Les maladies artérielles périphériques athéromateuses sont graves : l'atteinte des troncs supra-aortiques est à risque d'accident vasculaire cérébral et l'atteinte des artères des membres inférieurs est à risque d'amputation et de décès cardiovasculaire. Le développement de stratégies innovantes capables d'optimiser le diagnostic précoce et le traitement de ces maladies est un enjeu considérable.Nous montrons une corrélation entre deux biomarqueurs inflammatoires, les microparticules leucocytaires (MPL) et la lipoprotéine phospholipase A2, et l'instabilité de la plaque carotidienne définie histologiquement, dans une population de patients porteurs d'une sténose carotidienne serrée. Les MPL sont élevées de façon significative et indépendante y compris chez les patients asymptomatiques porteurs d'une sténose carotidienne serrée instable. Ainsi, le taux circulant de MPL aider à sélectionner les meilleurs candidats à une chirurgie carotidienne préventive parmi les patients ayant une sténose carotidienne serrée asymptomatique. Deuxièmement, nous montrons que l'administration ex vivo d'érythropoïétine (EPO) améliore les capacités proangiogéniques des progéniteurs endothéliaux circulants tardifs in vitro et in vivo sur un modèle d'ischémie de patte de souris nude. Ces effets semblent médiés par la sous-unité CD131 du récepteur à l'EPO. Si ces résultats se confirment chez l'homme, l'EPO pourrait être utilisée pour améliorer les capacités de revascularisation des progéniteurs endothéliaux circulants tardifs circulants humains avant réinjection autologue comme produit de thérapie cellulaire chez des patients atteints d'ischémie critique des membres inférieurs. / Atherosclerotic peripheral arterial diseases are frequent and severe. They undertake the functional and vital prognosis of patients: lesions of supra-aortic trunks are at risk of stroke and lesions of lower limb arteries are at risk of amputation and cardiovascular death. The development of innovative strategies that optimize early diagnosis and therapeutic management of these diseases is thus a considerable challenge.In this work, we show a correlation between inflammatory biomarkers, leukocyte microparticles and lipoprotein phospholipase A2, and carotid plaque instability defined histologically, in a population of patients with tight carotid stenosis with or without neurological symptoms. Leukocyte microparticles are elevated significantly and independently including asymptomatic patients with tight unstable carotid stenosis. Thus, the circulating levels of leukocyte microparticles could be a tool in the future to select the best candidates for carotid surgery among patients with asymptomatic carotid stenosis tight.Second, we show that ex vivo administration of erythropoietin improves the proangiogenic capacity of late circulating endothelial progenitor in vitro and in vivo in a mouse model of hindlimb ischemia. These effects appear mediated by CD131 subunit of the receptor for erythropoietin. If these results are confirmed in humans, erythropoietin could be used to improve the revascularization capacity of late circulating endothelial progenitor before reinjection as autologous cell therapy product in patients with critical ischemia of the lower limbs.

