The fast evolution of medical micro- and nanorobots in the endeavor to perform non-invasive medical operations in living organisms has boosted the use of diverse medical imaging techniques in the last years. Among those techniques, photoacoustic imaging (PAI), considered a functional technique, has shown to be promising for the visualization of micromotors in deep tissue with high spatiotemporal resolution as it possesses the molecular specificity of optical methods and the penetration depth of ultrasound. However, the precise maneuvering and function's control of medical micromotors, in particular in living organisms, require both anatomical and functional imaging feedback. Therefore, herein, the use of high-frequency ultrasound and PAI is reported to obtain anatomical and molecular information, respectively, of magnetically-driven micromotors in vitro and under ex vivo tissues. Furthermore, the steerability of the micromotors is demonstrated by the action of an external magnetic field into the uterus and bladder of living mice in real-time, being able to discriminate the micromotors’ signal from one of the endogenous chromophores by multispectral analysis. Finally, the successful loading and release of a model cargo by the micromotors toward non-invasive in vivo medical interventions is demonstrated.

After injury, dorsal root (DR) axons regenerate in the peripheral nervous system (PNS), but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the central nervous system (CNS). Examination of the dynamic axon regeneration that occurs following injury to the DR provides the opportunity to advance our understanding of what happens to sensory axons as they approach and arrive at the DREZ and expands our knowledge of sensory axon regeneration failure at the entrance to the spinal cord. Additionally, findings from these studies may offer potential avenues to provide insight into regeneration failure elsewhere in the central nervous system. Nevertheless, our understanding of the cellular and molecular processes underlying the failure of DR axons to regenerate through the DREZ is incomplete. The goal of my thesis work was to determine whether application of the time lapse-in vivo imaging technique is feasible and useful in studying dorsal root regeneration. I have also applied recently developed post-mortem analyses to the axons monitored in vivo, which provided additional insights into the mechanisms that prevent axon regeneration at the DREZ. Results in Chapters 2 and 3 demonstrate that wide-field microscopy is indeed feasible and useful for monitoring regenerating sensory axons immediately before, during, and in the days to weeks after lumbar (L5) DR crush. I was surprised to find that most axons were immobilized abruptly and chronically at the CNS portion of the DREZ, with their axon tips and shafts exhibiting features of differentiated nerve terminals. This observation raises the possibility, which has not been appreciated previously, that DR axons stop at the DREZ because their regeneration is terminated prematurely by forming synaptic contacts with unidentified postsynaptic cells. To confirm the immobilization of DR axons at the DREZ, I applied two-photon microscopy to examine the axon behavior at the DREZ at high resolution. Results described in Chapter 4 confirm those obtained with the time-lapse imaging performed with wide-field microscopy: axons arrested soon after their arrival at the DREZ did not exhibit even subtle movements. Light microscopic analyses of the failed axon tips monitored in vivo demonstrated that almost all axons stopped at the CNS territory of the DREZ, and that axon tips and adjacent shafts intensely immunolabeled with synapse markers. Ultrastructural analyses revealed that numerous axonal profiles had the characteristic features of pre- but not postsynaptic endings. Findings from these studies lead us to speculate that most, if not all, dorsal root axons become arrested as they enter the CNS territory of the DREZ by forming presynaptic terminals on non-neuronal cellular elements that differ from the dystrophic-like endings formed by a few axons. In the chapter 5, I discuss what I have found to be the key factors for successful monitoring of regenerating dorsal root axons in living animals; the feasibility, usefulness and limitations of the available techniques and future directions for studying spinal root injury and regeneration. My thesis work represents the first to employ in vivo imaging to study DR regeneration directly in living animals. This approach was more challenging to develop than we had anticipated but provided unexpected insights into the mechanisms preventing sensory nerve regeneration. Continuous application of the powerful in vivo imaging technique in combination with conventional analyses will elucidate critically important issues that previous static analyses could not decipher. / Cell Biology

BACKGROUND:Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model.RESULTS:The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected.CONCLUSIONS:The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.

