The major obstacle many promising drugs struggle to overcome is the barrier imposed by the outer cell membrane. In addition to technologies such as liposomes and cell-penetrating peptides, more attention is being given to the class of proteins known as lectins to deliver therapeutic and antigenic proteins to the interiors of cells. Lectins bind to but do not modify sugars, and provide an efficient route to endocytosis. The galactose/N-acetyl-galactosamine specific lectin ricin B-chain (RTB) is especially attractive in possibly fulfilling a carrier role due to its well-characterized endocytotic trafficking and its efficacy over a wide range of cell types. By producing RTB recombinantly in plants it is possible to create a fully active, non-toxic carrier that does not rely on the processing of large amounts of toxic material (e.g. castor bean). Payload molecules such as small molecules and proteins can be attached to RTB via chemical conjugation at primary amine groups, without the loss of lectin or uptake activities. The biotin/streptavidin interaction and direct genetic fusion of polypeptides also provide efficient mechanisms for the attachment of payload proteins to RTB. An immunoglobulin domain-based scaffolding mechanism bridges modified RTB and payload proteins when co-expressed in Agrobacterium-infiltrated plant leaves. Carrier and payload proteins expressed in plants and E. coli, respectively, and purified independently are not able to assemble into an efficient carrier/payload arrangement. These findings show that plant cells are able to correctly produce the two components of the carrier/payload system and assemble them into an efficient and flexible capture and carry technology. / Ph. D.

No description available.

