Le cilié Paramecium tetraurelia est un modèle intéressant pour l'étude de la diversité et de l'évolution des voies d'ARN interférence (ARNi) chez les eucaryotes. L'une des voies d'ARNi végétatives peut être induite en nourrissant les paramécies de bactéries produisant un ARN double-brin (ARNdb) homologue à un gène donné, dont l'expression est inactivée de manière post-transcriptionnelle par la production de siARN de 23 nt. Un crible de mutagénèse a permis d'obtenir des mutants mendéliens déficients pour l'ARNi, dont les génomes ont été séquencés afin d'identifier sans a priori des gènes impliqués dans cette voie. 6 gènes ont été identifiés: un Dicer, deux ARN polymérases ARN-dépendantes (RDR1 et 2), une nucléotidyl-transférase (CID1) et deux gènes codant de nouvelles protéines (PDS1 et 2). Pour étudier leur rôle dans la biosynthèse ou l'action des siARN, ces derniers ont été séquencés à partir de cellules sauvages ou mutantes, nourries d'un ARNdb homologue à un gène non essentiel. L'analyse bio-informatique a montré que des siARN dits 'primaires' sont produits à partir de l'ARNdb bactérien, tandis que des siARN dits 'secondaires' sont produits à partir de la totalité de l'ARNm endogène ciblé, et sont majoritairement de polarité anti-sens. Alors que la production des siARN primaires dépend de tous les gènes étudiés, les résultats n'impliquent que RDR2 dans celle des siARN secondaires. Enfin, j'ai montré que certains clusters de siARN endogènes dépendent de RDR1 et de CID1, tandis que d'autres dépendent de RDR2. La paramécie produit également des siARN antisens aux ARN ribosomaux bactériens, suggérant de nouvelles hypothèses quant à la fonction naturelle de cette voie. / The ciliate Paramecium tetraurelia is an interesting model to study the diversity and evolution of RNA interference (RNAi) pathways. One of the vegetative RNAi pathways is induced by feeding cells with bacteria producing double-stranded RNA (dsRNA) homologous to a given gene, which is then post-transcriptionally silenced through the production of 23-nt siRNAs. A forward genetic screen allowed us to obtain Mendelian mutants deficient in dsRNA-induced RNAi, and mutated genes were identified by whole-genome resequencing. 6 genes were identified: one Dicer, two RNA-dependent RNA polymerases (RDR1 et 2), one nucleotidyl-transferase (CID1) and two genes encoding novel poteins (PDS1 and 2). To study their roles in siRNA biosynthesis or action, we sequenced small RNAs from wild-type or mutants cells fed with a dsRNA homologous to a non-essential endogenous gene. Bioinformatic analyses showed that 'primary' siRNAs are produced from the bacterial dsRNA trigger, while 'secondary' siRNAs, predominantly of antisense polarity, are produced from the whole length of the targeted endogenous mRNA. While primary siRNA production requires all of the genes studied, the results only implicate RDR2 in the production of secondary siRNAs. Finally, I showed that some clusters of endogenous siRNAs depend on RDR1 and CID1, whereas others depend on RDR2. Paramecium was also shown to produce siRNAs that are antisense to bacterial ribosomal RNAs, suggesting new hypotheses about the possible natural functions of this pathway.

