A lesão medular traumática é uma patologia incapacitante, ainda sem tratamento eficaz. As terapias celulares representam uma nova estratégia para o tratamento destas lesões. As células-tronco adultas são fontes potenciais para o transplante celular com o objetivo de minimizar a lesão e promover a recuperação de tecidos lesados, como a medula espinhal. O objetivo desta tese foi avaliar a eficácia do transplante de células-tronco adultas na recuperação funcional e regeneração da lesão medular traumática em modelo experimental de lesão medular contusa em ratas fêmeas Wistar. Os principais objetivos foram: a) comparar os efeitos do transplante da fração mononuclear de sangue de cordão umbilical humano e de células-tronco mesenquimais dos vasos da parede do cordão umbilical humano; b) determinar a janela terapêutica deste tipo de intervenção, comparando os implantes de células- tronco realizados 1 hora, 24 horas e 9 dias após a lesão; c) demonstrar a possível diferenciação das células-tronco implantadas, bem como sua integração no tecido lesado. Os resultados obtidos demonstraram que o transplante de células foi mais eficaz para a recuperação funcional da lesão medular em ratas Wistar quando realizado pela via de administração local 1h após a lesão, quando comparado com a administração na cisterna magna e a aplicação 9 dias a lesão. O tratamento com a fração de células mononucleares ou com as células-tronco mesenquimais do sangue do cordão umbilical 24h após a lesão, não apresentou resultado funcional significativo.Observou-se a neuroproteção do tecido medular quando foi realizado o transplante de células-tronco mesenquimais 1h após a lesão medular. As células humanas transplantadas migraram e sobreviveram no local da lesão quando administradas na cisterna magna ou quando administradas diretamente no local da lesão, porém não se diferenciaram em células gliais ou neurônios. Concluímos que o transplante de células-tronco adultas promoveu a recuperação funcional após a lesão medular contusa, principalmente quando realizado 1h após a lesão diretamente no local da lesão. Apesar das células transplantadas sobreviverem na área da lesão, não foi evidenciada diferenciação celular. / Spinal cord injury is a debilitating disease and yet no effective treatment is available. In this framework cell therapy represents a new strategy to treat this condition. Adult stem cells are potential sources for cell transplantation in order to minimize injury and promote the recovery of damaged tissues, such as the spinal cord. The purpose of this Thesis was to evaluate the action of adult stem cells in the regeneration and functional recovery of spinal cord injury in experimental contusion spinal cord injury in female Wistar rats. Main goals were: a) to compare the effects of transplantation of the mononuclear cells of human umbilical cord blood and mesenchymal stem cells of the vessel wall of human umbilical cord; b) to determine the therapeutic window of this type of intervention, comparing the stem cell implants performed 1 hour, 24 hours and 9 days after injury; c) to demonstrate the possible differentiation of cells implanted, as well as their integration into the damaged tissue. Results reported demonstrate that the transplantation of stem cells was more effective for functional recovery of spinal cord injury when performed into the site of the lesion 1 h after injury, as compared with administration in the cisterna magna 9 days after injury. Treatment with mononuclear cells and mesenchymal cells from umbilical cord blood 24 hours after injury, not showed functional outcome. Neuroprotection was observed when mesenchymal stem cells were transplanted 1 hour after spinal cord injury. The transplanted human cells survived and migrated to the site of injury either when administered in the cisterna magna or directly onto the injury site, but did not differentiated into glial cells or neurons. It is suggested that the transplantation of adult stem cells promotes functional recovery after spinal cord injury when performed 1 hour after injury directly at the injury site, however differentiation of transplanted cells was not detected.