La Polarité Cellulaire Planaire (PCP) est une voie de signalisation originellement identifiée chez les invertébrés pour son rôle dans l’établissement d’une asymétrie cellulaire perpendiculaire à l’axe apico‐basal. Elle définit une polarité dans le plan d’un épithélium et coordonne cette polarité dans tout l'épithélium. L'activation de la voie PCP conduit à une réorganisation ducyto squelette en passant par une modulation des zones d'adhésion, régulant ainsi la forme et les mouvements des cellules. La voie de signalisation de la PCP est conservée tout au long de l'évolution jusqu'au mammifères, et contrôle la morphogénèse de divers tissus dont les tissus épithéliaux et mésenchymateux, ainsi que pour les tissues cardiaques, osseux, pulmonaire ou encore rénaux, mais aussi le système nerveux pour n'en citer que quelques‐uns.Afin d'identifier le rôle de vangl2, un des gènes centraux de la PCP, dans la mise en place de la circuiterie hippocampale, nous avons créé un modèle murin où vangl2 est supprimé de façon conditionnelle (cKO) dans le télencéphale à des stades précoces de l’embryogénèse. J’ai d'abord montré que Vangl2 est enrichi dans les neurones immatures de la zone sous granulaire du DG, ainsi que dans l’arborisation des neurites (axones et dendrites) des cellules granulaires (CG) du gyrus denté (DG) de l’hippocampe. Ainsi, Vangl2 est enrichi dans le stratum lucidum (sl), une région dense en contacts synaptiques entre le DG et le CA3. Dans cette région a lieu une synapse très particulière entre l'axone des CG, la fibre moussue (Mf) qui forme des boutons géants (MfB) et les excroissances épineuse (TE) issues de la partie proximale des dendrites apicaux. L'analyse structurale et ultra structurale de ces épines démontre que l'élargissement et la complexification de la synapse MfB/TE est bloquée dans nos mutants, alors que les zones actives (PSD) des épines sont présentes, mais réorganisées. De façon intéressante,dans une zone plus distale des dendrites des neurones du CA3 (sl), les épines sont, elles, plus grosses, suggérant un remodelage complexe du réseau en l'absence de vangl2. Enfin, j’ai pu montrer que ces défauts morphologiques étaient corrélés à des problèmes de mémoire complexe (mémoire déclarative) qui dépendent de l’hippocampe mais aussi du cortex. Cette étude montre pour la première fois l’importance du signal PCP dans maturation in vivo d’un circuit hippocampique spécifique ainsi que ces conséquences cognitives. D'autres résultats in vitro montrent que la suppression de vangl2 augmente la vitesse de déplacement des cônes de croissance sur des substrats de N‐cadhérine. J’ai utilisé la microscopie en super résolution spt‐PALM‐TIRF pour montrer que cette augmentation de croissance est inversement proportionnelle à la vitesse du flux rétrograde d’actine. Des expériences de FRAP permettent de suggérer que les molécules de N‐cadhérine engagées dans des interactions hémophiliques (adhésion) est plus importante dans les mutants vangl2 Je propose que Vangl2 contrôle le recyclage et la stabilité des protéines N‐cadhérine dans les sites d’adhésion afin de réguler localement les dynamiques d’actine et par conséquent la croissance neuronale. / Planar Cell Polarity (PCP) is a signaling pathway originally known for its role in the establishment of cellular asymmetry perpendicular to the apico‐basal axis, in the plane of an epithelium. PCPsignaling has been shown to be crucial for many tissue patterning, including epithelial and mesenchymal tissue, but also cardiac, lung, bone, or kidney tissues, to cite a few. PCP signaling controls the regulation of cellular movement via the control of adhesion turnover and cytoskeleton reorganization. Vangl2 is one of the most upstream core PCP proteins that has been implicated in the recent years in various neuronal mechanisms, such as axonal guidance, dendrite morphogenesis or synaptogenesis. However, most of these studies rely on acute downregulation of the gene in vitro or in the use of a mouse presenting a spontaneous mutation of this gene, called Loop‐tail (Vangl2Lp) which causes the death of the embryo at birth. Moreover, the Vangl2Lp form of this protein has been described has a dominant‐negative form, making it difficult to untangle the molecular mechanism leading to the many phenotypes (included neuronal ones) reported inhomozygotes Looptail mice. To bypass this problem we created a conditional knockout (cKO) mouse in which vangl2 is deleted in the telencephalon during early embryogenesis. First, I analyzed the profile of expression of the protein during the first 3 weeks after birth, and I show that Vangl2 is specifically targeted to the arborization of granular cells (GC) of the dentate gyrus (DG) of the hippocampus, and excluded from cell bodies. Also, the protein was highly enriched in immature neurons of the subgranular zone of the DG, and in the stratum lucidum, a region of high‐density contacts between the GC and the CA3. In this region, a special type of synapse is formed: the Mossy Fiber Bouton (MfB) / Thorny Excrescence (TE) synapse. These synapses are bigger and more complex than conventional synapses. I then performed a structural and ultrastructural analysis of the DG/CA3 circuit in the Vangl2 cKO mice in order to understand the role of Vangl2 in the hippocampus maturation. For this, I used stereotaxic mice infection viruses, and Serial block face scanning electron microscopy (SBFsEM) with 3D reconstruction. Results show that in cKO mice, Mfs fasciculation is mildly impacted, and that the enlargement and complexification of the MfB/TE synapse is arrested, with TEs almost absent. I was able to link these morphological abnormalities to deficits in complex hippocampal‐dependent learning tasks. This work demonstrates for the first time the importance of PCP signaling for the in vivo maturation of a specific hippocampal circuit and its specific cognitive consequences. Next, I attempted to identify the functional consequences of vangl2 deletion on young hippocampal neuron maturation. My results confirm that Vangl2 is expressed in young hippocampal neurons and that the deletion of the gene affected neurite outgrowth on Ncadherin substrate. I used spt‐PALM‐TIRF super‐resolution microscopy to show that this increased neurite outgrowth was inversely proportional to a decrease in actin retrograde flowand to a decrease in the number of directed actin trajectories. These results strongly suggest that N‐cadherin adhesions are affected by Vangl2 deletion. FRAP experiments demonstratedthat in Vangl2 cKO neurons the recovery of N‐cadherin molecules engaged in homophilicbindings (adhesion) was decreased, suggesting that the turnover of N‐cadherin involved inadhesion is reduced. Altogether, I propose that Vangl2 controls the turnover/stability of Ncadherin proteins at adhesion sites to regulate local actin dynamics and consequently neuronal outgrowth

Dans l'introduction, diverses voies de trafic intracellulaire et endocytose seront discutées. Je familiarise le lecteur avec des protéines inactivant les ribosomes, en mettant l'accent sur la structure, l'endocytose, et le trafic intracellulaire de la toxine bactérienne Shiga toxin (STX). STx et la ricine suivent la voie rétrograde pour exercer leur effet toxique sur les cellules. Ils sont respectivement, une menace maladie infectieuse pour la santé humaine et des outils potentiels pour le bioterrorisme pour lequel aucun antidote n’existe actuellement. D'un criblage à haut débit, Retro-1 et Retro-2 avaient déjà été identifiés comme de puissants inhibiteurs de la voie rétrograde à l'interface des endosomes précoces-TGN, et Retro-2 a été démontré pour protéger les souris contre la ricine. Parmi les facteurs de trafic analysés, seule la protéine SNARE syntaxine-5 a été ré- localisée dans les cellules traitées avec Rétro - 2. / In the introduction, various endocytic and intracellular trafficking pathways will be discussed. I acquaint the reader with ribosome-inactivating proteins, with emphasis on the structure, endocytosis, and intracellular trafficking of the bacterial toxin Shiga toxin (STx). STx and ricin follow the retrograde route to exert their toxic effect on cells. They are respectively, an infectious disease threat to human health and potential tools for bioterrorism for which no antidote currently exists. From a high throughput screening, Retro-1 and Retro-2 had previously been identified as potent inhibitors of the retrograde route at the early endosomes-TGN interface, and Retro-2 was demonstrated to protect mice against ricin. Of the trafficking factors analyzed, only the SNARE protein syntaxin-5 was re-localized in Retro-2 treated cells. Yet, whether syntaxin-5 is the direct target of Retro-2 and whether its re-localization was directly responsible for retrograde transport inhibition remained to be established.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.

