the W/T MPR codes is carried out and found to be superior to other codes. In unipolar 1-D Optical Orthogonal Codes (OOCs) proposed by Salehi et al., the ratio of code length/code weight grows rapidly as the number of users is increased for a reasonable weight. Hence, for a given pulse width, the data rate decreases or in other words for a given data rate very narrow pulses have to be used, because of which dispersion effects will be dominant. To overcome the drawbacks of non-linear effects in large spread sequences of 1-D unipolar codes in FO-CDMA networks, several two-dimensional codes have been proposed. Wavelength-time (W/T) encoding of the two-dimensional codes is practical in FO-CDMA networks. W/T codes reported so far can be classified mainly into two types: 1) hybrid sequences, where one type of sequence is crossed with another to improve the cardinality and correlation properties and 2) matrix codes, 1-D sequences converted to 2-D codes or 2-D codes by construc- tion, to reduce the ’time’ spread of the sequences/codes. Prime-hop and eqc/prime W/T hybrid codes have been proposed where one type of sequence is crossed with another to improve the cardinality and correlation properties. Other constructions deal with conversion of 1-D sequences to 2-D codes either by using Chinese remainder theorem or folding GoLomb rulers. W/T single-pulse-per-row (W/T SPR) codes are 2-D codes constructed using algebraic method Addition Modulo Group operation. Motivation for this work: To design a family of 2-D codes which have the design choice of length of one dimension over the other, and also have better cardinality, spectral efficiency and also low cross-correlation values (thereby have low BER) than that of the reported unipolar 2-D codes. In this thesis, we describe the design principles of W/T MPR codes, for in- coherent FO-CDMA networks, which have good cardinality, spectral efficiency and minimal cross-correlation values. Another feature of the W/T MPR codes is that the aspect ratio can be varied by trade off between wavelength and temporal lengths. We lay down the necessary conditions to be satisfied by W/T MPR codes to have minimal correlation values of unity. We analytically prove the correlation results and also verify by simulation (of the codes) using Matlab software tool. We also discuss the physical implementation of the W/T MPR FO-CDMA network with optical encoding and decoding. We show analytically that when distinct 1-D OOCs of a family are used as the row vectors of a W/T MPR code, it will have off-peak autocorrelation equal to ‘1’. An expression for the upper bound on the cardinality of W/T MPR codes is derived. We also show that 1-D OOCs and W/T SPR codes are the limiting cases of W/T MPR codes. Starting with distinct 1-D OOCs, of a family, as row vectors, we propose a greedy algorithm, for the construction of W/T MPR codes and present the repre- sentations of the results. An entire W/T MPR code family, generated using greedy algorithm, is simulated for various number of interfering users. Performance analysis of the W/T MPR codes and their limiting cases is carried out for various parameter variations such as the dimensions of wavelength, time and weight of the code. We evaluate the performance in terms of BER, capacities of the networks, temporal lengths needed (to achieve a given BER). Multiple access interference (MAI) signal can be reduced, by using a bistable optical hard-limiter device in the W/T MPR code receiver, by eliminating those signal levels which exceed a certain preset level. Performance analysis of the W/T MPR codes and their limiting cases is studied for various parameter variations. For given wavelength × time dimensions, we compare various W/T codes, whose cardinalities are known, and show that W/T MPR family of codes have better cardinality and spectral efficiency than the other (reported) W/T codes. As W/T MPR codes are superior to other W/T codes in terms of cardinality, spectral efficiency, low peak cross-correlation values and at the same time have good performance, makes it a suitable coding scheme for incoherent FO-CDMA access networks.