A doação de plaquetas por aférese é um procedimento automatizado que permite a obtenção deste hemocomponente em grande quantidade e com ato grau de pureza; deste processo obtém-se um subproduto chamado Câmara de Leucorredução (CLR) que é descartado ao final da doação. São permitidas até 24 doações/ano; porém as possíveis consequências de doações frequentes para esses doadores são pouco investigadas. Assim, foram identificados e quantificados os leucócitos de doadores de plaquetas frequentes e de 1ª vez. Também foi avaliada a viabilidade do uso das células mononucleares da CLR para pesquisas. Observou-se mais células na CLR que no sangue e que a frequência das populações é similar. O estado de ativação e a capacidade funcional (proliferação e produção de citocinas) foram similares entre CLR e sangue, assim como a taxa de apoptose espontânea. Entre doadores frequentes e de primeira vez não houve diferença no número de leucócitos, sugerindo que doações recorrentes não alteraram as populações leucocitárias. / Plateletpheresis is an automatized procedure to obtain high purity platelet for transfusions. From this procedure its possible to obtain a byproduct: The Leukoreduction system chamber (LRSC), which is discarded at the end of donation process. This type of donation allows 24 donation/year, but the consequences of frequent donations are poorly investigated. Therefore, we identified and quantified leukocytes of frequent and first time platelet donor. Also, was evaluated the viability, for research, of mononuclear cells recovery from LRSC. The total number of mononuclear cells was higher in LRSC than in peripheral blood samples, but the frequencies were similar in all the samples. Activation state and functional capacity (measured by cell proliferation and cytokine production) were similar in both, blood and LRSC mononuclear cells, as well as spontaneous apoptosis. Among frequent (6 or more donations in 1 year) and first time donor, there was no difference in the leukocyte total number, suggesting that frequent donation do not modify these cells.

This work deals with studying a molecular mechanism of anticancer effect of a new platinum - based drug. The qualities of still unknow dinuclear platinum komplex (BBR3571-DACH) were parallelly studied together with clinically time-tested and used mononuclear platinum complex DACH. Earlier essays demostrated, that DNA is the critical target for the cytostatic activity of platinum compounds. Altered properties of DNA and binding characteristics of these two platinum compounds were monitored by several different bioanalytical methods (differential pulse polarography, UV-VIS spectrophotometry, fluorescence spectrophotometry, CD spectroscopy and electrophoresis) after modification of DNA by both of platinum complexes. For the compounds BBR3571-DACH and DACH it was determined that the DNA binding is rapid and bifunctional. The stabilizing effect on DNA was significant particularly after modification of DNA by dinuclear komplex, while denaturating effect wasn´t proved at all. The results also indicate that dinuclear platinum complex BBR3571-DACH probably does not participate on formation of long-range cross-links like other early studied polynuclear platinum complexes.

Ziele der hier vorliegenden Arbeit waren eine immunhistochemische Analyse von GFAP (‚glial fibrillary acidic protein’) und GLUT-1 (Glukosetransporter-1) nach fokaler zerebraler Ischämie sowie deren mögliche Beeinflussung durch eine intravenöse Transplantation autologer mononukleärer Knochenmarkzellen (mKMZ) im Schafmodell. Eine differenzierte Analyse der Zielstrukturen in grauer und weißer Substanz (GS bzw. WS) sollte Aufschluss über eventuell unterschiedliche Reaktionsmuster liefern. Das Gehirnmaterial von zehn Tieren der bereits 2006/2007 stattgefundenen Studie, welche mit PET und MRT-Untersuchungen sowie der Durchführung von Verhaltenstests einherging, wurde retrospektiv im Rahmen der vorliegenden Arbeit untersucht. Je fünf gehörten zu einer Kontroll- bzw. Therapiegruppe (KG bzw. TG). Bei allen