This thesis addresses the three principal questions concerning the development of CT urography for investigating haematuria and each question is the subject of a separate chapter. The questions are: What is the reasoning behind using CT urography? What is the optimum diagnostic strategy using CT urography? What are the problems with using CT urography and how may solutions be provided? Haematuria can signify serious disease such as urinary tract stones, renal cell cancer, upper tract urothelial cancer (UTUC) and bladder cancer (BCa). CT urography is defined as contrast enhanced CT examination of kidneys, ureters and bladder. The technique used here includes unenhanced, nephrographic and excretory-phases for optimized diagnosis of stones, renal masses and urothelial cancer respectively. The reasoning behind using excretory-phase CT urography for investigating haematuria is based on results showing its high diagnostic accuracy for UTUC and BCa. Patients with haematuria are classified as low risk or high risk for UTUC and BCa, by a risk score, determined by the presence/absence of risk factors: age > 50 years, visible or nonvisible haematuria, history of smoking and occupational exposure. The optimum diagnostic strategy for patients at high risk for urothelial cancer, uses CT urography as a replacement test for ultrasonography and intravenous urography and as a triage test for flexible and rigid cystoscopy, resulting in earlier diagnosis and potentially improving prognosis. For patients at low risk, ultrasonography, unenhanced and nephrographic-phase CT urography are proposed as initial imaging tests. Problems with using CT urography include false positive results for UTUC, which are eliminated by retrograde ureteropyelography-guided biopsy, an innovative technique, for histopathological confirmation of diagnosis. Recommendations for the NHS and possible future developments are discussed. CT urography, including excretory-phase imaging, is recommended as the initial diagnostic imaging test before cystoscopy for patients with haematuria at high risk for urothelial cancer.

<div>As destinations of missions in both human and robotic spaceflight become more exotic, a foundational understanding the dynamical structures in the gravitational environments enable more informed mission trajectory designs. One particular type of structure, quasi-periodic orbits, are examined in this investigation. Specifically, efficient computation of quasi-periodic orbits and leveraging quasi-periodic orbits as trajectory design alternatives in the Earth-Moon and Sun-Earth systems. First, periodic orbits and their associated center manifold are discussed to provide the background for the existence of quasi-periodic motion on n-dimensional invariant tori, where n corresponds to the number of fundamental frequencies that define the motion. Single and multiple shooting differential corrections strategies are summarized to compute families 2-dimensional tori in the Circular Restricted Three-Body Problem (CR3BP) using a stroboscopic mapping technique, originally developed by Howell and Olikara. Three types of quasi-periodic orbit families are presented: constant energy, constant frequency ratio, and constant mapping time families. Stability of quasi-periodic orbits is summarized and characterized with a single stability index quantity. For unstable quasi-periodic orbits, hyperbolic manifolds are computed from the differential of a discretized invariant curve. The use of quasi-periodic orbits is also demonstrated for destination orbits and transfer trajectories. Quasi-DROs are examined in the CR3BP and the Sun-Earth-Moon ephemeris model to achieve constant line of sight with Earth and avoid lunar eclipsing by exploiting orbital resonance. Arcs from quasi-periodic orbits are leveraged to provide an initial guess for transfer trajectory design between a planar Lyapunov orbit and an unstable halo orbit in the Earth-Moon system. Additionally, quasi-periodic trajectory arcs are exploited for transfer trajectory initial guesses between nearly stable periodic orbits in the Earth-Moon system. Lastly, stable hyperbolic manifolds from a Sun-Earth L₁ quasi-vertical orbit are employed to design maneuver-free transfer from the LEO vicinity to a quasi-vertical orbit.</div>