Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T19:11:15Z (GMT). No. of bitstreams: 1 Morari_Joseane_D.pdf: 2485057 bytes, checksum: 88660e44d267d2bb162402326080d56d (MD5) Previous issue date: 2013 / Resumo: Nas últimas décadas tornou-se claro que indivíduos e animais obesos apresentam um quadro subclínico de inflamação sistêmica. Estudos desenvolvidos em nosso laboratório ao longo dos últimos 10 anos revelaram que a inflamação do hipotálamo está presente em roedores obesos e sua inibição por métodos genéticos e farmacológicos resulta na correção do fenótipo obeso e dos distúrbios metabólicos comumente associados à obesidade. Ácidos graxos saturados de cadeia longa presentes na dieta parecem ser os principais responsáveis pela ativação da resposta inflamatória no hipotálamo. Os primeiros sinais de inflamação podem ser detectados 24 h após a introdução de uma dieta rica neste tipo de gordura. Pelo menos dois mecanismos moleculares foram identificados como potenciais desencadeadores desta resposta inflamatória, sendo eles; a ativação de receptores TLR4, e a indução do estresse de retículo endoplasmático. Durante a exposição precoce a uma dieta rica em gordura saturada, células da micróglia localizadas no hipotálamo, tornam-se ativas e passam a expressar citocinas que, eventualmente, levam a ativação de vias inflamatórias em neurônios da região. A ativação de JNK e IKK em neurônios resulta na indução de resistência hipotalâmica à leptina e insulina, e, subsequentemente na perda do controle coordenado da ingestão alimentar e do gasto calórico. O fato de se observar ativação inflamatória no hipotálamo poucas horas após a exposição aos nutrientes desencadeadores sugere que células da micróglia residentes participem deste processo. Entretanto, com a manutenção da exposição ocorre uma modificação do padrão inflamatório ao longo do tempo, o que sugere que novas células estejam sendo recrutadas. Na primeira etapa deste estudo avaliamos a migração de células monocíticas da medula óssea para o hipotálamo em animais alimentados com dieta rica em gordura saturada. Para tal, transplantes de medula óssea foram realizados para gerar quimeras expressando TLR4 funcional somente em células da medula óssea ou somente em tecidos não originários da medula óssea. A parecença de TLR4 funcional em células de medula óssea foi necessária para expressão completa do fenótipo obeso. Além disso, somente quando TLR4 estava expresso em células da medula óssea, ocorreu infiltração hipotalâmica com células de micróglia oriundas da periferia. Como o recrutamento de células monocíticas da medula óssea para um determinado sítio anatômico depende invariavelmente da expressão de quimiocinas, nós aventamos a hipótese de que nas etapas iniciais da ativação da inflamação hipotalâmica na obesidade alguma(s) quimiocina(s) poderia(m) desempenhar um papel determinante na progressão do processo inflamatório e, consequentemente do fenótipo obeso. Tanto humanos como animais experimentais não consanguíneos apresentam grande variabilidade na expressão fenotípica da obesidade quando expostos a condições ambientais que favoreçam a mesma. Desta forma, buscamos por diferenças no padrão de inflamação do hipotálamo e na expressão de quimiocinas em duas cepas de camundongos com perfis metabólicos e predisposição a obesidade diametralmente opostos, sendo eles: o camundongo Swiss, com grande predisposição a obesidade e diabetes; e, o camundongo Balb-c, virtualmente protegido da obesidade e dos distúrbios metabólicos afins. Identificamos a quimiocina CX3CL1 (fractalkina) como aquela, dentre as avaliadas, com maior distinção de sua expressão no hipotálamo entre as duas cepas de camundongos, encontrando-se mais expressa em Swiss que em Balb-c. Na segunda parte do estudo mostramos que a expressão da fractalkina é induzida em neurônios do hipotálamo frente a um estímulo com nutrientes ricos em ácidos graxos saturados. Em cultura de neurônios, a expressão de fractalkina é induzida preferencialmente por TNF, mas também por palmitato. A inibição da fractalkina no hipotálamo com siRNA protege camundongos Swiss da intolerância à glicose e da obesidade induzidas por dieta rica em gordura saturada, além de reduzir o recrutamento de células monocíticas originárias da medula óssea para o hipotálamo. Assim, concluímos que durante as etapas iniciais da indução da obesidade, células da micróglia residentes ativam a produção de fractalkina por neurônios do hipotálamo a qual desempenha papel importante no recrutamento de novas células monocíticas da medula óssea, que perpetuarão o processo inflamatório do hipotálamo caso a exposição à dieta rica em gordura saturada seja mantida. A fractakina constitui-se, portanto, num dos mais precoces sinalizadores durante a instalação da inflamação hipotalâmica associada à obesidade / Abstract: Studies performed during the last 20 years have shown that obese subjects and experimental models of obesity present a systemic subclinical inflammation. Our group has shown that in experimental obesity the hypothalamus is affected by an inflammatory response and the employment of genetic and pharmacological means to inhibit diet-induced hypothalamic inflammation rescues the obese phenotype and the metabolic disarrangements commonly associated with obesity. Long-chain saturated fatty acids present