Versuchstieren wurde durch die permanente Okklusion der linken mittleren Zerebralarterie (pMCAO) eine fokale zerebrale Ischämie im Bereich des Neokortex hervorgerufen. Die Tiere der Therapiegruppe erhielten 24 Stunden nach dem Eingriff eine Transplantation autologer mKMZ (4x106/kg KGew). Nach sieben Wochen wurden die Versuchstiere getötet, ihre Schädel perfundiert und ihre Gehirne fixiert. Eine Lamelle der Gehirne wurde für die anschließende histologische Untersuchung in 30% Saccharose konserviert. Nach der Etablierung der Antikörper GFAP und GLUT-1 wurden vier Regionen der Gehirn-lamellen immunhistochemisch markiert und abschließend qualitativ und quantitativ analysiert. Die Regionen I (infarktnah) und III (infarktfern) lagen in der ipsilateralen Hemisphäre, die Regionen II (korrespondierend zu Region I) und IV (korrespondierend zu Region III) in der kontralateralen Hemisphäre. Durch den höheren Substanzverlust an Gehirnmasse in der ipsi-lateralen Hemisphäre der KG, wurden in dieser Tiergruppe die Regionen III und IV nicht ausgewertet. Vor der Analyse sind die physiologischen Markierungsmuster der vier Regionen in grauer und weißer Substanz an zwei gesunden Tieren (Prozesskontrolle) aufgezeigt worden. Durch die elektronenmikroskopische Untersuchung von Präparaten und anhand von GFAP/GLUT-1 doppelmarkierten Präparaten konnte festgestellt werden, dass die Astrozytenendfüßchen durch den hier verwendeten GLUT-1 Antikörper nicht markiert wur-den, sondern dass alleinig die gefäßständige, 55 kDa schwere Isoform detektiert worden ist. Die fokale zerebrale Ischämie führte in beiden Gruppen zu einer hochgradigen reaktiven Astrogliose mit Ausprägung einer Glianarbe in Region I. Protoplasmatische Astrozyten der grauen und fibrilläre Astrozyten der weißen Substanz zeigten hypertrophe Veränderungen. Die reaktive Astrogliose von Region I spiegelte sich in einer erhöhten GFAP-Dichte wider (p<0,05 in der Therapiegruppe). Region III hatte die gleiche GFAP-Dichte wie die Regionen II und IV. Der direkte Vergleich zwischen den Regionen I der beiden Gruppen zeigte Veränderungen der GFAP-Dichte durch die Zelltherapie auf: In der GS der Therapiegruppe lag eine geringere GFAP-Dichte vor, in der WS eine höhere (≠ p<0,05; GS und WS). Die Ergebnisse der GLUT-1-Analyse sind denen der GFAP-Analyse sehr ähnlich. Durch den Schlaganfall ist es zu einer erhöhten GLUT-1-Expression in GS und WS (p<0,05 WS) von Region I der Kontrollgruppe gekommen. Auch in Region I der Therapiegruppe konnten er-höhte GLUT-1-Dichten in GS und WS (p<0,05 WS) detektiert werden, zusätzlich dazu lag in der GS von Region III der Therapiegruppe eine erhöhte GLUT-1-Dichte vor (p<0,05). Der Vergleich zwischen beiden Gruppen zeigte Veränderungen durch die Therapie für die Regio-nen I und II auf. Die GLUT-1-Dichte der WS war in beiden Regionen in der TG erhöht (p<0,05), die GS von Region I zeigte in der Therapiegruppe eine geringere GLUT-1-Dichte. Ein Schlaganfall führt zu einer Erhöhung der GFAP sowie GLUT-1-Dichten in WS und GS im infarktnahen Gebiet. Durch die Transplantation von 4x106 autologen mononukleären Knochenmarkzellen pro kg KGew 24 Stunden nach dem Schlaganfall können diese Strukturen in ihren Expressionsmustern beeinflusst werden, dabei reagieren graue und weiße Substanz unterschiedlich: Die GS mit einer Verringerung, die WS mit einer Erhöhung der GFAP- bzw. GLUT-1-Dichte (p<0,05 WS, GLUT-1). Die Funktionskreisläufe in infarktfernen Regionen sind sieben Wochen nach dem Schlaganfall auf Astrozytenebene normalisiert (vgl. Region III). Die erhöhte GLUT-1-Dichte (p<0,05) in der GS der infarktfernen Region ist möglicherweise mit einem erhöhten Glukosemetabolismus in Verbindung zu setzen. Dies kann jedoch erst durch die Auswertung der FDG-PET-Daten beantwortet werden. Ob die durch Transplantation autologer mKMZ festgestellten Veränderungen der GFAP- und GLUT-1-Dichte in der Therapiegruppe zusätzlich mit einer verbesserten motorischen Leistung der Tiere einhergingen, wird erst durch die Analyse der Daten aus den Verhaltenstests festgestellt werden können.