Ce manuscrit présente le rayonnement de second harmonique provenant de plusieurs milieux diélectriques allant de la réponse interfaciale à la réponse volumique. Ce travail de thèse constitue une première étape dans le but d'étudier une bulle de savon dans un piège optique par génération de second harmonique. Deux axes de recherche sont ainsi explorés : d'une part l'étude de la génération de second harmonique sur deux tensioactifs le TTAB et le SDS, constituants de base d'une bulle de savon, et de l'autre l'élaboration d'un nouveau dispositif expérimental permettant d'allier un piège optique à une excitation de second harmonique par un laser femtoseconde. Dans un premier temps, on explicite le montage optique reposant sur le principe d'un microscope confocal inversé et les mesures associées de la réponse harmonique d'interface air/verre et air/eau. Dans un second temps, on s'intéresse comme objet modèle, en lieu et place de la bulle, à une microparticule de silice. Ainsi, on commence par analyser sa réponse harmonique en microscopie non linéaire quand celle-ci se trouve déposée sur une matrice de verre. L'étude de cet objet met en avant l'interdépendance entre les aspects d'optique linéaire mesurés à la longueur d'onde fondamentale et l'intensité harmonique mesurée. Par la suite, cette microsphère est placée au sein du piège optique réalisé par un second faisceau. Malgré la faible taille et la symétrie de la particule diélectrique, on parvient à mettre en évidence la réponse harmonique d'une particule unique piégée. Enfin, dans une dernière partie, on s'intéresse à caractériser la génération de second harmonique pour des solutions savonneuses, première étape dans la réalisation d'une bulle. Les mesures de diffusion de second harmonique en volume pour des solutions de TTAB et SDS de diverses concentrations montrent un comportement similaire et une singularité lors du passage de la concentration micellaire critique. Lors de l'étude du signal de second harmonique à l'interface air/eau savonneuse en revanche, on mesure des comportements différents pour des tensioactifs anioniques et cationiques, provenant de la charge de surface et de l'orientation de l'eau sous-jacente / This thesis reports an experimental study of harmonic radiation from various dielectric media. We both study second harmonic generation on interfaces and hyper Rayleigh scattering in solutions. We present the first step for the realization of the following project : the study of the second harmonic generation on an optically trapped soapy bubble. To do so, two main axis of investigation are developped. Firstly, we study the harmonic response of two commonly used surfactants such as TTAB, a cationic one and SDS carrying a negative charge. This study consits of understanding the main component of a soapy bubble. We investigate how harmonic intensity is modified with an increase of soap concentration. These experiements are carried both in volume and at the air/water interface. The harmonic scattered intensity shows similar features for SDS and TTAB, as a sudden drop appearing at the critical micelar concentration. However, on the surface, the two oppositely charged surfactants give a radically different evolution of second harmonic signal versus their concentration. Indeed, the water molecules of the surroundings behaves in an opposite manner with negative or positive charged surfactants. The second axis of research that we work on in this thesis, is on a novel experimental development. We combine an optical tweezer to trap microsized particles with a femtosecond laser probe to generate the second harmonic. This is a first step in order to trap a bubble and study it with second harmonic generation. This non linear inverted confocal microscope is used to study harmonic profil of an silica bead stuck on a sample. This study gives a glance of the complexity of the correlation between linear optics effects on the laser probe beam and the created harmonic intensity. We also perform experiments on a trapped particle and we measure the harmonic response of an unique particle

Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

Submitted by Biblioteca do Instituto Biomédico BIB (uffbib@gmail.com) on 2017-06-05T19:47:11Z No. of bitstreams: 1 Helena Naly Miguens Rocha.pdf: 1130470 bytes, checksum: 43146823dc28af52b34aa41dd863ff95 (MD5) / Made available in DSpace on 2017-06-05T19:47:11Z (GMT). No. of bitstreams: 1 Helena Naly Miguens Rocha.pdf: 1130470 bytes, checksum: 43146823dc28af52b34aa41dd863ff95 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mecanismos de ativação e reparo endoteliais em resposta ao fluxo sanguíneo retrógrado (FSR) exacerbado ainda não foram completamente elucidados, nem em condições fisiológicas, nem na hipertensão arterial sistêmica (HAS). O objetivo deste estudo foi determinar os efeitos do FSR exacerbado sobre biomarcadores endoteliaisem indivíduos saudáveis e com HAS. Oito homens saudáveis (grupo CT; 36±3) e oito pacientes com HAS(grupo HAS;39±5) foram submetidos a manobra de indução de FSR em um dos braços, através da insuflação de dois manguitos, um no antebraço a 75mmHg e outro manguito próximo ao ombro a 40 mmHg, por 30 minutos. A avaliação do fluxo sanguíneo (ultrassom vascular) e a coleta de sangue foram realizadas no momento basal e no 30º minuto de manobra em ambos os braços (contralateral e ipsilateral). Ativação endotelial, micropartículas endoteliais (MPE) e células progenitoras endoteliais (CPE) foram mensuradas por citometria de fluxo. Nitrito foi mensurado por NOA Sievers. Em condições basais, fluxo sanguíneo médio, condutância vascular, taxa de cisalhamento média (p<0,01) e MPE (p=0,03) foram maiores no grupo HAS quando comparado ao grupo CT. Níveis basais de CPE estavam reduzidos no grupo HAS, permanecendo assim durante a manobra (p<0,01). Ambos os grupos apresentaram redução no fluxo sanguíneo médio e na condutância vascular (p≤0,01), bem como aumento na taxa de cisalhamento retrógrado (p<0,01) e no índice de cisalhamento oscilatório (p<0,01) durante a manobra. Somente o grupo HAS aumentou o número de MPE (p=0,02) e a ativação endotelial (p=0,04) durante a manobra. A razão MPE/CPE foi maior em ambos os momentos no grupo HAS (p<0,02). A resposta dos níveis séricos de nitrito a manobra foi menor no grupo HAS (p=0,03). Conclui-se que pacientes com HAS apresentam um quadro subclínico de disfunção endotelial com comprometimento no reparo vascular, o que foi agravado pela indução de FSR. / Endothelial activation and repair mechanism in response to increased retrograde blood flow (RBF) have not beenfully elucidated, neither in physiological conditions nor in hypertension. We aimed to determinethe effects of increased RBF on endothelial biomarkers in healthy individuals and hypertensive patients. Eight healthy subjects (CT group; 36±3) and eight hypertensive men (HT group; 39±5) underwent a maneuver to increase RBF, using two pneumatic cuffs: one in the forearm, inflated to 75 mmHg and one near the shoulder, inflated to 40 mmHg, for 30 minutes.Blood flow measures (ultrasound doppler) and blood samples were obtained at baseline and during the last minute of the maneuver from both arms (ipsilateral and contralateral). Endothelial activation, endothelial microparticle (EMP) and endothelial progenitor cell (EPC) were measured by flow cytometry. Nitrite was measured through NOA Sievers.At baseline, mean blood flow, vascular conductance, mean shear rate (p<0.01) and EMP (p=0.03) were higher in HT group than CT group. Baseline EPCs levels were reduced in HT group, which was sustained during the maneuver (p<0.01). Both groups presented decreased mean blood flow and vascular conductance (p<0.01), along with increased retrograde shear rateand oscillatory shear index (p<0.01), during the maneuver. Only the HT group showed increased EMP (p=0.02) and endothelial activation levels (p=0.04). EMP/EPC was higher in HT group in both moments (p<0.02).Nitrite levels in response to the maneuver were lower in HT group (p=0.03).Hypertensive patients present a subclinical endothelial dysfunction along with impaired endothelial repair, which was worsened by the RBF induction.

L’objectif de ce travail fut de développer et d’étudier les performances d’un microprocédé basse pression à écoulement élongationnel pour la production de nanoémulsions polymérisables de tailles contrôlées et de distributions de taille étroites. Le diamètre des nanogouttelettes a pu être précisément ajusté dans la gamme 50-300 nm en modifiant simplement les paramètres de procédé : le débit réciproque au travers du micromélangeur, le nombre de cycles et la dimension caractéristique du microcanal. Les nanoémulsions produites furent, dans une seconde étape, polymérisées par voie thermique ou par irradiation UV afin de générer des suspensions colloïdales de nanoparticules de polymère de tailles également contrôlées (87-360 nm). Un monomère, un agent de réticulation ainsi qu’un amorceur thermique ou photochimique appropriés furent par la suite ajoutés au milieu continu de ces nanosuspensions. Les solutions résultantes servirent comme phases dispersées dans des générateurs microfluidiques de gouttelettes à capillaires. Les microgouttelettes de taille contrôlée ainsi produites furent polymérisées en ligne par irradiation UV pour donner lieu à des microsphères ou à des microparticules coeur-écorce composites de polymère toutes deux dopées avec des nanoparticules de polymère. Des microparticles composites et hydrides comportant des nanoparticules d’or dans le coeur et d’argent dans l’écorce furent également obtenues grâce à la réduction photochimique in situ des sels précurseurs lors de la photopolymérisation des microgouttelettes. Ce travail a démontré l’efficacité d’un nouveau dispositif microfluidique basse énergie pour la production de nanoémulsions et leur emploi pour la synthèse de matériaux polymères morphologiquement complexes. / The aim of this work was to develop and to study the performances of a low pressure elongational-flow microprocess for the production of size-controlled polymerizable nanoemulsions with narrow size distributions. Nanodroplets diameter was easily tuned in the size range 50-300 nm by varying the process parameters, namely the reciprocating flow rate through the micromixer, the number of cycles and the characteristic dimension of the microchannel. Obtained nanoemulsions were in a second step thermally or UV-assisted polymerized to give colloidal suspensions of size-tunable polymer nanoparticles (87-360 nm). Then, a proper monomer, crosslinker and thermal- or photo-initiator were added to the continuous phase of these nanosupensions. The resulting mixtures were used as the dispersed phases of two different capillaries-based microfluidic droplet generators. The produced sizecontrolled microdroplets were finally UV polymerized online and plain as well as core-shell composite polymeric microparticles doped with lower scale polymer nanoparticles were obtained. Composite/hybrid polymeric core-shell microparticles were also synthesized for which gold nanoparticles in the core and silver nanoparticles in the shell were synthesized in situ from their salt precursors during microdroplets polymerization. This work has demonstrated the high efficiency of a novel low energy microfluidic emulsification device for the production of nanoemulsions which were used for the synthesis of morphologically complex polymeric materials.

