This proposal in the Mosaic group in the Fresnel Institute (Marseille), concerns the development of a new nonlinear microscopy technique based on the spectral coherent control, in phase and polarization, of the optical excitation. This technique will be dedicated to the observation of the dynamic of local orientational orders in molecular and biomolecular samples, from ordered organic crystalline systems to the living cell. The goal of this work is to answer to the need of a more refined knowledge of molecular arrangements in processus where they are crucial, such as the dynamics of crystals formation or the characteristics of lipid domains formation in cell membranes contributing to cell recognition. This experiment will be mainly based on two-photon (second harmonic generation: SHG) and three-photon (coherent anti-stokes Raman scattering: CARS) nonlinear processes, chosen for the complementary information they bring on molecular systems. These phenomena are also well known in the team responsible for this project, through the benefit of experience I gained in my previous works on SHG and of the Mosaic group in CARS. The originality of this experience is to combine on the one hand recent advances in terms of multiphotonic microscopy and on the other hand in coherent control. In particular, it is recognized today that a polarimetric measurement of nonlinear responses is rich in information on molecular organization, and that the spectral phase profile of a short pulse can greatly influence the nonlinear processes efficiency. In this experience, we benefit from an excitation of molecular systems by a large spectrum (~120nm) from an ultra-short laser pulse (~10fs). The coherent control will be based on the shaping of the spectral profile of the pulses, in phase and polarization, using a spatial light modulator after spectral dispersion. This set-up allow to (i) optimise the excitation pathways of molecules by multiphotonic intra-pulse interferences, and (ii) profit from a polarization shaping of the excitation for a direct read-out, local and in real time, of complex molecular orientational distributions. The studied samples will be first based on simple symmetries such as in crystals, then on more complex structures such as in molecular monolayers and lipid bilayers. These structures will be doped with active molecules defined in collaboration with chemists in ENS Lyon. The last part of the project concerns the application of this imaging technique to cell membranes with the study of the dynamics of molecular order within lipid domains. This last part will be developed in collaboration with the Centre d'Immunologie de Marseille Luminy (CIML).

Le projet DrosActinCut vise à développer des méthodes spécifiques pour mesurer et déterminer le rôle des forces de tension corticale du réseau d'acto-myosine associées à la morphogenèse tissulaire in vivo. Nous étudions l'intercalation cellulaire, un processus conservé reposant sur le remodelage polarisé des interfaces adhérentes de cellules épithéliales. Le système étudié est l'embryon précoce de drosophile, un système remarquablement adapté aux méthodes récentes d'imagerie permettant de suivre la dynamique cellulaire et moléculaire in toto, et en raison des approches fonctionnelles génétiques propres au modèle drosophile. Le réseau d'acto-myosine gouverne la forme et le comportement dynamique des cellules. Notre projet vise à étudier la dynamique et les propriétés mécaniques du réseau d'actomyosine au cours du remodelage des interfaces cellulaires pendant l'intercalation. Nous suivrons la dynamique de l'actine (polymérisation et dépolymérisation) avec des techniques de fluorescence (Fluorescence Loss Induced by Photobleaching et Fluorescence Recovery after Photobleaching) et à l'aide de variants fluorescents de l'actine. Cette étude nous permettra d'observer une anisotropie possible de la dynamique de l'actine en relation avec le remodelage des jonctions adhérentes durant l'intercalation. Par ailleurs, nous approfondirons l'étude de cdGAP, une protéine Gap régulatrice de Rac1 et Cdc42, interagissant avec le réseau d'acto-myosine et impliquée dans l'intercalation cellulaire. Pour étudier les propriétés mécaniques des jonctions adhérentes et le rôle du réseau d'acto-myosine, nous développerons et utiliserons des outils optiques spécifiques. Tout d'abord, nous tenterons de modifier l'équilibre des forces agissant sur les jonctions avec un système dynamique de pinces optiques. Nous chercherons à perturber la balance des forces contradictoires du réseau contractile d'acto-myosine et d'adhésion, en exerçant des forces sur plusieurs billes placées aux jonctions. Nous développerons en outre un microscope pour réaliser la nanoablation du réseau d'acto-myosine, à l'aide d'impulsions laser femtosecondes dans le proche infra-rouge. L'étude du mouvement des filaments fragmentés et l'observation des rearrangements cellulaires nous permettront d'identifier les mécanismes moteurs de l'intercalation et probablement la distribution des «générateurs» de force responsables du remodelage des jonctions adhérentes.