Les rétinopathies ischémiques prolifératives telles que la rétinopathie diabétique proliférative et la rétinopathie du prématuré sont les principales causes de la perte de la vision dans la population active et pédiatrique des pays industrialisés. Malgré le fait que les événements initiateurs sont différents et propres à chacune des pathologies, les rétinopathies ischémiques prolifératives sont le résultat d’un processus biphasique. On a d’abord une phase initiale de dégénérescence microvasculaire suivie d’une néovascularisation excessive et pathologique de la rétine hypoxique qui tente de réinstaurer l’apport en nutriments et en énergie. Mais au lieu d’aller revasculariser les zones avasculaires de la rétine, ces nouveaux vaisseaux sanguins sont mal orientés et se dirigent plutôt vers le vitré normalement avasculaire. Ceci provoque des tensions physiques dans la rétine et mène à long terme à son détachement et une perte de vision conséquente. Les traitements actuels ne viennent pas sans effets secondaires majeurs. Par exemple, la formation de la cataracte et l’augmentation de la pression intraoculaire avec l’utilisation des corticostéroïdes ou la perte de la vision partielle dans le cas du traitement au laser sont fréquemment observées. De même, la thérapie anti-VEGF (Vascular endothelial growth factor) apporte aussi son lot de complications, telles que la thromboembolie veineuse et l’augmentation de la neurotoxicité après un long usage, vu les propriétés neuro- et vaso-protectrices du VEGF. Le développement d’une nouvelle approche thérapeutique pour les rétinopathies ischémiques prolifératives est donc nécessaire afin de contrer ces limitations thérapeutiques. Dans notre première étude, nous mettons en évidence un nouveau mécanisme par lequel les cellules neuronales sous stress diabétique sont à l’origine d’une forte inflammation oculaire. Nos résultats démontrent que le co-récepteur multi-ligand Neuropiline-1, le VEGF et la Sémaphorine-3A agissent de concert afin d’attirer une sous-population particulière de phagocytes mononucléaires susceptibles d’activer le processus de croissance vasculaire pathologique dans la rétine diabétique. De plus, notre étude propose une base pour de futures recherches sur l’impact des phagocytes mononucléaires exprimant Neuropiline-1 dans les pathologies du système nerveux central caractérisées par une inflammation excessive. Nos résultats permettent aussi de mettre en lumière le caractère anti-inflammatoire potentiel des thérapies actuelles anti-VEGF (à cause du rôle de VEGF dans la mobilisation des phagocytes mononucléaires via Neuropiline-1) au niveau oculaire. Dans notre deuxième étude, nous mettons en évidence l’activation du facteur HIF1α dans les phagocytes mononucléaires présents dans la rétine hypoxique. L’utilisation d’une approche protéomique non biaisée de spectrométrie de masse en tandem nous a permis d’identifier les partenaires interagissant avec HIF1α dans un milieu déficient en oxygène. Nous avons pu ainsi déterminer pour la première fois l’association entre la voie d’HIF1α et celle d’IRE1α (un des trois senseurs de la voie de l’UPR « unfolded protein response ») dans le processus d’adaptation à l’oxygène des phagocytes mononucléaires. Nos résultats révèlent ensuite l’importance d’IRE1α (plus principalement son activité kinase) dans la production d’HIF1α. Nous démontrons finalement que la synergie entre les signalisations d’IRE1α et HIF1α pourrait être responsable du comportement pathogénique des phagocytes mononucléaires via leur libération de cytokines inflammatoires; ce qui participerait ainsi à la progression des rétinopathies. Collectivement, nos travaux ont permis d’identifier d’importants régulateurs de l’activité pathogénique des phagocytes mononucléaires. Nous montrons : 1) le rôle de Neuropiline-1 dans l’infiltration des phagocytes mononucléaires au niveau des zones endommagées de la rétine et 2) l’impact du mécanisme convergent entre les voies