Les tumeurs primitives du système nerveux central les plus fréquentes sont les gliomes et se classent en deux grandes catégories : les astrocytomes et les oligodendrogliomes. La survie des patients porteurs d'oligodendrogliomes est de 70 % à 5 ans alors qu'elle ne dépasse pas une année en moyenne pour les glioblastomes. La divergence de pronostic observée entre ces deux types de gliomes résulte d'une différence de sensibilité aux traitements conventionnels. La chimiothérapie classique appliquée à ces tumeurs permet en effet d'obtenir une réponse pour 90 % des oligodendrogliomes contrastant avec moins de 10 % pour les glioblastomes. La chimio-sensibilité particulière des oligodendrogliomes peut être la conséquence des altérations géniques survenant lors du processus oncogène. Elle résulterait de l'absence d'expression de gènes de résistance des régions chromosomiques 1p et 19q, gènes ainsi distinctement exprimés entre les deux entités tumorales. Les gènes de réparation de l'ADN peuvent rendre compte de la résistance aux traitements, puisque responsables de la correction des adduits créés par la chimiothérapie. Nous avons recherché grâce à une évaluation fonctionnelle in vitro, quels gènes de réparation de la région 1p/19q sont impliqués dans la correction des adduits du CDDP. Quatre gènes ont ainsi été identifiés : ercc1, ercc2, mutyh et pnkp. Ces résultats ont été vérifiés avec du témozolomide, la drogue de référence dans le traitement des gliomes. L'expression des ces gènes a été mesurée dans des extraits de tumeurs de patients, résistantes (glioblastomes) et sensibles (oligodendrogliomes) à la chimiothérapie, et les 3 premiers sont effectivement surexprimés dans les glioblastomes. Ces gènes définissent ainsi les cibles d'un traitement chimio-sensibilisant. Nous avons entrepris l'étude préclinique de ce traitement basé sur l'utilisation concomitante de siRNA dirigés contre les gènes d'intérêt et de chimiothérapie. Des modèles de gliomes humains sous-cutanés et intra-cérébraux résistants à la chimiothérapie ont été utilisés chez les souris. Le traitement chimio-sensibilisant ciblant ercc1 a significativement augmenté l'effet du témozolomide sur les tumeurs, permettant ainsi de diminuer leur volume. Il pourrait faire prochainement l'objet d'essais cliniques. Nos travaux, menés de l'in silico au stade préclinique, d'une part démontrent la validité de notre hypothèse, c'est-à-dire l'implication des gènes de réparation du 1p/19q dans la chimio-résistance, et conduisent surtout à une opportunité thérapeutique nouvelle de chimio-sensibilisation.

Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.

Plasmon-resonant nanoparticle complexes show promising potential for light-triggered, controllable delivery of deoxyribonucleic acids (DNA) for research and therapeutic purposes. For example, the approach of RNA interference (RNAi) ‒ using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein ‒ is very useful in dissecting genetic function and holds promise as a molecular therapeutic. Herein, we investigate the mechanism and probe the in vitro therapeutic potential of DNA light-triggered release from plasmonic nanoparticles. First, we investigate the mechanism of light-triggered release by dehybridizing double-stranded (dsDNA) via laser illumination from two types of nanoparticle substrates: gold (Au) nanoshells and Au nanorods. Both light-triggered and thermally induced releases are distinctly observable from nanoshell-based complexes. Surprisingly, no analogous measurable light-triggered release was observable from nanorod-based complexes below the DNA melting temperature. These results suggest that a nonthermal mechanism may play a role in light-triggered DNA release. Second, we demonstrate the in vitro light-triggered release of molecules non-covalently attached within dsDNA bound to the Au nanoshell surface. DAPI (4',6-diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellular light-induced release process. Illumination through the cell membrane of the nanoshell-dsDNA-DAPI complexes dehybridizes the DNA and releases the DAPI molecules within living cells. The DAPI molecules diffuse to the nucleus and associate with the cell’s endogenous DNA. This work could have future applications towards drug delivery of molecules that associate with dsDNA. Finally, we demonstrate an engineered Au nanoshell (AuNS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer coated onto the AuNS surface (AuNS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotide, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and GFP gene silencing mediated by AuNS-PLL delivery vector. The light-triggered release of oligonucleotides could have broad applications in the study of cellular processes and in the development of intracellular targeted therapies.

Nucleic acids are macromolecules in cells for storing and transferring genetic information. Moreover, nucleic acids, especially RNAs, can fold into well-defined 3D structures and catalyze biochemical reactions. As ubiquitous biological molecules in all living systems, nucleic acids are important drug targets, and they can also be used in diagnostics and therapeutics. Structural information of nucleic acids provides the foundation for DNA and RNA function studies. X-ray crystallography has been a useful tool for structural studies of bio-macromolecules at atomic level. There are two major problems in macromolecular crystal structure determination: phasing and crystallization. Although selenium derivatization is routinely used for solving novel protein structures through the MAD phasing technique, the phase problem is still a critical issue in nucleic acid crystallography. The covalent selenium-derivatization of nucleic acids has been proven to be a useful strategy for solving the phase problem in nucleic acid X-ray crystallography. Besides the facilitation of nucleic acid crystallography, there is also a wide range of other applications for selenium-derivatized nucleic acids (SeNA). The investigation presented in this dissertation mainly focuses on the following research subjects (1) Synthesis and characterization of selenium-derivatized nucleic acids for X-ray crystallography, especially phosphoroselenoate RNAs. They are generated and used for crystallization. (2) Application of selenium-derivatized RNA for RNA interference. Phosphoroselenoate RNAs are tested for RNAi activities. (3) Synthesis and characterization of the uridine 5’-triphosphate modified with selenium at position 4. (4) Facile synthesis and antitumor activities of selenium modified deoxyribonucleosides. MeSe-thymidine nucleosides have shown antitumor activity in cell assays.