Since decades, investigations of pathological and physiological events using spectroscopic techniques became more and more important. Nowadays, such events are investigated at the molecular level and require techniques exhibiting high levels of sensitivity, resolution and specificity of the bioprocess. Furthermore, the ultimate aim of such techniques is to be applied to human being. However, at this time, no techniques exhibit these requirements. Thus, tremendous efforts are performed to develop bioprobes aiming to improve the requirements mentioned above. For spectroscopic applications, beside their biospecificity, these bioprobes must afford a typical spectroscopic signal. This has been partly reached for many bioprocess investigated by MRI, the most common and suitable technique to investigate bioprocess in human beings and animals. Despite many successes, a lot of events still escape to this technique. Hence, the aim of this project is to develop new bioprobes exhibiting high bio-specificity and a very typical signal. For this, we plan to develop molecules suitable to investigate non-radical bioprocess using a radical reaction. Indeed, we plan to prepare a bioprobe (bioalkoxyamine R1R2NOR3) stable, which, in the presence of a specific enzyme, hydrolysis into an alkoxyamine (R1R2NOR4), the latter decomposes spontaneously into alkyl radical (R4.) and nitroxide (R1R2NO.) at physiological temperature. Then, the nitroxide can be easily detected by EPR or by DNP-MRI by its effect on the surrounding molecules. Our project is divided in 3 phases: i) preparation of bioprobes, ii) investigation of their hydrolysis (bioactivation by enzymes) and homolyse (spontaneous fragmentation into radicals at physiological temperature) as well as the resistance of nitroxides to bio-reductants, and iii) application in DNP-MRI to investigate in a non-invasive way the proteolysis in living mice. A Smart Spin Probe is then defined as a radical-type probe bearing a typical signal which appears only in the presence of the investigated biological event.

Introduction The present demand is a resubmission of project RBF4MED submitted to the ANR within the 2007 'Blanc' call for proposals, in which it was placed on the complementary list, but ultimately not financed. Taking into account the entirely positive evaluation by the reviewers and the recommendations made by the ANR committee, the project was reduced in scale, and its costs are therefore significantly diminished in terms of MRI exams. Perfusion MRI measures capillary blood flow that is actually used by the tissue. In present clinical practice, perfusion MRI protocols are used on patients with macrovascular lesions after stroke or myocardial infarction. Beyond relative perfusion assessment comparing different territories, the existing perfusion MRI methods can in theory be used to absolutely quantify regional capillary blood flow. Reliable perfusion MRI methods, however, are still under development, and post-processing is still too complicated to be used by MR technologists or clinicians without interaction with MR physicists. Reliable and easy perfusion quantification would allow diagnosis and study of pathologies that are presently inaccessible by MRI. The application field of MRI would benefit from a considerable enlargement if quantitative perfusion MRI techniques entered clinical protocols. Concerned pathologies across all organs are Alzheimer as well as diabetes with its cardiovascular implications. Using quantitative approaches, longitudinal follow-up of pathologies in patients and animals becomes possible. Research objectives Methodology -Establish an organ-dependent method comparison for quantitative perfusion MRI. This will concern pure methodological healthy volunteer studies. Comparative animal studies will precede the work on humans. -Establish an original spin-labeling method for human myocardial perfusion quantification. -Develop optimized quantitative perfusion MRI techniques by including recent hardware developments such as dedicated multi-channel coil arrays, high field strengths and fast gradient systems. -Evaluate the practical impact of high-field clinical MRI devices on the comparison of perfusion MRI methods. This will be done using the 3 Tesla facility implemented at CRMBM in june 2008. Arterial spin labeling MRI techniques are in the focus of this project and particularly benefit from higher fields. Applied research -Increase the accessibility of quantitative perfusion MRI methods with respect to clinical teams and technologists by the development of reliable and sensitive imaging techniques and highly automated post-processing software. -Strengthen small animal MRI by adding quantitative perfusion MRI techniques to already established longitudinal protocols and by extending CRMBM's experience on cardiac small animal MRI to other organs. Project outline With respect to the structure of the CRMBM, this project is transversally organized between heart and brain, and also going from animal to human