d’IRE1α et HIF1α sur leur sécrétion de facteurs pro-inflammatoires durant les rétinopathies. Nos résultats offrent une base pour le développement de nouvelles stratégies thérapeutiques (ciblant Neuropiline-1, IRE1α et HIF1α) dans le traitement de maladies oculaires et d’autres pathologies caractérisées par une inflammation excessive. / Proliferative ischemic retinopathies such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are the principal causes of vision loss in working age and pediatric populations of industrialized countries. Although they display different initial triggers, proliferative ischemic retinopathies are biphasic ocular diseases that affect retinal vessels. There is an initial degeneration of the microvasculature, followed by a hypoxic stress on the retina. This triggers a second phase of deregulated and destructive blood vessel growth within the retina. Given this sequence of events and prominent clinical features, the currently most widely used local ocular therapeutic interventions directly target pathological blood vessel growth, yet present a number of non-desirable off-target effects such as the destruction of the retina itself. In fact, currently available treatments for proliferative ischemic retinopathies present non-negligible side effects, such as cataract formation with intravitreal use of corticosteroid or reduced visual field with laser-based photocoagulation surgery. Similarly, the anti-VEGF (Vascular endothelial growth factor) therapy may be associated with thromboembolic events, neuronal toxicity and atrophy when used as frequent long-term treatment given the fact that VEGF serves a vaso- and neuro-protective factor in the retina. Overcoming these therapeutic limitations and exploring novel pharmacological avenues are therefore required to ameliorate the safety profiles of current interventions. In our first study, we describe a novel mechanism by which severely stressed neuronal cells in the diabetic retina provoke destructive inflammation in the eye. We demonstrate that the multi-ligand co-receptor Neuropilin-1, VEGF and Semaphorin3A act as potent attractants for a specialized population of immune cells (mononuclear phagocytes) that later promote the exaggerated pathological vessel growth associated with the disease progression. Importantly, we provide evidence for a novel pharmacological intervention that reduces the inflammation associated with pathological retinal vessel growth. Our findings also suggest that current anti-VEGF therapies (a popular treatment for ocular vascular diseases) may in part be effective by reducing destructive ocular inflammation. In our second study, we provide evidence that those mononuclear phagocytes activate HIF1α in the hypoxic and inflamed retina. After using the unbiased proteomic approach of tandem mass spectrometry, we were able to identify HIF1α partners and found a novel link between HIF1α and the UPR (unfolded protein response) sensor IRE1α. Our data next established the crucial role of IRE1α (precisely via its kinase activity) in HIF1α production. We also suggested that the synergy between IRE1α and HIF1α pathways may be responsible of the pathogenic activity of the hypoxic mononuclear phagocytes via their secretion of inflammatory cytokines, thus contributing to the progression of the retinopathy. Collectively, our work identifies important regulators of the pathogenic activity of mononuclear phagocytes. We show that: 1) Neuropilin-1 promotes the infiltration of mononuclear phagocytes in the retina and 2) the convergent mechanism between IRE1α and HIF1α pathways is responsible for their release of pro-inflammatory factors during retinopathy. Our results could be used as a basis for the development of alternative therapeutic strategies (targeting Neuropilin-1, IRE1α and HIF1α) to treat ocular diseases or other pathologies characterized by an excessive inflammation.

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.

