Les microfaisceaux d’ions ont, au cours de ces dernières décennies, montré leur efficacité dansl’étude des effets des rayonnements ionisants sur le vivant notamment concernant les effets des faiblesdoses ou l’étude de l’effet de proximité. Le CENBG dispose depuis 2003 d’un dispositif permettant la micro-irradiation ciblée d’échantillons biologiques vivants. Les applications des microfaisceaux dans ce domainese sont récemment diversifiées et des études plus fines sur les mécanismes de réparation desdommages ADN radio-induits aux échelles cellulaire et multicellulaire sont devenues possibles via lesévolutions en imagerie par fluorescence et en biologie cellulaire. Ces approches ont nécessité une évolutionimportante de l'instrumentation de la ligne de micro-irradiation du CENBG qui a été entièrementredessinée et reconstruite dans un souci d’optimisation d’apport de nouvelles fonctionnalités. Les objectifsde mes travaux ont été i) la mise en service du dispositif, ii) la caractérisation des performances dusystème, iii) la mise en place de protocoles pour l’irradiation ciblée à dose contrôlée aux échelles cellulaireet multicellulaire, in vitro et in vivo, et le suivi en ligne des conséquences précoces de cette irradiation,iv) la modélisation des irradiations afin d’interpréter les observables biologiques au regard des donnéesphysiques calculées.Ces travaux ont permis i) de caractériser les performances du dispositif : une taille de faisceau d’environ2 μm sur cible et une précision de tir de ± 2 μm, de développer des systèmes de détection d’ions pour uncontrôle absolu de la dose délivrée, ii) d’induire des dommages ADN fortement localisés in vitro, et devisualiser en ligne le recrutement de protéines impliquées dans la réparation de ces dommages,iii) d’appliquer ces protocoles pour générer des dommages ADN in vivo au sein d’un organisme multicellulaireau stade embryonnaire, Caenorhabditis elegans.Ces résultats ouvrent la voie vers des expériences plus fines sur la ligne de micro-irradiation ciblée duCENBG pour étudier les effets de l’interaction des rayonnements ionisants avec le vivant, aux échellescellulaire et multicellulaire, in vitro et in vivo. / The main goal of radiobiology is to understand the effects of ionizing radiations on the living.These past decades, ion microbeams have shown to be important tools to study for example the effects oflow dose exposure, or the bystander effect. Since 2003, the CENBG has been equipped with a system toperform targeted micro-irradiation of living samples. Recently, microbeams applications on this subjecthave diversified and the study of DNA repair mechanisms at the cellular and multicellular scales, in vitroand in vivo, has become possible thanks to important evolutions of fluorescence imaging techniques andcellular biology. To take into account these new approaches, the CENBG micro-irradiation beamline hasbeen entirely redesigned and rebuilt to implement new features and to improve the existing ones. My PhDobjectives were i) commissioning the facility, ii) characterizing the system on track etch detectors, and onliving samples, iii) implementing protocols to perform targeted irradiations of living samples with a controlleddelivered dose, at the cellular and multicellular scales, and to visualize the early consequencesonline, iv) modelling these irradiations to explain the biological results using the calculated physical data.The work of these past years has allowed us i) to measure the performances of our system: a beam spotsize of about 2 μm and a targeting accuracy of ± 2 μm, and to develop ion detection systems for an absolutedelivered dose control, ii) to create highly localized radiation-induced DNA damages and to see onlinethe recruitment of DNA repair proteins, iii) to apply these protocols to generate radiation-induced DNAdamages in vivo inside a multicellular organism at the embryonic stage: Caenorhabditis elegans.These results have opened up many perspectives on the study of the interaction between ionizing radiationsand the living, at the cellular and multicellular scales, in vitro and in vivo.