in the diet are amongst the most important inducers of the hypothalamic inflammation in obesity. The first signs of hypothalamic inflammation can be detected as early as 24 h after the initial exposure to dietary fats. At least two molecular mechanisms have been shown to play a role in the induction of the hypothalamic inflammation in obesity; i.e., activation of TLR4 and induction of endoplasmic reticulum stress. During the early exposition to dietary fats, resident microglia cells are activated and express cytokines that eventually can lead to the induction of inflammatory signaling in the neighboring neurons. The activation of JNK and IKK in neurons results in the induction of hypothalamic resistance to leptin and insulin and, thus, to the loss of the coordinated control of food intake and energy expenditure. As hypothalamic inflammation can be induced a few hours after the initial exposure to dietary fats, it has been suggested that resident microglia are involved in this process. However, as the exposure to dietary fats persists, changes in the pattern of inflammation take place, which may indicate the recruitment of new cells to the inflamed site. In the first part of this study, we evaluate the migration of bone marrow-derived monocytic cells to the hypothalamus of obese animals. Chimera mice were generated by the transplantation of TLR4-expressing bone marrow into TLR4 defective mice and vice-versa. The presence of functional TLR4 in bone marrow-derived cells was required for the complete expression of the obese phenotype. In addition, diet-induced hypothalamic infiltration with bone marrow-derived microglia was obtained only in the presence of functional TLR4 in bone marrow-derived cells. Because the recruitment of bone marrow-derived monocytic cells to a given anatomical site depends invariably on the expression of chemokines, we have raised the hypothesis that, during the initial steps of diet-induced hypothalamic inflammation a (some) chemokine (s) could play an important role in the progression of the inflammatory activity and, consequently, of the obese phenotype. Both humans and outbreed experimental animals display a huge phenotypic variability whenever exposed to environmental conditions that favor obesity. With this concept in mind, we searched for differences in the patterns of hypothalamic inflammation in two strains of mice with different predispositions to obesity and metabolic diseases. The Swiss mice which are prone to obesity, and the Balb-c mice, which are protected from obesity and related metabolic conditions. We identified the chemokine CX3CL1 (fractalkine) as the one, among those that we evaluated, with highest distinction on its hypothalamic expression levels between the two mouse strains; being increased in the hypothalamus of Swiss mice. In the second part of the study we demonstrate that fractakine is induced in hypothalamic neurons by fat-rich diet. In culture, neurons express fractalkine in response to a TNF stimulus preferentially, but also in response to palmitate. The inhibition of hypothalamic expression of fractalkine by siRNA protects mice from glucose intolerance and diet-induced obesity. Moreover, under hypothalamic fractalkine inhibition, diet-induced recruitment of monocytic cells from the bone marrow is severely impaired. We conclude that during the initial steps of diet-induced hypothalamic inflammation, resident microglia cells induce the expression of fractalkine by neighboring neurons, which in turn recruit additional monocytic cells from the bone marrow to maintain the inflammatory process under continuous dietary fatty acid exposure. Thus, fractalkine is one of the earliest signals generated in the hypothalamus during the installation of diet-induced obesity / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Fisiopatologia Médica

Tumour-associated macrophages (TAMs) have been shown to play an important role in tumour survival and progression. Thus, high numbers of macrophages in the tumour tissue are often associated with a poor prognosis. Identification of factors responsible for recruiting macrophages to the sites of different types of tumours might help to develop more effective cancer treatment. Burkitt's lymphoma (BL) is characterised by uncontrolled cell proliferation, high rate of spontaneous apoptosis and significant macrophage infiltration. Although BL cells undergo extensive apoptosis, in situ their corpses are cleared very effectively by macrophages infiltrating the tumour. It is now widely believed that dying cells are themselves able to release chemotactic molecules to ensure macrophage chemotaxis and subsequent clearance of their site of death. Previous work carried out in this laboratory identified fractalkine/CX3CL1 (FKN) released from dying BL cells to be an important player in macrophage chemotaxis to BL. Yet, these results have also indicated that FKN may not be the only chemokine involved in this process. Following from those observations, the first part of this work focused on examination of the potential role of monocyte chemoattractant protein-1 (MCP-1) in macrophage recruitment to BL. Despite the initial promising results, careful analysis of the data obtained by various techniques led to the conclusion that MCP-1 is, probably, not expressed by BL cells. Subsequently, effort