Le CD36 est un récepteur éboueur de classe B exprimé par plusieurs types cellulaires dont les macrophages et les cellules endothéliales de la microvasculature. Le CD36 présente une haute affinité de liaison pour les ligands lipidiques tels que les lipoprotéines oxydées de basse densité (LDLox). De part sa capacité à internaliser les LDLox au niveau des macrophages et de son implication dans la formation des cellules spumeuses, le CD36 joue un rôle critique dans le développement des lésions athérosclérotiques. Nous avons testé l'hypothèse selon laquelle le EP 80317, un ligand synthétique sélectif du CD36, exerce des effets anti-athérosclérotiques chez les souris déficientes en apolipoprotéine E. Un traitement prolongé (12 semaines) avec le EP 80317 réduit fortement (de 51%) la surface des lésions athérosclérotiques par comparaison aux souris témoins. L'effet anti-athérosclérotique est associé à une diminution des taux de cholestérol plasmatique, à une réduction de l’internalisation des LDLox au niveau des macrophages et à une augmentation de l’expression des protéines impliquées dans le transport inverse du cholestérol. De plus, un traitement par le EP 80317 est également associé une diminution de l’expression aortique et plasmatique de protéines pro-inflammatoires. Nos études ont aussi montré un rôle pour le CD36 dans le recrutement des phagocytes mononucléés au niveau des lésions athérosclérotiques, tel que démontré par une réduction de l’accumulation des phagocytes mononucléés radiomarqués CD36–/– par rapport aux cellules CD36+/+. À l’échelle moléculaire, nous avons montré que les phospholipides oxydés induisent la phosphorylation de la kinase Pyk2 des podosomes des monocytes/macrophages de manière dépendante de l’expression du CD36 et de Src. Cette phosphorylation est atténuée par un traitement par le EP80317. Nos résultats appuient le rôle important du CD36 dans l’athérosclérose et suggèrent que les ligands synthétiques qui modulent la fonction du CD36 représentent potentiellement une nouvelle classe d'agents anti-athérosclérotiques. Le CD36 exprimé par les cellules endothéliales de la microvasculature est un récepteur de l’hétérodimère protéique S100A8/A9. Ces protéines s’associent à l’acide arachidonique intracellulaire (AA) des neutrophiles polymorphonucléaires (PMN) et le complexe S100A8/A9/AA peut être sécrété par les PMN activés au contact de l’endothélium. Nous avons vérifié l’hypothèse selon laquelle le CD36 exprimé par la microvasculature est impliqué dans le métabolisme transcellulaire de l’AA par la liaison du complexe S100A8/A9/AA et la réponse inflammatoire. Chez deux modèles murins d'inflammation aiguë (ischémie/reperfusion des membres inférieurs et poche d’air dorsale), nous avons observé que la réponse inflammatoire, notamment l’accumulation des PMN au niveau des sites inflammatoires, est diminuée en moyenne de 63% chez les souris CD36-/-. De même, un traitement par le EP 80317 ou par les anticorps anti-S100A8/A9 diminue chacun de 60% en moyenne l’extravasation des PMN vers les tissus inflammatoires. L’administration simultanée des deux traitements n’a aucun effet supplémentaire, et ces traitements n’exercent aucun effet chez les souris CD36-/-. Nos résultats appuient le rôle du récepteur CD36 de la microvasculature dans la régulation de la réponse inflammatoire. L’utilisation des ligands synthétiques du CD36 pourrait représenter une nouvelle avenue thérapeutique dans le traitement des réponses inflammatoires aiguës. / CD36 is a class B scavenger receptor expressed by multiple cell types such as macrophages and microvascular endothelial cells. CD36 shows a high affinity binding towards lipid-based ligands such as oxidized low-density lipoproteins (oxLDL). Macrophage CD36 has been shown to play a critical role in the development of atherosclerotic lesions by its ability to internalize oxLDL and to lead to foam cell formation. We tested the hypothesis that EP 80317, a selective CD36 ligand, exerts anti-atherosclerotic effects in apolipoprotein E-deficient (apoE–/–) mice fed on atherogenic diet. Long term treatment (12 weeks) with EP 80317 results in a striking reduction (51%) of lesion areas in EP 80317-treated apoE–/– mice. This effect was associated with a decrease in plasma cholesterol, a reduced oxLDL internalization within macrophages and an up-regulation of proteins involved in cholesterol efflux. Additionally, treatment with EP 80317 was associated with a reduced expression of vascular and plasma pro-inflammatory proteins. Our studies also showed a role of CD36 in modulating the recruitment of mononuclear phagocytes to the arterial wall, as shown by a reduced migration of radiolabeled CD36-/- macrophages into atherosclerotic lesions compared to CD36+/+ cells. At the molecular level, our studies showed that oxidized phospholipids induced the phosphorylation of the adhesion kinase Pyk2 in monocytes/macrophages, in a CD36- and Src-dependent manner. The Pyk2 phosphorylation is attenuated by treatment with EP80317. Our results strongly support the role of CD36 in atherosclerosis development and suggest that synthetic ligands featuring modulatory effect on CD36 function may represent a novel class of anti-atherosclerotic agents. CD36 expressed by microvascular endothelial cells is a receptor for the heterodimer S100A8/A9. These proteins bind intracellular arachidonic acid (AA) within polymorphonuclear neutrophils (PMN) and the complex S100A8/A9-AA may be secreted at sites of inflammation where it exerts chemotactic activities. We aimed to delineate the role of microvascular CD36, as a receptor for the S100A8/A9, in the AA transcellular metabolism and the regulation of the associated PMN trafficking to inflammatory sites. In two mouse models of acute inflammation (hind limb ischemia/reperfusion and dorsal air pouch), CD36 regulated trafficking of PMN to inflammatory sites, as shown by a mean of 63% reduction of PMN accumulation in CD36-/- mice. Treatment with EP 80317 or with S100A8/A9 antibodies reduced, each by ~ 60%, the recruitment of PMN to inflammatory sites. The combined administration of anti-S100A8/A9 and EP 80317 did not exert any additional inhibitory effect and neither treatment featured a modulatory effect in CD36-/- mice. Our results strongly support a role for microvascular CD36 in regulating PMN trafficking to inflammatory sites. Targeting CD36 might represent a novel therapeutic avenue for the treatment of acute inflammatory responses.