Systemisch applizierte Therapeutika können erhebliche Nebenwirkungen auslösen, welche auf Grund eines unspezifischen Transports oder einer hohen Dosis von appliziertem Wirkstoff auftreten. Daher bedarf es der Entwicklung neuartiger Wirkstoff-Transportsysteme (Drug Delivery Systems) welche in der Lage sind, Wirkstoffe in genau definierbaren Dosen gezielt in die adressierte Zelle zu transportieren. Ein vielversprechender Ansatz, welcher diesen Anforderungen nachkommt, findet sich in der Layer-by-Layer-Technik (LbL), d.h. der wechselseitigen Assemblierung von Polymeren/Wirkstoffen auf soliden sphärischen Templaten, eines funktionellen Supported Lipid Bilayers sowie der Oberflächenfunktionalisierung mit spezifischen Antikörpern. Dabei ist die Homogenität und Regularität des Supported Lipid Bilayers von großer Bedeutung, um in biomedizinischen Anwendungen eine ungewollte Interaktion mit Serumkomponenten sowie eine Opsonierung zu verhindern. Insbesondere die funktionelle Lipidkomponente besitzt allerdings maßgebliche Auswirkungen auf diese Parameter. In dieser Arbeit wurde die Idee der Oberflächenfunktionalisierung von LbL-beschichteten Silica-Mikropartikeln (SiO2) mit einem funktionellen Supported Lipid Bilayer aufgegriffen und weiterentwickelt, wobei insbesondere die Homogenität des Supported Lipid Bilayers auch auf sehr kleinen Längenskalen (wenige Nanometer) bestimmt wurde. In einem letzten Schritt konnte anhand zweier verschiedener Zelllinien (3T3 und Vero) die Adaptivität und Effektivität des entwickelten Drug Delivery Systems nachgewiesen werden.

No description available.

<p>Miniaturization of technologies to milli-, micro- and nanoscale offers numerous advantages for diagnostic and therapeutic biomedical applications. In comparison to their macro-scale counterparts, these small-scale systems are more portable, less invasive and less costly. They can facilitate rapid, sensitive and high throughput detection of abnormalities, help track disease progression, reduce sample consumption and improve therapeutic efficacy of drug delivery while decreasing systemic toxicity. Thus, there is clearly a need for creating innovative milli-, micro- and nanoscale tools that can uncover new possibilities in detection and treatment of various types of diseases. The overall objective of this dissertation was to develop novel small-scale technologies that could help enhance diagnostic and/or therapeutic outcomes in patients with cancer, opioid addiction and inflammatory bowel disease. First, we developed an echogenically stable nanodroplet ultrasound contrast agent with potential applications in extravascular molecular imaging of tumors and targeted cancer therapies. Then, we created a polymer blend microsphere system that could be integrated in prescription opioid tablets to develop an abuse-deterrent formulation against smoking. Finally, we designed a release system for localized delivery of aminosalicylates from magnetically actuated millirobots in the colon to improve therapeutic outcomes in patients suffering from inflammatory bowel disease. Overall, the technologies we developed could serve as a basis for designing diagnostic and therapeutic tools that are superior to currently existing platforms.</p>

