Shiga toxin-producing Escherichia coli (STEC) are extensive contributors to foodborne illness, causing renal and central nervous system damage due to production of Shiga toxin (Stx). Rapid Stx detection is important to distinguish STEC from other enteric pathogens. Current detection techniques are time consuming, expensive, and lack sensitivity. We have developed and evaluated a novel targeted mass spectrometry-based assay for detection of Stx using parallel reaction monitoring (PRM). The PRM assay used 11 target tryptic peptides and was validated using STEC and non-STEC bacterial cultures. Stx was detected in 56 of 62 STEC isolates and did not detect Stx in any of the 29 non-STEC isolates. The PRM assay successfully determined the Stx2 subtype in 32 of 46 Stx2-positive isolates. By applying a targeted proteomics assay, we were able to simultaneously detect Stx toxins 1 and 2 and subtype Stx2 into six toxin subgroups in Stx2-positive isolates. / February 2017

A novel series of near-infrared fluorescent contrast agents was developed and characterized. Their physicochemical and optical properties were measured. By altering functional groups of cyanine fluorophores, the selective targeting of endocrine glands, exocrine glands, cartilage and bone using NIR fluorescence to visualize the targeted tissue has been reported. These agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection these agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. The results lay the foundation for future improvements in optical imaging in endocrine surgery, tissue engineering, joint surgery, and cartilage-specific drug development.

Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies.  Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties. In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules.   Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours. The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults, affecting thousands of people worldwide every year, with a life expectancy, post diagnosis of 12 months. Surgery, radiotherapy and chemotherapy together, result in an overall mean survival not exceeding 15 months. Targeted therapeutic agents sorafenib, an oral multi kinase inhibitor, and lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, used in combination have been shown to kill GBM cells be through inhibition of major growth mediating signaling pathways that are frequently over expressed in gliomas, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT). Sorafenib can restore lapatinib induced cytotoxicity by down regulation of myeloid cell leukaemia-1 (Mcl-1) expression. Prior studies have shown Mcl-1 to play an important role in resistance to lapatinib. Furthermore, data indicated that this drug combination is able to trigger activation of autophagic and apoptotic pathways and induce endoplasmic reticulum (ER) stress response in GBM cells, collectively resulting in cell death. In conclusion, data presented here demonstrates that the combination of sorafenib and lapatinib can kill GBM cells in a greater than additive fashion, through induction of autophagy, apoptotic events (extrinsic and intrinsic) and ER stress.

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...