Qualitativ unterschiedliche genomische und nichtgenomische Mechanismen vermitteln die starken anti-inflammatorischen und immunmdulatorischen Eigenschaften der Glucocorticoide (GC). Der genomisch vermittelte Mechanismus ist bereits gut untersucht und dokumentiert, während der nichtgenomisch vermittelte Mechanismen noch einen Gegenstand vielseitiger Untersuchungen darstellt. Wir haben uns daher die Frage gestellt, ob Beclometason und Clobetasol besonders geeignet für die topische Applikation sind, weil sie sich in ihrem Wirkungsspektrum von systemisch zu applizierenden GC wie Dexamethason unterscheiden. Wir verglichen dazu die Effekte auf den Sauerstoffverbrauch mittels der Clark-Elektrode (nichtspezifisch nichtgenomischer Mechanismus), auf die IL-6-Synthese mittels ELISA (genomischer Mechanismus) und auf die Apoptose mittels Durchflusszytometrie (nichtgenomischer und genomischer Mechanismus) in ruhenden und stimulierten humanen PBMC. Dabei zeigten Beclometason und Clobetasol in sehr niedrigen Konzentrationen (10-10, 10-8 M) einen stärkeren Effekt auf den Sauerstoffverbrauch, waren aber in hohen Konzentrationen (10-5, 10-4 M) weniger potent im Vergleich zu Dexamethason. Auch hinsichtlich ihrer genomischen Potenz waren die topischen GC in einer Konzentration von 10-10 M und 10-8 M effektiver als Dexamethason, in höheren Konzentrationen unterschieden sie sich aber nicht. Alle drei GC induzierten Apoptose konzentrationsabhängig und unterschieden sich nicht in Konzentrationen zwischen 10-8 M und 10-5 M. In einer Konzentration von 10-4 M war die Induktion von Apoptose durch die topischen GC in PBMC und Jurkat-T-Zellen aber signifikant stärker im Vergleich zu Dexamethason. Diese Ergebnisse zeigen, dass sich topische und systemische GC in ihrer genomischen und nichtgenomischen Potenz signifikant unterscheiden. Es ist daher davon auszugehen, dass nichtgenomische Effekte eine deutlichere klinische Relevanz besitzen als bisher angenommen. / Several different genomic and non-genomic mechanisms mediate the important anti-inflammatory and immunomodulatory effects of glucocorticoids (GCs). The genomic effects are the most important while the clinical relevance of non-genomic actions is still a matter of debate. We therefore investigated whether beclomethasone and clobetasol are particularly suitable for topical application because they differ in their spectrum of activity from systemically administered GCs such as dexamethasone. We compared effects on oxygen consumption as measured with a Clark electrode (nonspecific non-genomic glucocorticoid effects), on interleukin-6 synthesis by means of ELISA (genomic effects) and on apoptosis using flow cytometry (non-genomic and genomic effects) in quiescent and mitogen-stimulated PBMCs. Beclomethasone and clobetasol had stronger effects on the oxygen consumption of quiescent and stimulated cells at lower concentrations (10-10, 10-8 M) but were less potent at higher concentrations (10-5, 10-4 M) in comparison with dexamethasone. Also in terms of genomic potency, topical GCs were more effective than dexamethasone at 10-10 M and 10-8 M but gave similar results at higher concentrations. The ability of all three GCs to induce apoptosis was found to be concentration-dependent and similar at concentrations between 10-8 and 10-5 M but, compared with 10-4 M dexamethasone, 10-4 M beclomethasone or clobetasol was significantly more effective at inducing apoptosis in both PBMCs and Jurkat T cells. These results show that systemic and topical GCs differ significantly in their ability to induce genomic and non-genomic effects. This suggests that non-genomic effects are more therapeutically relevant in certain clinical conditions than currently assumed.