Pyrazoloanthrone and its analogues form the central core of the thesis and the work is focused on the evaluation of chemical and biological applications of pyrazoloanthrones. Selective and sensitive detection of biologically, environmentally and industrially important molecular species such as fluoride, cyanide and picric acid by using pyrazoloanthrones as sensors form the first part while the second part deals with selective and specific kinase inhibition by pyrazoloanthrones to moderate inflammation associated disorders like septic shock. All the investigations are based on extensive crystallographic studies of the participating molecules. Chapter 1 provides a brief review on the history and biological importance of 1,9-pyrazoloanthrones. The potential of these molecules as probes in sensor chemistry and protein kinase inhibition is envisaged. A short account of the techniques employed for the investigations along with a preamble is presented. Chapter 2 is divided into two parts. Part A deals with the design of a colorimetric and “turn-on” fluorescent chemosensor based on 1,9-pyrazoloanthrone specifically for cyanide and fluoride ion detection. A remarkable solid state reaction indicated by the development of intense red color occurs when crystals of tetrabutylammonium cyanide/fluoride are brought in physical contact with 1,9¬pyrazoloanthrone resulting in corresponding molecular complexes (Figure 1). X-ray crystal structures of these complexes and also of 1,9-pyrazoloanthrone have been determined and the ion sensing activity has been substantiated on the basis of spectroscopic (absorption, fluorescence and NMR) and structural analyses. The crystal structure of the parent compound exhibits a disorder as a consequence of tautomerism and the disorder gets carried on to the complexes as well with even the cyanide and the fluoride ions showing partial occupancy sites. The presence of the –NH group and associated intramolecular charge transfer upon complex formation is attributed to the extreme sensitivity of 1,9-pyrazoloanthrone for cyanide and fluoride (detection limits of 0.2 ppb and 2 ppb) ions respectively. Figure 1. Development of intense red color during the solid state reaction (shown on left) and the turn on fluorescence behavior (shown to the right) Part B demonstrates the utilization of electron rich N-alkyl substituted pyrazoloanthrones to design sensors for detecting explosive and electron deficient nitro aromatics such as picric acid (PA). The N-alkyl derivative of 1,9-pyrazoloanthrone has been synthesized, characterized by single crystal X-ray diffraction studies and evaluated as a potent sensor for picric acid. NMR and fluorescence lifetime measurements validate that the fluorescence quenching of sensor compound by PA (Figure 2) as due to the formation of excited state charge-transfer complex resulting in dynamic quenching. Figure 2. Fluorescence quenching measurements demonstrating the dynamic quenching in the charge transfer complex. Chapter 3 deals with the biological evaluation of 1,9-pyrazoloanthrone and its alkyl derivatives towards the inhibition of a decisive protein kinase called c-Jun N-terminal Kinase (JNK), an important member of MAP kinase family. JNK controls crucial cellular processes like apoptosis and cell proliferation and is implicated in disorders associated with inflammation such as septic shock, arthritis, inflammatory bowel disease, etc. Therapeutic inhibition of JNK activity by small molecules has proven to be advantageous in the treatment of diseases coupled with derailed inflammation. In this context, it is already established that 1,9-pyrazoloanthrone (SP600125) effectively and selectively inhibits JNK at concentrations beyond 10 M. A series of alkyl isomers of pyrazoloanthrone derivatives have been synthesized to evaluate the structural implications of inhibition and to elevate both selectivity and sensitivity at lower concentrations. The crystal structures of these isomers have been characterized and their utility as inhibitors has been tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). The minimum inhibitory concentrations required by these molecules to inhibit JNK was found to be lesser as compared to 1,9-pyrazoloanthrone (<5 µM; Figure 3). Critically, it turns out that among the various inhibitors synthesized, the lead candidates SPP1 and SPB1 display specific inhibition of JNK among other LPS activated MAP kinases like ERK1/2 and p38. These results suggest that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders. Figure 3. Inhibition of JNK in macrophages by the SPP1 and SPB1 compared to the known SP600125. Inspired by the results reported in the previous chapter, Chapter 4 is devoted to the generation of a library of compounds based on SPP1 and SPB1 with a purpose to design inhibitors of JNK which perform at the lowest possible concentrations and the consequent evaluation of their potential on endotoxin induced septic shock. Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. Figure 4. Two selected molecules for specific inhibition studies of JNK at lower concentrations. It is demonstrated that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Based on the results from both in vitro with macrophages and in vivo with the mouse model of septicemia, the potential role of two selected molecules D1 and D2 (Figure 4) in regulating endotoxin induced inflammation is firmly established. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the in vitro as well as the in vivo data clearly potentiates the selective inhibitory capacity of small molecule inhibitors like D1 and D2 which can facilitate the treatment of current inflammatory disorders when used in combination with the available drugs having varied efficacies. The results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.