The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 123-135.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 108-125.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: References located at the end of each chapter.

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 45-48.

Le sujet de thèse porte sur la maîtrise et la mesure des formes neuronales, “maîtrise” du fait de l’emploi de micropatterns adhésifs permettant un contrôle des formes cellulaires en deux dimensions, “mesure” du fait de notre volonté d’accéder au volume ainsi qu’à la masse sèche de la cellule par l’emploi de deux techniques complémentaires faisant appel à l’interférométrie ou à des mesures de fluorescence en espace confiné.La question biologique au cœur de cette thèse est celle de la régulation par le neurone de diverses caractéristiques morphologiques comme sa longueur, son volume en lien avec l’établissement de la polarité axo-dendritique. Ces aspects sont développés et approfondis au cours de cette thèse des points de vue expérimentaux mais aussi théoriques (coll. Nir Gov, Institut Weizmann).Ce sujet de thèse multidisciplinaire porte ainsi des aspects de biologie et d’instrumentation physique. / The thesis deals with the control and the measurement of neuronal shapes, "control" by using adhesive micropatterns allowing to constrain cells shape in two dimensions, "measurement" by using either interferometry or fluorescence measurements in confined spaces to gain knowledge on cell dry mass and volume.The biological question at the heart of this thesis is the regulation by the neuron of its various morphological characteristics such as length, volume, in association with the establishment of the axo-dendritic polarity. These aspects are developed and deepened in the course of this thesis on experimental but also theoretical (coll. Nir Gov, Weizmann Institute) point of views.This multidisciplinary thesis topic thus builds on biological aspects and physical instrumentation.

La conception des systèmes multimédia embarqués présente de nombreux défis comme la croissante complexité du logiciel et du matériel sous-jacent, ou les pressions liées aux délais de mise en marche. L'optimisation du processus de débogage et validation du logiciel peut aider à réduire sensiblement le temps de développement. Parmi les outils de débogage de systèmes embarqués, un puissant outil largement utilise est l'analyse de traces d'exécution. Cependant, l'évolution des techniques de traçage dans les systèmes embarqués se traduit par des traces d'exécution avec une grande quantité d'information, à tel point que leur analyse manuelle devient ingérable. Dans ce cas, les techniques de recherche de motifs peuvent aider en trouvant des motifs intéressants dans de grandes quantités d'information. Concrètement, dans cette thèse, nous nous intéressons à la découverte de comportements périodiques sur des applications multimédia. Donc, les contributions de cette thèse concernent l'analyse des traces d'exécution d'applications multimédia en utilisant des techniques de recherche de motifs périodiques fréquents. Concernant la recherche de motifs périodiques, nous proposons une définition de motif périodique adaptée aux caractéristiques de la programmation parallèle. Nous proposons ensuite une représentation condensée de l'ensemble de motifs périodiques fréquents, appelée Core Periodic Concepts (CPC), en adoptant une approche basée sur les relations triadiques. De plus, nous définissons quelques propriétés de connexion entre ces motifs, ce qui nous permet de mettre en oeuvre un algorithme efficace de recherche de CPC, appelé PerMiner. Pour montrer l'efficacité et le passage à l'échelle de PerMiner, nous réalisons une analyse rigoureuse qui montre que PerMiner est au moins deux ordres de grandeur plus rapide que l'état de l'art. En plus, nous réalisons un analyse de l'efficacité de PerMiner sur une trace d'exécution d'une application multimédia réelle en présentant l'accélération accompli par la version parallèle de l'algorithme. Concernant les systèmes embarqués, nous proposons un premier pas vers une méthodologie qui explique comment utiliser notre approche dans l'analyse de traces d'exécution d'applications multimédia. Avant d'appliquer la recherche de motifs fréquents, les traces d'exécution doivent être traitées, et pour cela nous proposons plusieurs techniques de pré-traitement des traces. En plus, pour le post-traitement des motifs périodiques, nous proposons deux outils : un outil qui trouve des pairs de motifs en compétition ; et un outil de visualisation de CPC, appelé CPCViewer. Finalement, nous montrons que notre approche peut aider dans le débogage des applications multimédia à travers deux études de cas sur des traces d'exécution d'applications multimédia réelles. / Increasing complexity in both the software and the underlying hardware, and ever tighter time-to-market pressures are some of the key challenges faced when designing multimedia embedded systems. Optimizing software debugging and validation phases can help to reduce development time significantly. A powerful tool used extensively when debugging embedded systems is the analysis of execution traces. However, evolution in embedded system tracing techniques leads to execution traces with a huge amount of information, making manual trace analysis unmanageable. In such situations, pattern mining techniques can help by automatically discovering interesting patterns in large amounts of data. Concretely, in this thesis, we are interested in discovering periodic behaviors in multimedia applications. Therefore, the contributions of this thesis are focused on the definition of periodic pattern mining techniques for the analysis of multimedia applications execution traces. Regarding periodic pattern mining contributions, we propose a definition of periodic pattern adapted to the characteristics of concurrent software. We then propose a condensed representation of the set of frequent periodic patterns, called core periodic concepts (CPC), by adopting an approach originated in triadic concept approach. Moreover, we define certain connectivity properties of these patterns that allow us to implement an efficient CPC mining algorithm, called PerMiner. Then, we perform a thorough analysis to show the efficiency and scalability of PerMiner algorithm. We show that PerMiner algorithm is at least two orders of magnitude faster than the state of the art. Moreover, we evaluate the efficiency of PerMiner algorithm over a real multimedia application trace and also present the speedup achieved by a parallel version of the algorithm. Then, regarding embedded systems contributions, we propose a first step towards a methodology which aims at giving the first guidelines of how to use our approach in the analysis of multimedia applications execution traces. Besides, we propose several ways of preprocessing execution traces and a competitors finder tool to postprocess the mining results. Moreover, we present a CPC visualization tool, called CPCViewer, that facilitates the analysis of a set of CPCs. Finally, we show that our approach can help in debugging multimedia applications through the study of two use cases over real multimedia application execution traces.