was concentrated on understanding mechanisms regulating FKN processing during cell death. The studies performed before in this laboratory identified a new form of FKN to be present in apoptotic BL cells and showed that this is the form that is, most likely, responsible for mediating macrophage migration. Here, this apoptosis-related 60 kDa FKN was found to be a likely caspase-3 cleavage product. Moreover, it was demonstrated that FKN and active caspase-3 are released together in apoptotic BL cell-derived microparticles, suggesting that the proteolytic events could take place also extracellularly. In the final results chapter the differences between BL cell lines in the way they process FKN during cell death were revealed and a new cell death-associated 55 kDa FKN was observed. Through several lines of evidence, this new form was identified to be a possible product of calpain-mediated proteolysis. To conclude, this work provides the first evidence for a possible direct participation of the two major cell death executioner proteases – caspases and calpains, in production of ‘find me’ signals for macrophages and thus, ensuring effective clearance of dying cells. These results indicate that FKN cleavage and release might be of key importance during cell death. Moreover, the studies presented here contribute to better understanding of the process of FKN secretion.

In the central nervous system, the axons of neurons are protected from damage and aided in electrical conductivity by the myelin sheath, a complex of proteins and lipids formed by oligodendrocytes. Loss or damage to the myelin sheath may result in impairment of electrical axonal conduction and eventually to neuronal death. Such demyelination is responsible, at least in part, for the disabling neurodegeneration observed in pathologies such as Multiple Sclerosis (MS) and Spinal Cord Injury. In the regenerative process of remyelination, oligodendrocyte precursor cells (OPCs), the resident glial stem cell population of the adult CNS, migrate toward the injury site, proliferate and differentiate into adult oligodendrocytes which subsequently reform the myelin sheath. Existing research indicates that OPC migration is directed by chemomigratory signals released from the site of injury and that the absence of OPCs is a feature of some MS lesions, suggesting that increased recruitment of OPCs to injury sites might improve remyelination, eventually leading to treatments of patient pathologies. I hypothesized that as yet undiscovered migration cues for OPCs might be released at sites of demyelination, diffuse through the CNS tissue, activate distal OPCs and guide them back to sites of demyelination. In this thesis, I performed bioinformatics analysis of gene expression arrays and identified upregulated cell surface receptors on OPCs activated in a cuprizone model, and upregulated secreted factors in whole lesion sites from an LPC induced MS type injury model and a Spinal Cord Injury model. I then optimised the X-celligence system for the quantification of OPC migration in response to secreted factors identified in my bioinformatics screen. By combination of these techniques with immunofluorescent staining I discovered novel expression of the cell surface receptor CX3CR1 on OPCs, increased expression of the corresponding ligand CX3CL1 in both MS type injury and Spinal Cord Injury, increased directional migration of OPCs in response to low concentrations of CX3CL1, and increased maturation of OPCs into adult oligodendrocytes at high concentrations of CX3CL1. Taken together these results propose a system in which an increasing gradient of CX3CL1 released from the site of injury directs the recruitment, then maturation of OPCs, making CX3CL1 a master regulator of OPC led CNS regeneration.

Spinal cord injury (SCI) often leads to irreversible damage, and permanent paralysis inferior to the injury is common (Leibinger et al., 2013). Injury to the spinal cord occurs in two phases. In the first phase, components of the spinal cord are subject to mechanical trauma causing direct damage. In the second phase, damage spreads from the area of injury through molecular processes. Several studies have linked M1 "pro-inflammatory" macrophages to exacerbation of damage by inducing dieback of dystrophic axons, but not healthy axons, through direct cellular contact. Several studies have identified the presence of macrophage subtypes at specific time. A literature review was conducted in order to summarize these findings (Busch, Horn, Silver, & Silver, 2009; Evans et al., 2014; Horn, Busch, Hawthorne, van Rooijen, & Silver, 2008; Kigerl et al., 2009; Shechter et al., 2013). Although the full mechanism behind the process of M1 macrophage-mediated dieback of dystrophic axons is unclear, matrix metalloproteinase-9 (MMP-9) produced by these macrophages has been shown to play a role. However, the specific interaction between MMP-9 and neurons is under investigation. The research described explores the relationship between MMP-9 and fractalkine (CX3CL1), a surface protein expressed by CNS neurons. SDS-PAGE and western blot were used to determine whether the presence of MMP-9 increases the cleavage of fractalkine at several time intervals. At a concentration of 300ng/ml, MMP-9 was not found to demonstrate cleavage of fractalkine.

