Anticancer therapeutics are often limited to suboptimal doses due to their lack of selectivity for tumor cells and resultant damage to healthy tissue. These limitations motivated researchers to develop tumor-specific delivery systems for improved therapeutic efficacy and reduced unintended cytotoxicity. Polyamidoamine dendrimers offer an ideal platform for designing targeted therapeutics with tunable characteristics that optimize pharmacokinetic behavior and targeting specificity. Ligand conjugation to dendrimer provides the biochemical interaction necessary to activate tumor-specific receptors for receptor-mediated endocytosis and effective internalization of polyplexes. Tumor-specific receptors overexpressed in carcinomas, like folate receptor-alpha (FOLRα), are targeted by ligand-conjugated dendrimer to allow enhanced internalization of dendrimer and its therapeutic cargo. We examined the cellular trafficking dynamics and potential of folate-conjugated dendrimer for nucleic acid delivery in vitro. Results show folate-conjugation to G4 PAMAM dendrimer (G4FA) confers enhanced uptake in FOLRα-positive tumor cells. Cells internalize G4FA in a receptor-dependent manner with specificity for FOLRα-positive tumor cells.

Apoptosis (Type I) and autophagy (Type II) represent two major forms of programmed cell death. Numerous anticancer agents employed in standard chemotherapy or novel targeted therapy induce both apoptosis and autophagy. Of note, a cytoprotective autophagic response often counteracts apoptosis triggered by such agents, potentially contributing to drug-resistance. Mechanistically, autophagy and apoptosis share molecular regulatory mechanisms primarily governed by the Bcl-2 family proteins. However, since autophagy acts as the double-edge sword in cancer, whether autophagy should be inhibited or activated in cancer treatment remains the subject of debate. Here we report a) a novel autophagy-targeted strategy that targeting the adaptor SQSTM1/p62 induces “inefficient” autophagy due to cargo-loading failure and converts cytoprotective autophagic response to apoptosis via the BH3-only protein NBK/Bik (Part 1); and b) a new mechanism for acquired drug-resistance in which the BH3-only protein Bim acts as a dual-agent regulating both autophagy and apoptosis (Part 2).

The cancers of liver, colon and breast are amongst the top five most prevalent and most fatal worldwide. As the Raf/MEK/ERK pathway is frequently deregulated in hepatocellular carcinoma (HCC), sorafenib, a Raf kinase inhibitor, became the first systemic therapy approved for the treatment of patients with HCC. However, sorafenib only produced modest effects with low response rates in the clinic. Similarly, regorafenib, which was approved for the treatment of metastatic colorectal cancer (CRC), has had a poor response rate in the clinic. Since phosphodiesterase type 5 has been reported to be overexpressed in HCC and CRC, we hypothesized that sildenafil, a phosphodiesterase type 5 inhibitor, could enhance the toxicities of sorafenib and regorafenib in HCC and CRC cells, respectively. Our in vitro data indicated that the drugs interacted strongly to kill cancer cells via induction of ER stress, autophagy and apoptosis. In accordance with these findings, our in vivo data demonstrated a significant reduction in tumor growth. The second study in this manuscript was conducted based on the growing body of evidence about the significant contribution of EGFR and JAK/STAT signaling to the breast tumorigenesis. Our preliminary in vitro data demonstrated that the concurrent inhibition of these two pathways by lapatinib, a dual ERBB1/2 inhibitor, and ruxolitinib, a JAK1/2 inhibitor, synergistically killed breast cancer cells of all types, including the resistant triple negative subtype. Our mechanistic studies showed that the combination of ruxolitinib and lapatinib triggered cytotoxic mitophagy, and autophagy-dependent activation of BAX and BAK leading to the mitochondrial dysfunction.