Los peces carnívoros presentan baja capacidad para utilizar carbohidratos provenientes de la dieta y controlar los niveles de glucosa en sangre. En comparación con los mamíferos, estos animales tras la ingesta de glucosa o de dietas con alto contenido en carbohidratos, muestran una hiperglucemia mas prolongada. La alanina aminotransferasa (ALT) constituye un nexo de interacción entre el metabolismo de aminoácidos y el de carbohidratos al catalizar la reacción de la transaminación reversible entre L-alanina y 2-oxoglutarato para formar piruvato y L-glutamato. Estudios previos de nuestro grupo indicaron la presencia de tres isoformas ALT en dorada (Sparus aurata): las isoenzimas citosólicas cALT1 y cALT2 y una isoforma mitocondrial, mALT. En hígado de dorada, la expresión de cALT2 incrementa en situación de gluconeogénesis mientras que cALT1 predomina durante el período postprandial para la utilización de los nutrientes de la dieta. El objetivo general del presente estudio es comprender a nivel molecular los efectos metabólicos derivados de la inhibición de ALT en peces para ayudar a establecer nuevas aplicaciones biotecnológicas orientadas a mejorar la utilización de los nutrientes de la dieta. Así, en acuicultura, identificar los efectos metabólicos asociados a la modulación de la actividad ALT constituye un punto de interés para conocer si es posible efectuar una sustitución parcial de las proteínas de la dieta por carbohidratos u otros nutrientes, a fin de reducir el coste de la producción en acuicultura y disminuir la eutrofización de las aguas del entorno. Nuestros estudios muestran que la inyección intraperitoneal de doradas con nanopartículas del complejo pCpG-siRNA-quitosán resultó adecuada para promover la expresión de un siRNA para bloquear la expresión de cALT1 en hígado de Sparus aurata. La inyección intraperitoneal de nanopartículas de pCpGsi1sh1-quitosán promovió la silenciación de cALT1 a nivel de mRNA y actividad enzimática en hígado de dorada. Por otra parte, hemos analizado la inhibición postranscripcional de la actividad ALT in vivo e in vitro con el compuesto aminooxiacetato (AOA) y analizado los cambios promovidos en metabolitos y enzimas clave en el metabolismo intermediario de carbohidratos y proteínas en hígado de Sparus aurata, tras la ingesta del AOA con dietas de diferente composición. In vitro, el AOA ejerce una inhibición dependiente de dosis sobre la actividad ALT hepática citosólica y mitocondrial. In vivo, el AOA se comportó como inhibidor de la actividad ALT citosólica hepática, pero no de la mitocondrial. Una exposición a largo plazo a AOA promovió un aumento de la actividad piruvato quinasa en el hígado de dorada, independientemente de la composición de la dieta suministrada a los peces. Los estudios de 1H-RMN mostraron que la inclusión de AOA en la dieta promueve una disminución en los niveles hepáticos de alanina, glutamato y glucógeno. Adicionalmente, los análisis de 2H-RMN indicaron una tasa de renovación más alta de alanina en el hígado de los peces alimentados con una dieta con un contenido alto en carbohidratos y bajo en proteínas y que el AOA disminuye el enriquecimiento de alanina en 2H independientemente de la composición de la dieta. Los estudios derivados de esta tesis indican que la inhibición dependiente de AOA de la actividad de la ALT citosólica podría contribuir a aumentar el uso de nutrientes por carbohidratos de la dieta de Sparus aurata. / Carnivorous fish have low ability to utilize dietary carbohydrates and controlling blood glucose levels. Compared with mammals, these animals after ingestion of glucose or diets high in carbohydrates show a more prolonged hyperglycemia. Alanine aminotransferase (ALT) links carbohydrate and amino acid metabolism through catalysing the reversible transamination between L-alanine and 2-oxoglutarate to form pyruvate and L-glutamate. Previous studies from our group indicated the presence of three isoforms ALT bream (Sparus aurata): cALT1 cytosolic isoenzymes and cALT2 and a mitochondrial isoform, mALT. In the liver os Sparus aurata cALT2 expression increases gluconeogenesis situation prevails while cALT1 during the postprandial period for the utilization of dietary nutrients. The overall objective of this study is to understand at a molecular level the resulting metabolic effects from the inhibition of ALT in fish to help establish new biotechnological applications aimed to improve the use of dietary nutrients. Thus in aquaculture, identifying metabolic effects associated with ALT activity modulation is a point of interest to know if it is possible to perform a partial replacement of dietary proteins by carbohydrate or other nutrients, to reduce the cost of Aquaculture production and reduce eutrophication of the environment. Our studies show that intraperitoneal injection of a chitosan-pCpG-siRNA nanoparticles complex to Gilthead seabream proved to be suitable for the expression of a siRNA to silence the expression of liver cALT1 in Sparus aurata. Intraperitoneal injection of pCpGsi1sh1-chitosan nanoparticles complex promoted cALT1 silencing at the mRNA level and a decrease of liver enzyme activity of Gilthead seabream. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients by the Sparus aurata fish.