Le cancer épithélial de l’ovaire (CEO) est une cause majeure de mortalité parcancer gynécologique. Il est associé à un mauvais pronostic car il est souventdécouvert à un stade tardif. Mieux comprendre les causes et les mécanismesmoléculaires et cellulaires associés à la progression de ce cancer représente unenjeu majeur.Les deux chimiokines CXCL12/SDF-1 et fractalkine (FKN)/CX3CL1 ont étéimpliquées dans diverses tumeurs. La chimiokine SDF-1, a un effetimmunosuppresseur dans le CEO. Elle est aussi impliquée dans l’angiogenèsetumorale. L’effet de SDF-1 médié par CXCR4 est également impliqué dans larégulation de la prolifération, la survie, la migration et l'invasion des cellulescancéreuses. La FKN, a largement été mise en évidence dans les tissusépithéliaux et dans divers cancers où elle peut avoir soit un rôle anti-tumoral soitun rôle pro-tumoral. Jusqu’à présent la FKN n’a pas été étudié dans le CEO.Dans notre étude, nous avons démontré l’expression de SDF-1 et de la FKNdans l’épithélium de surface de l’ovaire sain et dans les tumeurs bénignes etmalignes. Ces résultats montrent que l’expression de SDF-1 et de la FKNpréexiste à la tumorigenèse. Nous avons démontré une expression hétérogènedes deux chimiokines dans les cellules du CEO. Les niveaux d’expression deSDF-1 dans les cellules tumorales sur une cohorte de 183 patientes n’ont aucunevaleur pronostique sur la survie globale et sur la survie sans progressiontumorale des patientes atteintes par le CEO. L’étude de la corrélation del’expression de la FKN avec les deux marqueurs de prolifération, Ki-67 etGILZ, sur une autre cohorte de 54 patientes, complétée par des expériences invitro, a montré que GILZ augmente l’expression de la FKN et d’autre part que laFKN elle-même augmente la prolifération. Cette étude contribue à élucider lerôle de SDF-1 et de la FKN dans le CEO. / Little is known about the molecules that contribute to tumor growth ofepithelial ovarian carcinomas (EOC) that remains the most lethal gynecologicalneoplasm in women.The two chemokines CXCL12/SDF-1 and fractalkine (FKN)/CX3CL1 havebeen widely studied in tumorigenesis. In epithelial ovarian cancer (EOC), SDF-1enhances tumor angiogenesis and contributes to the immunosuppressivenetwork. SDF-1 also acts on tumor cell proliferation and survival and, throughits main receptor CXCR4, governs the migration of malignant cells and theirinvasion of the peritoneum. The chemokine FKN has been documented inepithelial tissues and in various cancers. FKN have paradoxical effects intumors: anti-tumoral effect in some tumor entities and pro-tumoral effect inother tumor entities.In our study, we demonstrated the expression of SDF-1 and FKN on thesurface epithelium of normal ovaries and benign and malignant tumors,suggesting that the expression of these chemokines preexists to tumorigenesis.We also demonstrated an heterogeneous expression of both chemokines in EOC.In our large and homogeneous cohort (183 specimens of EOC), SDF-1expression levels had no effect on overall survival or progression-free survival.Thus, SDF-1 expression by tumor epithelial cells is not in itself a valuableprognostic factor in patients with advanced EOC. FKN immunostaining scores(in 54 specimens of EOC) correlated positively with the two proliferationmarkers : Ki-67 and GILZ. In vitro, we demonstrated that GILZ increases theexpression of FKN and that FKN itself increased proliferation. This studycontributes in elucidating the role of the two chemokines SDF-1 and FKN inEOC.