Unmanned combat aircraft systems (UCAS) represent a certain type of modern technology the States use to counter the growing number of security threats coming from the various non-state actors. The thesis focuses on the most common use of UCAS in the fight against these threats, on the practice called targeted killing. The purpose of the thesis is to identify all conditions amongst the norms of international law pertaining to the use of force between States, international human rights law and international humanitarian law, which are relevant for the targeted killing through UCAS. Furthermore, the thesis seeks to set out the circumstances, under which this practice can satisfy the relevant conditions, and when, on the other hand, it is never lawful. The thesis is composed of four chapters. The First Chapter defines the key terms used in the thesis and clarifies the terminology relating to UCAS. It also offers a short overview of their technical specifications and capabilities and identifies the States, which own, develop and sell the technology. The following three chapters represent the analytical core of the thesis and set out the conditions for the use of UCAS found in three systems of international law. The Second Chapter examines the use of UCAS from the perspective of the prohibition on the...

Dans l’arsenal thérapeutique anticancéreux, les agents anti-microtubules (MTA) occupent une place essentielle dans le traitement de tumeurs solides et d’hémopathies malignes. Néanmoins, leur utilisation est limitée par l’induction d’une toxicité neurologique qui affecte la qualité de vie des patients et dont les mécanismes d’action demeurent peu compris. L’absence de solutions préventives ou curatives réellement efficaces, reflète la complexité des mécanismes d’action des MTA. Dans le cadre du projet « Mitotarget » (7ème PCRD) porté par le partenaire industriel Trophos, notre objectif était de préciser le mécanisme à l’origine de la neurotoxicité des MTA et d’évaluer le potentiel neuroprotecteur de l’olesoxime, composé ayant fait la preuve de son efficacité neuroprotectrice dans différents modèles de pathologies neurodégénératives. Nous montrons ici que les réseaux microtubulaire (dynamique des microtubules, localisation de la protéine EB1) et mitochondrial (motilité des mitochondries), cibles des MTA dans les cellules cancéreuses, sont aussi affectés dans les cellules de type neuronal. Leur préservation par l’olesoxime est nécessaire à l’établissement d’une neuroprotection. Ce travail met en évidence l’originalité du mécanisme d’action de l’olesoxime, premier neuroprotecteur capable d’agir tout à la fois sur les microtubules et les mitochondries, et souligne l’importance des liens étroits existant entre ces deux compartiments. Deux axes d’étude ont été initiés à la suite de ce projet afin de (i) déchiffrer les interconnexions microtubules-mitochondries dans la réponse des cellules cancéreuses aux MTA; (ii) préciser l’importance et la régulation post-traductionnelle de la protéine EB1 dans l’efficacité anti-migratoire des MTA. L’ensemble des données obtenues appelle à poursuivre la caractérisation des mécanismes de réponse aux agents anti-microtubules afin d’optimiser les stratégies thérapeutiques existantes. / Nowadays, the so-called Microtubule Targeting Agents (MTAs) remain benchmark clinical treatments displaying high efficiency and are still widely used against a broad spectrum of tumors and hemopathies. The new compounds in clinical development and the discovery of their anti-angiogenic properties make them a family booming. However, MTAs treatment is limited by the occurrence of neurological toxicities that greatly impair patients quality of life and which mechanisms of action are still poorly understood. The current absence of really efficient curative of preventive strategies underline the complexity of MTA mechanisms of action. In the framework of the “MitoTarget” project from the 7th PCRD,lead by the industrial partner Trophos, we aimed to precise MTA neurotoxic mechanisms and to evaluate neuroprotective potential of olesoxime, a compound that already showed to be efficient in various models of neurodegenerative diseases. Our data show that microtubular (microtubule dynamics parameters, EB1 protein localization) and mitochondria (mitochondria) networks, MTA targeted compartments in cancer cells, are damaged in neuronal-like cells. Interestingly, olesoxime neuroprotective activity implies preservation of both microtubule and mitochondria from MTA-induced damages. This work highlights the original mechanism of action of olesoxime as the first neuroprotective agent able to act on both microtubule and mitochondria and underlines the strengthened link existing between these compartments. It thus gave rise to two side projects with the aim to (i) decipher microtubule-mitochondria interconnections in response to MTA treatment; (ii) precise the importance and regulation of EB1 in the anti-migratory efficacy of MTA by looking at EB1 post-translational modifications. Altogether, the data obtained incite to keep on characterizing mechanisms involved in response to MTA in order to optimize the existing therapeutic strategies.