Hydrogel micro- and nanoparticles (microgels and nanogels) are a promising class of drug delivery vehicles. Composed of hydrophilic polymers arranged into a cross-linked network structure, nanogels show several attractive features for the delivery of macromolecule therapeutics. For instance, the hydrated, porous internal cavity of the nanogel may serve as a high capacity compartment for loading macromolecules, whereas the periphery of the nanogel may be used as a scaffold for conjugating cell-specific targeting moieties. This dissertation presents recent investigations of nanogels as targeted delivery vehicles for oligonucleotides to cancer cells, while exploring new nanogel chemistries that enable future in vivo applications. For instance, synthetic efforts have produced particles capable of erosion into low molar mass constituents, providing a possible mechanism of particle clearance after repeated administration in vivo. In another example, the microgel network chemistry was tuned to promote the encapsulation of charged proteins. In parallel with those synthetic efforts, new light scattering methodologies were developed to accurately quantify the particle behaviors (e.g. loading, erosion). Using multiangle light scattering (MALS), changes in particle molar mass and radius were measured, providing a quantitative and direct approach for monitoring nanogel erosion and macromolecule encapsulation. The new particle chemistries demonstrated, together with enabling light scattering methods, will catalyze the development of improved delivery vehicles in the near future.

Over the past several decades, gene therapy technologies have been developed for a diverse number of applications ranging from DNA-based vaccines to gene silencing with RNAi. While all are powerful tools, a common limitation for these technologies is the need for effective and safe delivery to target sites within the body. Such delivery vectors are necessary for retention of bioactivity and stability, while also providing a method of cellular and tissue uptake and distribution, which may require endosomal escape. Although, viral and lipid-based technologies have shown promise as nucleic acid delivery vectors, both have inherent issues such as cytoxicity, oncogenicity, and immunogenicity. Thus, the development of polymer-based non-viral vectors has been an area of great focus over the past decade. While many polymeric vectors have been developed for plasmid DNA (pDNA) delivery, very few have shown effective delivery of short interfering RNA (siRNA), a powerful tool for gene silencing via the RNA interference mechanism. Furthermore, very few prospective delivery vectors have shown versatility for the administration of siRNA through multiple routes of administration. The overall goal of this research was to develop a biocompatible non-viral delivery system for the delivery of plasmid DNA, siRNA, and contrast agents through the modification of the natural biopolymer chitosan. We have synthesized an imidazole modified chitosan (chitosan-IAA) by conjugation of imidazole acetic acid to chitosan. Extensive evaluation and characterization of the modified polymer demonstrates enhanced solubility and buffering capacity within the physiological and endosomal pHs, thus providing enhanced endosomal escape by exploiting the "proton sponge" effect. We have demonstrated effective in vitro gene expression and gene silencing with chitosan-IAA mediated delivery of pDNA and siRNA, respectively. Furthermore, we have demonstrated in vivo gene silencing by delivery of siRNA through both intranasal and intravenous routes of delivery with chitosan-IAA/siRNA nanocomplexes. We have also demonstrated delivery of contrast agents up to 45 nm in size through mucosal tissue following treatment with chitosan and no contrast agent modification in both human and animal tissue. In conclusion, we have successfully developed a versatile and highly effective delivery vector for both nucleic acids and contrast agents. / text

No description available.

No description available.