Ce travail de recherche vise à mettre au point la première gamme de sondes magnétogéniques à base de Fer(II) répondant à un analyte biochimique. L’objectif est de créer les premières sondes résolument silencieuses en IRM qui ne génèrent un signal qu’après leur rencontre avec l’analyte, une caractéristique hautement désirable dans le domaine de l’IRM moléculaire. Au cours d’un travail doctoral précédent, une paire de complexes ferreux modèles, hydrosolubles et chargés positivement, a été identifiée et a permis de valider l’idée de ce concept OFF-ON in vitro. Dans un premier temps nous avons démontré qu’un tel concept pouvait être également validé in vivo chez la souris. Ceci a nécessité le développement de stratégies de synthèse organique inédites et notamment la mise au point de synthons tétrazolyleméthyle protégés. Une telle méthodologie a notamment démontré son efficacité dans la synthèse du premier analogue totalement azoté de l’EDTA. Par la suite nous avons démontré qu’il était possible sous certaines conditions, non physiologiques, de réaliser les exigences du concept de magnétogénèse. En particulier nous avons démontré que des unités amidines peuvent être modifiées, en ayant recours à des concepts de type prodrogue, et utilisées pour éteindre et allumer le spin électronique du Fer(II) après rencontre avec l’analyte. Enfin nous avons également démontré au cours de ce travail, avec une seconde stratégie, que le concept de magnétogénèse était possible dans des conditions physiologiques et constantes et avons construit un modèle biologique afin d’évaluer une molécule candidate prometteuse in cellulo. / This PhD project aims to develop the first line of Iron(II) based magnetogenic probes that respond to bio-chemical analytes. It sets out to address one of the main limitations of responsive probes by rendering the initial probe completely MRI silent. During the previous investigations of the Bio-organic chemistry group, a duo of Iron(II) low spin-high spin parent complexes has been identified as the basis for a magnetogenic design. In the current work we have validated this OFF-ON approach, in vivo, by ensuring the electroneutrality of the final contrast agent. Such a feature required the development of protected synthons for the convergent introduction of tetrazolylmethyl chelating motifs. And such a synthetic methodology was also applied for the synthesis of the first full nitrogen analog of EDTA. In a second part of this work, a first magnetogenic concept was explored exploiting amidine moieties to silence or awaken the electronic spin of ferrous complexes. We demonstrated that this magnetogenic concept was valid, after a short chemical stimulation, though at the expense of harsh acidic conditions to trigger the paramagnetism of the final complex. Finally we successfully explored a second magnetogenic concept operating in physiological conditions and responding to bio-chemical stimulations.We then evaluated the most promising candidate in cellulo by developing a biological model expressing the nitroreductase enzyme.

During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.

Resumo:A leishmaniose visceral é uma importante doença parasitária com distribuição mundial e que afeta homens e animais. Existem relatos de envolvimento encefálico durante a infecção, mas sua patogenia ainda não foi completamente elucidada. Com o presente estudo, objetivamos a avaliação de quimiocinas relacionadas com a resposta inflamatória no encéfalo e marcadores da integridade da barreira hematoencefálica em cães naturalmente infectados. Também foi estabelecido um modelo experimental murino que permite a avaliação da dinâmica da infecção, bem como localização do parasita e resposta inflamatória in vivo. Cães naturalmente infectados apresentaram um perfil específico de quimiocinas no encéfalo, altamente quimioatrativo para linfócitos T, além de evidente perda da integridade da barreira hematoencefálica, com elevado quociente de albumina e altas concentrações de anticorpos anti-Leishmania no líquido cefalorraquidiano. Utilizando inovadoras técnicas moleculares e de imagiologia in vivo, foi possível determinar a presença do parasita no encéfalo de camundongos Balb/c experimentalmente infectados, bem como sua localização intra-craniana. A inflamação no encéfalo, avaliada nos primeiros quatro meses pós-infecção, apresentou duas fases distintas, uma fase aguda nos primeiros quatorze dias, e uma fase de reinflamação após três meses de infecção. Conjuntamente, os dados desse estudo permitiram confirmar o envolvimento encefálico durante a leishmaniose visceral, e reforçam o fato de que o comprometimento encefálico tem sido negligenciado durante a leishmaniose visceral / Abstract:Visceral leishmaniasis is an important parasitic disease, with worldwide distributions that affects humans and animals. There are reports of brain involvement during the infection; however, its pathogenesis has not been completely elucidated. Therefore, with this study, we aimed the evaluation of the inflammatory response in the brain and the integrity of the blood-brain barrier in naturally infected dogs, and the establishment of an experimental mice model that could allow the evaluation of the infection dynamics, as well as the localization of the parasite and the inflammatory response in vivo. Naturally infected dogs presented a specific chemokine profile in the brain, highly attractive to T-lymphocytes, besides the evident blood-brain barrier disruption, with increased albumin quota and elevated concentrations of anti-Leishmania antibodies in the cerebrospinal fluid. Using molecular techniques and state-of-the-art in vivo imaging tools, we could determine the presence of parasites in the brain of experimentally infected Balb/c mice, as well as its intracranial localization. Brain inflammation, evaluated during four months post-infection, presented two distinct phases, one acute phase in the first fourteen days, and one phase of re-inflammation after three months of infection. Altogether, the data from this study allow us to confirm that the brain is affected during visceral leishmaniasis, and reinforce the fact that the brain involvement has been neglected during visceral leishmaniasis / Orientador:Gisele Fabrino Machado / Banca:Karen Regina Lemos / Banca:Valéria Marçal Felix de Lima / Banca:Cáris Maroni Nunes / Banca:Antonio Carlos Alessi / Doutor