In recent decades we can watch heated debates on the legal and moral permissibility of State-sponsored targeted killings involving representatives from Governments, academic circles, military and police forces, intelligence services, human rights groups, humanitarian institutions, intergovernmental organizations a and the mass media. These debates are even more intense after the killing of Osama bin Laden, leader of the terrorist group Al Qaeda, in May 2011. The aim of the thesis is to answer to the question: Are targeted killings a permissible method of fight against terrorism? The legality of targeted killings is analyzed in term of lex lata international law, namely under human rights law, international humanitarian law and law of international security. The thesis is composed of six chapters. Chapter One defines basic terminology used in the thesis. The Chapter is subdivided into two parts. Part One defines the concept of targeted killings. Part Two defines the notion of terrorism. Chapter Two examines the legality of targeted killings in term of law of international security. Chapter Three describes the parallel application of human rights law and international humanitarian law. Chapter Four analyzes the legality of targeted killings under human rights law. The Chapter is subdivided into two...

La croissance d’une tumeur requiert la formation de son propre réseau vasculaire par angiogenèse. Ce processus est orchestré par une multitude de médiateurs dont le VEGF (Vascular Endothelial Growth Factor) qui interagit avec des récepteurs surexprimés à la surface des cellules endothéliales des vaisseaux avoisinant la tumeur. Les thérapies ciblées anticancéreuses jouent sur cette surexpression pour concevoir de nouvelles molécules spécifiques de la zone tumorale. Un nouvel agent de ciblage a ainsi vu le jour au sein du laboratoire. Il s’agit d’un cyclopeptide de 17 acides aminés, nommé CBO-P11 qui présente une haute affinité pour les récepteurs du VEGF. Afin de prolonger le temps de circulation du peptide dans le sang, des nanoparticules de poly(fluorure de vinylidène) (PVDF), polymère biocompatible, ont été utilisées comme vecteur. Les nanoparticules ont tout d’abord été synthétisées par une polymérisation radicalaire en émulsion dans l’eau, puis radio-greffées avec de l’acide acrylique (AA) pour obtenir du PVDF-g-PAA, ce qui a permis par la suite de les fonctionnaliser avec le CBO-P11 et un agent d’imagerie par « click » chemistry via un bras espaceur de type oligo(éthylène glycol). Différents marqueurs ont été étudiés à savoir deux sondes fluorescentes pour l’imagerie optique et un agent de contraste pour l’imagerie par résonance magnétique à bas champ. Chaque étape de synthèse a été caractérisée par de nombreuses techniques telles que la diffusion statique et dynamique de la lumière, la diffusion de neutrons aux petits angles, la spectrométrie photoélectronique de rayons X, la spectrométrie infrarouge, la spectrométrie de fluorescence et la polarisation dynamique nucléaire. Des études in vitro sur des cellules endothéliales ont permis de montrer que les nanoparticules n’étaient pas cytotoxiques et que le CBO-P11 lié au vecteur conservait sa spécificité pour les récepteurs du VEGF. Ces outils multifonctionnels présentent un grand potentiel pour cibler et imager les zones tumorales. / Tumor growth requires the formation of its own vascular network by angiogenesis. This process is mediated through numerous factors including VEGF (Vascular Endothelial Growth Factor) which interacts with receptors overexpressed on endothelial cells of blood vessels neighboring tumor. Targeted anticancer therapies take advantage of this overexpression to design tumor-specific molecules. A new targeting agent, CBO-P11 was synthesized in the laboratory. It is a 17-mer cyclopeptide with a high affinity for VEGF receptors. In order to increase the blood circulation time of this peptide, nanoparticles of poly(vinylidene fluoride) (PVDF), biocompatible polymer, were used as carrier. Nanoparticles were synthesized by radical polymerization in aqueous emulsion, then radio-grafted with acrylic acid (AA) to obtain PVDF-g-PAA. Nanoparticles were functionalized with CBO-P11 and an imaging agent by “click” chemistry via an oligo(ethylene glycol)-like spacer arm. Different dyes were used: two fluorescent probes for optical imaging and a contrast agent for low-field magnetic resonance imaging. Each step of the nanoparticles preparation was monitored and successfully characterized by numerous techniques such as dynamic and static light scattering, small-angle neutron scattering, X-ray photoelectron spectroscopy, infrared spectroscopy, fluorescence spectroscopy and dynamic nuclear polarization. In vitro studies were performed on endothelial cells and have shown that nanoparticles were not cytotoxic and that CBO-P11 conjugated to nanoparticles kept its specificity for VEGF receptors. Those multifunctional tools offer a great potential to target and image tumor.

Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors. Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity. Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively. Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione. The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.

Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.

A leishmaniose é considerada uma doença tropical extremamente negligenciada, que afeta regiões de extrema pobreza. Trata-se de doença emergente com alta morbidade e mortalidade. Aproximadamente 20 diferentes espécies de parasitas intracelulares obrigatórios do gênero Leishmania têm sido identificadas como patogênicas. Estes protozoários são transmitidos ao hospedeiro humano por meio da picada de insetos hematófagos, conhecidos como flebotomíneos. A quimioterapia é escassa, limitada e muito cara. Os fármacos disponíveis apresentam elevada toxicidade, bem como suscitam número elevado de casos de resistência. Considerando-se que a descoberta e o desenvolvimento de novos agentes leishmanicidas são extremamente necessários o objetivo deste trabalho foi contribuir com essa busca utilizando-se o método de modificação molecular, a latenciação. Tendo em vista que os dendrímeros têm sido muito utilizados como transportadores de fármacos, propôs-se a síntese de fármacos dirigidos dendriméricos de primeira geração de hidroximetilnitrofural (NFOH), composto que apresenta potencial atividade leishmanicida. Assim, pretendeu-se desenvolver fármacos dirigidos de NFOH, que apresentem ação seletiva nos macrófagos, devido à presença de manose na estrutura, além de fármacos dirigidos, que apresentem ação seletiva no parasita, devido à presença de inositol em sua estrutura. Variedade de métodos sintéticos foi utilizada com o intuito de obter os dendrímeros dirigidos propostos. Os estudos sintéticos desenvolvidos indicam a provável obtenção dos dendrímeros dirigidos de manose e de inositol, embora os produtos estejam impuros e sem o agente bioativo incorporado. A maior dificuldade encontrada foi a purificação dos compostos obtidos. Em adição, métodos de modelagem molecular foram empregados para compreender os mecanismos de liberação dos fármacos dendriméricos dirigidos de primeira geração. Características moleculares, tais como disponibilidade espacial e potencial eletrostático, foram avaliadas para predizer a região mais suscetível à ação enzimática com vistas à liberação do composto ativo. / Leishmaniasis is considered a superneglected tropical disease and affects primarily areas of extreme poverty. It represents an emergent illness with high morbidity and mortality. About 20 different species of intracellular parasites of Leishmania spp. have been identified as pathogenic. Those protozoans are transmitted to human hosts by means of the female phlebotomine sandflies bite. The chemotherapy is scarce, limited, and expensive. The drugs available can cause undesirable side effects and there are already reports of the increased number of drug resistance. Considering that, the discovery and the development of new leishmanicide agents are urgently needed, the objective of this work as to contribute to this search using the method of molecular modification, prodrug design. Taken into account that dendrimers can be used as drug carriers, the purpose of this work was the synthesis of first generation dendrimer targeted drugs of hydroxymethylnitrofurazone (NFOH), that shows potential leishmanicide activity. Thus, we designed NFOH targeted drugs with selective action in macrophages, due to the presence of mannose in the structure, and also NFOH targeted drugs that present selectivity for the parasite, due to the presence of inositol in the structure. Several synthetic methods have been used with the aim of synthesizing the targeted dendrimers with NFOH. Synthetic studies suggested the targeted dendrimers of D-mannose and of myo-inositol have been obtained, although impure and without the bioactive agent. The major difficulty was the purification of the compounds. In addition, molecular modeling methods were applied to understand the bioactive compound releasing from the first generation leishmanicide targeted dendrimers. Molecular features as spatial hindrance and electrostatic potential were evaluated to predict which region would be the most available to an enzymatic action regarding the bioactive compound release.