Molecular imaging involves the development of probes which can specifically label a certain object in the body at cellular or subcellular level. This thesis consists of three parts, each involving the development of novel labelling methods for viruses or virus-like particles with specific applications. Virus-like particles (VLP) derived from the E. coli bacteriophage Qβ are widely employed as a nano-carrier for drugs and vaccines, but a powerful method for tracing its circulation without affecting its structure is yet to be developed. In the first part of the thesis, the electrophilic fluorine source ¹⁹F-Selectfluor^TM was employed for introducing single fluorine atoms on Qβ VLPs. For the 'tag-and-modify' approach, site-selective electrophilic C-F bond formation was achieved on the dehydroalanine (Dha) amino acid tag of VLPs under aqueous conditions. Chemoselective electrophilic aromatic fluorination on tyrosine residues were also achieved using the same reagent by manipulating the amino acid sequence. Similar results were observed in conditions required for ¹⁸F-Selectfluor™ reaction, indicating the potential of this technique for positron emission tomography (PET) imaging. In addition, there is a lack of in situ technique for tracking the functional status of Qβ VLPs and hence the release of cargos. In the second part of the thesis, a simple way to monitor the disassembly of ¹⁹F-labelled Qβ VLPs by ¹⁹F NMR spectrosocpy is reported. Analysis of resonances, using experiments under a range of conditions, allowed determination not only of the intact particle but also the disassembled multimeric species and even smaller peptides upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems. In the third part of the thesis, a new type of rhodamine B fluorescent dye functionalised with a 2-imino-2-methoxyethyl (IME) group is reported. The amidine linkage formed between the IME group and lysine residue retains the pKaH of the original side chain, which cannot be achieved using commercially available conjugating dyes. This in turn minimises the change in net charge hence virus infectivity following virus labelling. By employing adenovirus (AV) as an example, the IME dye was shown to be a better choice in retaining virus infectivity compared to dyes linked with other coupling groups. In addition, preliminary experiments on dengue virus with the synthesised dyes were also performed.

No description available.

Yes / Platinum drugs are widely used for cancer treatment. Other precious metals are promising, but their clinical progress depends on achieving different mechanisms of action to overcome Pt-resistance. Here, we evaluate 13 organo-Os complexes: 16-electron sulfonyl-diamine catalysts [(η6-arene)Os(N,N′)], and 18-electron phenylazopyridine complexes [(η6-arene)Os(N,N’)Cl/I]+ (arene = p-cymene, biphenyl, or terphenyl). Their antiproliferative activity does not depend on p21 or p53 status, unlike cisplatin, and their selective potency toward cancer cells involves the generation of reactive oxygen species. Evidence of such a mechanism of action has been found both in vitro and in vivo. This work appears to provide the first study of osmium complexes in the zebrafish model, which has been shown to closely model toxicity in humans. A fluorescent osmium complex, derived from a lead compound, was employed to confirm internalization of the complex, visualize in vivo distribution, and confirm colocalization with reactive oxygen species generated in zebrafish. / Wellcome Trust (grant no. 107691/Z/15/Z), ERC (grant nos. 247450, 324594), Science City (AWM and ERDF), WCPRS and Bruker Daltonics (Studentship for JPCC), Mike and Enfys Bagguley, and EPSRC (Studentship for HEB, and grant no. EP/F034210/1).

Ces travaux de recherche s’intéressent à la conception de nouvelles méthodologies analytiques destinées à mesurer le bénéfice d’une transplantation hépatique ou bien encore à l’évaluation de nouvelles voies thérapeutiques à l’essai pour des patients atteints de la polyneuropathie amyloïde familiale à transthyrétine (TTR). Cette maladie rare se caractérise par une déstabilisation structurale du tétramère de TTR qui aboutit à l’agrégation de fibrille dans les tissus du système nerveux autonome, au niveau des nerfs périphériques et autour de certains organes dont le cœur. Dans le cadre d’une collaboration entre hospitalo-universitaires, chimistes analystes, électrochimistes, physico-chimistes et technologues, nous nous sommes attachés à développer des séparations en électrophorèse capillaire couplée avec une détection optique de peptides natif et muté qui sont directement associés à une variente de cette maladie rare. La difficulté première de cette recherche concerne le choix même de ces biomarqueurs qui s’est finalement révélé pertinent grâce de la réalisation de cartes peptidiques à partir du sérum. Ensuite deux voies ont été explorées : une séparation électrocinétique avec une détection spectrométrique d’absorbance dans l’ultra-violet et l’autre nécessitant le marquage préalable de peptides par des molécules fluorophores ou fluorogènes pour ensuite faire une séparation en électrophorèse couplée LIF (laser induced fluorescence). Dans les deux cas le critère principal de séparation, la résolution, autorise une quantification et surtout les validations analytiques associées montrent une réelle robustesse des méthodologies développées. L’autre signe encourageant pour la transposition des ces méthodes à l’analyse de prélèvements issus de patients, concerne la limite de quantification qui est inférieure à celle couramment mesuré dans le sérum. La spectrométrie de masse, moyen d’investigation physico-chimique puissant à permis de suivre et de comprendre d’un point vue plus fondamental le produit des réactions de chimie organique de dérivation des peptides par trois marqueurs fluorescents : le TAMRA-SE, le NDA et le FQ. La possibilité de proposer un outil d’analyse miniaturisé et simple d’utilisation pour le monde hospitalier a également été étudiée. Un poste d’analyse sur puce microfluidique permettant l’analyse quantitative et qualitative a été installé pour permettre la réalisation de premiers essais expérimentaux de séparations électrocinétiques sur puce microfluidique. Ces travaux jettent les bases d’une nouvelle voie analytique pour séparer et quantifier les différents biomarqueurs caractéristiques de la polyneuropathie amyloïde familliale à TTR. / The purpose of our work was the development of new analytical methodologies to measure the benefit of liver transplantation and also the evaluation of new therapeutic approaches under testing on patients with Transtyretin (TTR) familial amyloid polyneuropathy. This rare disease is characterized by a structural destabilization of TTR tetramer leading to it’s aggregation into amyloïd fibrils that accumulate in the tissues of the autonomous nervous system, peripheral nerves and around certain organs, including the heart. As part of a collaboration between university, hospital, analytical chemists, electrochemist, physical chemists and technologists, we are committed to develop separations in capillary electrophoresis coupled with optical detection of native and mutated peptides that are directly associated with a variant of this rare disease. The first challenge of this research is the choice of these biomarkers that ultimately proved relevant with the realization of peptide maps from the serum. Then two approaches have been explored: electrokinetic separation with absorbance spectrometric detection in the ultraviolet and the other requiring the prior labeling peptides with fluorescent molecules and then to a separation on electrophoresis coupled with LIF (Laser induced fluorescence). In both cases the main criterion of separation, resolution, allows quantification and especially analytical validations show actual strength associated methodologies developed. Another encouraging sign for the transposition of these methods to the analysis of samples from patients regarding the quantification limit is lower than commonly measured in serum. Mass spectrometry, using physico-chemical investigation powerful allowed to follow and understand a more fundamental viewpoint the product of organic chemistry reactions bypass peptides by three fluorescent dyes: TAMRA-SE, the NDA and FQ. The ability to provide a miniaturized analysis and easy to use tool for the hospital environment was also studied. A post analysis on microfluidic chip for quantitative and qualitative analysis was installed to allow the realization of the first experimental tests of electrokinetic separations on microfluidic chip. These studies lay the foundation for a new analytical way to separate and quantify the different characteristics biomarkers family TTR amyloid polyneuropathy.

NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.

Fluoreszenzmarkierungstechniken für lebende Zellen ermöglichen es Biologen, einen Blick in eine komplexe biologische Umgebung zu werfen und Informationen über ein bestimmtes Ziel in einer nahezu natürlichen Umgebung zu erhalten. Dank der konzertierten Bemühungen der wissenschaftlichen Gemeinschaft gibt es eine Fülle von kommerziell erhältlichen, genetisch kodierbaren Markern und Reportern für die Fluoreszenzmikroskopie. Allerdings gibt es nur wenige Lebendzellmethoden, die eine direkte Konjugation von Nukleinsäuren mit Proteinen erlauben, obwohl es robuste DNA-Technologien gibt, die mit Oligo-Antikörper-Konjugaten auf Zelloberflächen durchgeführt werden. Ein weiterer, oft einschränkender Aspekt der Markierung ist die Fähigkeit, Ziele selektiv zu multiplexen. In dieser Studie wurde eine Methode der Tag-Probe-Markierung entwickelt, die eine selektive, gleichzeitige Markierung von zwei verschiedenen Zielen mit zwei Peptid-Nukleinsäure-Strängen (PNA) ermöglicht. Diese Methode verwendet ein Paar von Coiled-Coil-Peptiden, um die Konjugation einer PNA-Gruppe an ein Zielprotein zu steuern, das ein Peptid-Tag exprimiert. Die Verwendung orthogonaler Coiled-Coils ermöglicht Multiplexing. Die Markierung von synthetischen Tag-Peptiden, die mittels Flüssigchromatographie analysiert wurden, hat gezeigt, dass der orthogonale duale Transfer von PNA selektiv, quantitativ und schnell ist. Die PNA-Konjugation von exemplarischen Membranrezeptoren, gefolgt von der Hybridisierung mit komplementären Fluorophor-DNAs, ermöglichte eine unkomplizierte Visualisierung von dualen Rezeptoren in lebenden Zellen. Durch den Einsatz einfacher molekularer Hilfsmittel, die die Grundlage der DNA-Nanotechnologie bilden, konnte durch die Rekrutierung mehrerer DNAs eine zunehmend hellere Markierung erreicht werden und die löschbare Oberflächenmarkierung ermöglichte eine quantitative Untersuchung der Rezeptorinternalisierung. / Live-cell fluorescent labelling techniques allow biologists to glimpse into a complex biological environment and derive information about a specific target in a near-native environment. Thanks to a concerted effort from the scientific community, a plethora of commercially available, genetically encodable tags and reporters for fluorescence microscopy exist. However, few live-cell methods allow direct conjugation of nucleic acids with proteins despite the robust DNA technologies carried out on cell surfaces using oligo-antibody conjugates. Another aspect of labelling which is often limiting is the ability to selectively multiplex targets. In this study, a method of tag–probe labelling was developed that accomplishes selective, simultaneous labelling of two distinct targets with two peptide nucleic acid (PNA) strands. The technique uses a pair of coiled-coil peptides to guide conjugation of a PNA group to a target protein expressing a peptide tag and using orthogonal coiled-coil enables multiplexing. Initially, the labelling of synthetic tag-peptides analysed by liquid chromatography revealed the orthogonal dual transfer of PNA to be selective, quantitative, and rapid. PNA conjugation of exemplar membrane receptors followed by hybridization with complementary fluorophore-DNAs achieved straightforward live-cell dual receptor visualization. Finally, using simple molecular tools that form the basis of DNA nanotechnology, recruitment of multiple DNAs facilitated progressively brighter labelling, and erasable surface labelling allowed quantitative study of receptor internalisation.