L'hippocampe, structure cérébrale située dans le lobe temporal, est au coeur de la gestion de nombreuses fonctions cognitives comme l'encodage des informations spatiotemporelles ou encore la mémoire épisodique. A l'heure actuelle, l'hippocampe est étudié via de nombreuses méthodes notamment l'imagerie de fluorescence qui, utilisée sur des animaux éveillés, permet d'accéder au fonctionnement du réseau neuronal. Malgré cela, une sous-région : le gyrus denté a encore un rôle mal élucidé car profondément enfoui dans le cerveau. Son étude permettrait d'apporter de nouveaux éléments sur le fonctionnement de l'hippocampe. De part sa profondeur d’environs 1 mm, son imagerie demeure très difficile. En effet, la diffusion ainsi que les aberrations optiques introduites par les couches successives de matière dégradent fortement la qualité d'imagerie. Pourtant l'optique adaptative, une technique héritée de l'astronomie, pourrait changer cela. En l'intégrant à un microscope bi-photon, il serait possible de compenser les aberrations optiques introduites par le cerveau et ainsi d'arriver à effectuer l'imagerie in vivo du gyrus denté. Durant ma thèse, j'ai donc travaillé à la conception complète tant du point de vue matériel que logiciel d'un microscope bi-photon adapté à l'imagerie in vivo et équipé d'un dispositif de correction de front d'onde. J'ai également développé une méthode d'optimisation prometteuse basée sur l'approche modale de la correction des aberrations optiques couplée à l'utilisation d'une métrique adaptée à l'imagerie non-linéaire en profondeur. Enfin, j'ai pu appliquer cette méthode dans des conditions in vitro et in vivo permettant de montrer son efficacité. / The hippocampus, a cortical structure located in the temporal lobe, is at the heart of the management of many cognitive functions such as spatiotemporal information encoding or episodic memory. At present, the hippocampus is studied through many methods including fluorescence imaging, and used on awake animals, allows access for the study of the neural network function. Despite this, a sub-region: the dentate gyrus has still a poorly elucidated role because it is deeply buried in the brain. His study would bring new elements on the hippocampus functioning. Due to its depth of about 1 mm, its imagery remains very difficult. Indeed, scattering as well as optical aberrations introduced by the successive layers of matter strongly degrade the imaging quality. Yet adaptive optics, a technique inherited from astronomy, could change that. By integrating it into a bi-photon microscope, it would be possible to compensate optical aberrations introduced by the brain and thus to achieve the in vivo imaging of the dentate gyrus. During my PhD, I worked on the complete design both in hardware and software of a bi-photon microscope suitable for in vivo imaging and equipped with a wavefront correction device. I also developed a promising optimization method based on the modal approach of optical aberration correction coupled with the use of a metric adapted to nonlinear depth imaging. Finally, I was able to apply this method in in vitro and in vivo conditions to show its effectiveness.