Venous thromboembolism (VTE), defined as deep vein thrombosis and/or pulmonary embolism is the second leading cause of mortality in cancer patients, second only to cancer itself. A number of reports suggest that circulating extracellular vesicles (EVs) may be increased in cancer patients with VTE. The aim of this study was to examine circulating EVs in high-risk ambulatory cancer patients, determine if levels are associated with hematological outcomes (VTE, major bleeding event), and to assess the impact of prophylactic antithrombotic therapy (Apixaban). We hypothesized that elevated levels of circulating large EVs will be predictive of cancer associated VTE and/or bleeding events and that treatment with Apixaban will reduce EV levels and incidence of cancer VTE. Plasma samples from patients at baseline, and 90-days follow-up from the Apixaban for the Prevention of Venous Thromboembolism in High-Risk Ambulatory Cancer patients (AVERT) trial were investigated. Total EVs were quantified by their pro-coagulant activity using the Zymuphen MP-Activity kit. Platelet, endothelial and tissue-factor EV levels were quantified by flow cytometry. We observed that circulating EVs exhibited significant associations with sex, age, and cancer type, however we did not observe any relationships with clinical outcomes. Thus, it appears that circulating EVs may not have a role in risk stratification for VTE in in high-risk ambulatory cancer patients.

L'auteur n'a pas fourni de résumé en français / Nowadays in medicine and biotechnology a wide range of applications involves magnetic micro/nano-object manipulation including remote control of magnetic beads, trapping of drug vectors, magnetic separation of labelled cells and so on. Handling and positioning magnetic particles and elements functionalized with these particles has greatly benefited from advances in microfabrication. Indeed reduction in size of the magnet while maintaining its field strength increases the field gradient. In this context, arrays made of permanent micromagnets are good candidates for magnetic handling devices. They are autonomous, suitable for integration into complex systems and their magnetic action is restricted to the region of interest.In this thesis we have elaborated an original approach based on AFM and MFM for quantitative study of the magnetic force and associated force gradients induced by TMP micromagnet array on an individual magnetic micro/nano-object. For this purpose, we have fabricated smart MFM probes where a single magnetic (sub)micronic sphere was fixed at the tip apex of a non-magnetic probe thanks to a dual beam FIB/SEM machine equipped with a micromanipulator.Scanning Force Microscopy conducted with such probes, the so-called Magnetic Particle Scanning Force Microscopy (MPSFM) was employed for 3D mapping of TMP micromagnets. This procedure involves two main aspects: (i) the quantification of magnetic interaction between micromagnet array and attached microsphere according to the distance between them and (ii) the complementary information about micromagnet array structure. The main advantage of MPSFM is the use of a probe with known magnetization and magnetic volume that in combination with modelling allows interpreting the results ably.We conducted MPSFM on TMP sample with two types of microparticle probes: with superparamagnetic and NdFeB microspheres. The measurements carried out with superparamagnetic microsphere probes reveal attractive forces (up to few tens of nN) while MFM maps obtained with NdFeB microsphere probes reveal attractive and repulsive forces (up to one hundred of nN) for which the nature of interaction is defined by superposition of microsphere and micromagnet array magnetizations. The derived force and its gradient from MFM measurements are in agreement with experiments on microparticle trapping confirming that the strongest magnetic interaction is observed above the TMP sample interfaces, between the areas with opposite magnetization. Thanks to 3D MFM maps, we demonstrated that intensity of magnetic signal decays fast with the distance and depends on micromagnet array and microsphere properties.Besides the magnetic interaction quantification, we obtained new information relevant to TMP sample structure: we observed and quantified the local magnetic roughness and associated fluctuations, in particular in zones of reversed magnetization. The variation of detected signal can reach the same order of magnitude as the signal above the micromagnet interfaces. These results complete the experiments on particle trapping explaining why magnetic microparticles are captured not only above the interfaces, but also inside the zones of reversed magnetization.Quantitative measurements of the force acting on a single (sub)microsphere associated to the modelling approach improve the understanding of processes involved in handling of magnetic objects in microfluidic devices. This could be employed to optimize the parameters of sorting devices and to define the quantity of magnetic nanoparticles required for labelling of biological cells according to their size. More generally these experimental and modelling approaches of magnetic interaction can meet a high interest in all sorts of applications where a well-known and controlled non-contact interaction is required at micro and nano-scale.