Dans la première partie de ce travail de thèse, nous nous sommes intéressé à l’étude de l’anisotropie magnétique au sein de complexes mononucléaires de Ni(II) et de Co(II) pentacoordinés de géométrie allant de la pyramide à base carrée jusqu’à la bipyramide trigonale. Pour les complexes mononucléaires, nous avons montré que pour une géométrie donnée, la nature de l’ion métallique a une influence importante sur l’anisotropie magnétique.Nous avons étudié l’effet de la géométrie pour un même ion métallique. Dans le cas d’une géométrie bipyramidale trigonale (symétrie C3v), nous avons montré que le complexe de Co(II) possède un axe facile de l’aimantation et donc un blocage de l’aimantation qui conduisent à l’ouverture d’un cycle d’hystérèse à basse température. Ce type de complexe peut donc être utilisé pour le stockage de l’information. Dans la deuxième partie du travail, nous avons étudié les propriétés magnétiques de complexes binucléaires. Un complexe binucléaire de Co(II) pontés par deux Cl- présente un faible couplage ferromagnétique et un blocage de l’aimantation.Enfin, l’autre aspect de ce travail est de réaliser une molécule binucléaire où deux ions anisotropes, chacun possédant un axe facile de l’aimantation, soient faiblement couplés de manière antiferromagnétique. Pour ce faire, nous avons étudié des composés avec des ligands de type cryptant où la géométrie autour des Co(II) est bipyramide trigonale. Nous avons trouvé qu’avec un ligand pontant de type Cl- ou Br-, l’interaction d’échange est beaucoup plus importante que l’anisotropie locale des ions Co(II) conduisant à un comportement magnétique où les ions perdent leur caractère local. Ce travail ouvre la perspective de synthétiser le même type de complexes mais avec des ponts de plus grande taille pour diminuer l’intensité du couplage antiferromagnétique. / In the first part of this thesis, we studied the magnetic anisotropy of pentacoordinated mononuclear Ni(II) and Co(II) complexes possessing geometries from square pyramid to trigonal bipyramid. We have shown that, for a given geometry, the metal ion nature has an important influence on the magnetic anisotropy.Then, we studied for a given metal ion the effect of geometry on its magnetic anisotropy. In the case of a trigonal bipyramidal geometry (C3v symmetry), we showed that Co(II) has an Ising type anisotropy (easy axis of magnetization) and thus a blocking of magnetization that leads to an opening of a hysteresis cycle at low temperature. This type of complexes can be used for storing data albeit at low temperature.In the second part of the work, we studied the magnetic properties of binuclear complexes. A binuclear Co(II) complex bridged by two Cl- has a weak ferromagnetic coupling and a blocking of its magnetization.Finally, another aspect of this work was to design binuclear complexes, where two anisotropic ions having each one an easy axis of magnetization, are weakly antiferromagnetically coupled. To do this, we have studied compounds with cryptand ligands where the geometry around the Co (II) is trigonal bipyramid. We found that with a Cl- or Br- bridging ligand, the exchange interaction is much more important than the local anisotropy of Co(II) ions leading to a magnetic behavior where the ions lose their local character. This work opens up prospects for synthesizing the same type of complex but with larger bridges to decrease the intensity of the antiferromagnetic coupling.

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population