studies. Every « classical » team will benefit from the experience and developments carried out transversally. Developments on animals will not only contribute to accelerate human method development but also be available for the study of animal models. The research strategy proposed in this project starts with the optimal combination of the most recent approaches to quantitative perfusion MRI including recent CRMBM developments. The specific needs of each organ will stimulate further original sequence developments. Developments and comparisons on animals -Starting point: existing original arterial spin labeling methods on the animal heart -Optimization of spin labeling methods for brain applications -Optimization, simplification and automation of post processing software developed at CRMBM -Inclusion of quantitative perfusion MRI in animal model studies on heart and brain Developments and comparisons on humans, volunteer studies -Starting point: experience from animal studies and existing post-processing software -Implementation and development of spin labeling methods for brain -Implementation and development of cardiac arterial spin labeling at 3T(Siemens Verio, march 2008) -Comparison and validation of ASL techniques -Establishment of optimized protocols in collaboration with clinical teams Expected results This project aims at strengthening MRI as a medical imaging technique by extending its modal versatility as well as its capacity to be applied to other organs than the brain. Therefore, the major result expected from this work will be the feasibility of new clinical applications regardless of the organ concerned. Taking into account the framework of this project, we expect as endpoint new diagnostic protocols to emerge along with pathophysiological research applications on animals and humans (translational research).

In France and more recently in Spain, recent policies have sought to encourage men's involvement in fatherhood, promoting the norm of coparenthood after divorce or separation. One of the most problematic aspects of family transitions (increasing rates of divorce and separation, growing numbers of single-parent families and stepfamilies) is the decline of father-child relations. This fragility reveals the difficulty of conceiving the father's parenting role after divorce or separation, linked to a complex and incomplete reorganization of gendered statuses. Nevertheless, the current transformations of fatherhood cannot be completely understood if we do not consider the singularity of a new paternal character: the 'lone' father, whose links to his children are deprived of daily maternal mediation, and are embedded in a new structure of family space and time. Very few recent studies, in France or in Southern Europe, have tried to assess how the exercise of the paternal role after divorce or separation has changed in the wake of the new laws, in the context of increasing divorce and separation. Scientific collaboration between France and Spain will allow us to analyse the various cultural, political and legislative contexts of changing fatherhood in France and Spain. A new forum of exchange and research on family transitions could be developed in Europe. The research will be based on quantitative and qualitative studies (statistical studies of various national and international data bases, ethnographical and sociological approaches with regard to separated and divorced fathers), which will be interlinked, and enriched by the study of fatherhood in legal and political terms, in both France and Spain. To study the transformations of fatherhood through the situations of 'lone fathers', it is important to describe their private and daily routines, roles and representations, to analyse the connections between paternal and professional statuses, and to consider the influence of family policies, paying special attention to the social plurality of gender statuses in the family. An examination of the new forms of fatherhood in gay families, where men build parental projects without maternal mediation, will establish a rich comparative context to study the phenomenon of 'lone fatherhood'. In order to evaluate the changes introduced by the recent laws on divorce, a quantitative and qualitative analysis of legal practices will be conducted within the French and Spanish courts. Last, the rhetoric of coparenthood and equality in family statuses will be analysed from the angle of paternity as a stake in political debates, via the study of the father's rights movements. By combining these various approaches, this project will further the interdisciplinary study of gendered relations in new forms of family life. It will update the scientific knowledge on fatherhood after divorce or separation, and provide a better understanding of single mothers' family situations. From a theoretical point of view and thanks to empirical research, this project will improve the analysis of gender transitions and of the contemporary construction of masculinity and male identities.