L’ostéoporose est une maladie osseuse caractérisée par une perte importante de la masse osseuse et des altérations de la microarchitecture du tissu osseux. Aujourd’hui, en routine clinique, le diagnostic de l’ostéoporose est basé principalement sur une mesure de la densité minérale osseuse qui n’est pas suffisante, car elle doit être accompagnée par une analyse de la qualité de la microarchitecture osseuse. Les travaux présentés dans cette thèse concernent la caractérisation des images de radiographies osseuses pour le diagnostic précoce de l’ostéoporose. Pour ce faire, afin de mieux caractériser la texture osseuse sur radiographie, nous avons introduit une nouvelle technique de prétraitement des données pour réduire les redondances et éliminer le bruit issu des capteurs d’acquisition. Pour la caractérisation, nous avons proposé une nouvelle technique d’analyse inspirée des motifs binaires locaux (Local Binary Patterns, LBP). Le nouveau descripteur, appelé 1DLBP (One Dimensional Local Binary Patterns) s’applique de manière unidimensionnelle. Pour tester l’efficacité de notre approche, nous avons réalisé deux études cliniques où le nouveau descripteur LBP1D est comparé à la méthode classique, LBP afin de classifier des patients ostéoporotiques et des sujets sains. Les pourcentages de classification obtenus ont été améliorés de 72% avec la méthode classique LBP à 91% avec le nouveau descripteur 1DLBP. / Osteoporosis is characterized by a significant loss of bone mass and alterations in the microarchitecture of bone tissue. Actually, in clinical routine the diagnosis of osteoporosis is based mainly on measurement of bone mineral density. It turned out that this is not sufficient, it must be accompanied by an analysis of the microarchitecture of the bone to increase the efficiency of diagnosis. This thesis deals with the characterization of images of bone radiographs for the early diagnosis of osteoporosis. To do this, in order to better characterize the texture of bone radiography, we have introduced a new technique for data preprocessing to reduce redundancy and decrease the effect of the noise resulted by the acquisition sensors. For characterization, we propose a new analysis method inspired from the local binary patterns (LBP). The new descriptor called 1DLBP (One Dimensional Local Binary Patterns) applies in one-dimensionally manner. To evaluate the effectiveness of our approach, we conducted two clinical studies where the new descriptor (1DLBP) is compared with the conventional method (LBP) to classify osteoporotic patients and healthy subjects. The classification scores obtained were enhanced by 72% with the conventional LBP descriptor to 91% with 1DLBP descriptor.

La mucoviscidose est une maladie héréditaire monogénique grave en rapport avec une mutation du gène codant pour la protéine CFTR et dont la morbidité est due principalement aux atteintes pulmonaires. La greffe pulmonaire développée depuis quelques années semble être la seule thérapie curative efficace. Toutefois, la rareté des greffons disponibles, justifie de développer d’autres thérapies comme la thérapie génique. Les pseudovirions de papillomavirus sont capables de transférer des gènes dans de nombreuses lignées cellulaires. Cependant, le tropisme naturel des papillomavirus est l’épithélium malpighien et ces pseudovirions sont peu efficaces pour les cellules de l’épithélium pulmonaire. Afin de modifier le tropisme de nos pseudocapsides de papillomavirus, la première étape de mon travail a été d’identifier de nouveaux motifs de ciblage pulmonaire. La technique de phage display a permis l’identification des motifs PHPNRAQ et VDRLQQK par sélection sur les cellules épithéliales bronchique IB3-1 et S9. / Cystic fibrosis is an autosomal recessive genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Lung transplantation developed in recent years is the only effective curative treatment. However, due to the limitation in organ donors, it is necessary to develop other therapies such as gene therapy. Papillomavirus pseudovirions have the potential to deliver genes into different cells. However, the natural tropism of HPV is directed to the cervical epithelial cells and these pseudovirions have low transduction efficacy in pulmonary epithelial cells. The purpose of this thesis was to retarget and optimize the HPV-16 pseudovirions tropism to airway cells by insertion of short amino acid sequences within the major capsid protein L1.