Le danio zébré est un modèle bien établi pour étudier la biologie du développement des vertébrés. Ses larves transparentes sont favorables à des approches de microscopie non invasive, qui permettent de réaliser des observations à l’échelle d’un individu entier à des niveaux de résolution cellulaire et subcellulaire. Ces atouts font du danio zébré un excellent modèle pour étudier les infections virales in vivo. Au cours de mon projet, j’ai etudié l’entrée et la colonisation du système nerveux central (SNC) par le virus Sindbis (SINV) dans le modèle danio zébré. Mon projet présentait plusieurs axes: 1) développer un modèle d’infection du virus Sindbis chez le danio zébré, 2) caractériser l’invasion du SNC par le virus par des techniques d’imagerie à haute résolution, 3) définir la voie d’entrée du virus dans le SNC, 4) évaluer la dynamique de la réponse immunitaire innée par l’étude de la réponse interféron. Le suivi de la propagation du virus a été rendu possible par l’utilisation d’un ARN viral recombinant exprimant la protéine fluorescente verte ‘GFP’. L’utilisation de cette construction m’a permis de caractériser la progression de SINV chez le danio zébré et d’identifier les organes/tissus cibles que sont le vitellus, le foie, le cœur et enfin, le cerveau. Les données rassemblées jusqu'à présent m’ont aussi permis d’identifier le mécanisme par lequel SINV se propage vers le cerveau: le virus se propage par un transport axonal du system nerveux périphérique vers le SNC. Dans le cadre de la réponse immunitaire au niveau cellulaire, j’ai pu observer le rôle joué par les leucocytes, en particulier les neutrophiles, comme cellules productrices d'interféron. / The zebrafish (Danio rerio) is an important model organism, particularly for studies of development and more recently host pathogen interactions. As opposed to other vertebrate model organisms, its optical clarity and ease of genetic manipulations allow to visualize highly dynamic cellular processes in vivo at the whole organism scale. These assets make the zebrafish a perfect model for the study of viral infections in vivo, such as those caused by neurotropic viruses. The aim of this project has been to gain insights in some of the interactions that determine encephalitis, by characterizing the neurotropic Sindbis virus (SINV). This Thesis project has consisted therefore in: 1) the development of a SINV infection model in zebrafish larvae, 2) the characterization of SINV neuroinvasion upon its inoculation in the bloodstream, thanks to the use of high resolution microscopy, 3) the study of SINV mechanism of entry in the CNS, 4) the characterization of the innate immune response, both at the whole organism and organ specific level. Thanks to the use of a SINV recombinant strain, engineered to express the green fluorescent protein “GFP” in infected cells upon viral replication, we have been able to follow the onset and the progression of the infection. We have suggested infection of peripheral neurons and subsequent axonal transport to the CNS as SINV entry mechanism. At the cellular level, we have identified neutrophils as the main IFN producing cells.

La vaccination est considérée comme l’un des plus grandes découvertes de l’histoire des maladies infectieuses, ayant permis le déclin et l’éradication de plusieurs pathogènes. Cependant, nous ignorons encore tous les mécanismes impliqués dans la protection contre les pathogènes. Cette méconnaissance est la cause de notre incapacité à formuler des nouveaux vaccins contre le VIH, la tuberculose, le paludisme et les pathogènes émergents. Récemment, on note des efforts pour induire une réponse cellulaire efficace après une vaccination, qui joue un rôle crucial dans la clairance des pathogènes.Cette thèse s’appuie sur un modèle de vaccin vivant atténue issu du virus de la vaccine : le MVA (Modified Vaccinia Ankara) et sur le modèle de primate non-humain. Nous avons caractérisé la réponse cellulaire après une immunisation intradermique suivant un schéma en prime-boost homologue, avec un boost à 2, suivi d’un boost à 9 mois. Le MVA a induit une infiltration massive de Lymphocytes T CD8 au niveau du site d’injection, 7 jours après l’immunisation. La réponse cellulaire systémique était modérée et ne reflétait pas l’amplitude de la réponse locale. Les injections du prime et du boost ont orienté la réponse cellulaire de façon différente, ce qui a mené à une importante induction de cellules T CD4 et CD8, persistantes, spécifiques de l’antigène et polyfonctionnelles après l’injection du boost à 9 mois.Cette étude souligne la différence entre les réponses systémiques et locales, démontrant l’importance de se focaliser sur la réponse tissulaire. Elle a également mis en lumière l’impact du schéma d’immunisation sur la qualité de la réponse cellulaire. / Vaccination has been considered as one of the greatest discoveries in the history of infectious diseases by allowing pathogens decline or eradication. However, we still ignore all the mechanism that lead to protection and therefore, fail to elaborate new vaccines against HIV, tuberculosis, malaria and emergent pathogens. Recently, efforts have been made to elicit effective cellular response after vaccination, which is crucial for pathogen clearance.This thesis relied on live-attenuated vaccine model derived from the vaccinia virus: the MVA (Modified Vaccinia Ankara) and a non-human primate model. We characterized the cellular immune response triggered by a homologous prime-boost intradermal injection of MVA, with a 2 months and 9 months boost. The MVA induced a massive infiltration of CD8 T cells at the injection site 7 days post immunization. In comparison, the systemic cellular response was mild and did not reflect the magnitude of the local response. The prime and boost injections elicited distinct orientation of the systemic and local T cells, which led to an important induction of a persistent, antigen-specific and polyfunctional CD8 and CD4 T cell responses after the 9 months boost.This work emphasizes the difference between local and systemic response, demonstrating the importance of the focus on tissue immunity. It also highlights the impact of the immunization schedule on the quality of the cellular response.

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