Malgré les avancées thérapeutiques récentes, le pronostic des patients porteurs de métastases osseuses (MO) reste faible, ce qui incite à chercher des nouvelles stratégies thérapeutiques. Les chimiokines sont des acteurs majeurs de la réponse immune, et apparaissent comme des cibles potentielles de l’immunothérapie anti-cancéreuse. Nous avons recherché à définir si la chimiokine CX3CL1 pouvait représenter un axe thérapeutique efficace dans le contexte des MO. Pour cela nous avons développé des modèles murins de MO de cancer du rein et du poumon. Dans le modèle de MO de cancer du poumon, notre travail a démontré que l'expression de CX3CL1 inhibe la croissance tumorale. L’analyse transcriptomique des tumeurs a montré que CX3CL1 diminue (i) l’ostéloyse via un effet sur la triade OPG/RANKL/RANK (ii) l'expression de certains checkpoints, en faveur d’une réponse immune antitumorale. En revanche, dans le modèle de MO de cancer du rein, l’expression de CX3CL1 stimule le développement tumoral et l'ostéolyse via une action sur la triade OPG/RANKL/RANK et inhibe la réponse immune antitumorale via une augmentation de l'expression de certains checkpoints immunitaires. Les bisphosphonates (BPs) sont des agents utilisés pour le traitement des MO. Afin de réduire leurs effets indésirables, nous avons utilisé des ciments de phosphate de calcium (CPC), pour délivrer localement dans l’os des BPs (alendronate, ALN). Notre travail a mis en évidence que (i) ces ciments chargés en ALN relarguent en continue les BPs, (ii) le relarguage d’ALN est efficace pour induire des effets cytotoxiques et pro-apoptotiques vis à vis des cellules de cancer du sein / Despite recent therapeutic improvments, the prognosis for a patient with bone metastases (BM) remains poor, this situation prompting the research of new therapeutic strategies. Chemokines are central players in the immune response, and appear as potential targets in anti-cancer immunotherapies. We are interested to determine whether the CX3CL1 chemokine exerted pro or anti-tumor actions within the bone metastatic context. To address this issue, we developed mouse models of lung or renal cancer BM. In lung cancer BM model, our work demonstrated that CX3CL1 expression led to tumor growth inhibition. Tumors transcriptomic analysis revealed that CX3CL1: (i) impacted bone metabolism by modulating the OPG/RANKL/RANK triad (ii) decreased the expression of certain immune checkpoints, this up-regulating the anti-tumor immune response. By contrast, in renal cancer BM model, CX3CL1 expression stimulated bone tumor development and transcriptomic analysis showed that CX3CL1 (i) promoted osteolysis through an action on the OPG/RANKL/RANK triad (ii) -induced tumor development correlated with an increased expression of certain immune checkpoints, this down-regulating the anti-tumor immune response. Bisphosphonates (BPs) are targeted agents used for BM treatment. In order to reduce their side effects, we used resorbable calcium phosphate cements (CPC), which are frequently used as bone void fillers, as platform for a local delivery of BPs (alendronate, ALN). As a whole, our in vitro data demonstrated that: (i) ALN-CPC cements continuous released ALN; (ii) this ALN release was effective in inducing cytotoxic and pro-apoptotic effects in breast cancer cells

Contrairement aux autres chimiokine, CX3CL1 a la particularité d'exister sous deux formes protéiques fonctionnelles : une forme soluble chimio-attractante impliquée dans le recrutement leucocytaire comme toutes les chimiokines et une forme membranaire qui confère au couple CX3CL1/CX3CR1, une propriété surprenante de molécule d'adhésion participant à l'arrêt et à la transmigration des leucocytes circulants. Le CX3CR1 appartient à la famille des Récepteurs Couplés aux Protéines Gi, c'est-à-dire qu'il inhibe l'enzyme de synthèse de l'AMP cyclique (AMPc), l'adénylate cyclase. L'étude de la cinétique et du rôle du signal AMPc déclenché en aval du CX3CR1, activé par la forme soluble ou membranaire du CX3CL1 font l'objet de ce travail de thèse. La réponse à la forme membranaire est de même amplitude que celle induite par la forme soluble, mais présente une cinétique considérablement ralentie. Ce ralentissement corrèle avec une moindre internalisation du CX3CR1, qui semble être retenu en surface par le ligand membranaire. Un recrutement plus lent des ? arrestines sur le CX3CR1 activé par le CX3CL1 membranaire renforce cette hypothèse d'une internalisation plus tardive du CX3CR1 comparé à une activation par la forme soluble. Le rôle physiologique de l'AMPc a été également exploré sur les deux fonctions principales du couple CX3CL1/CX3CR1 : le chimiotactisme et l'adhésion. Le rôle inhibiteur de l'AMPc sur ces deux fonctions, confirme une action immunosuppressive de ce messager secondaire. Ces résultats indiqueraient que, grâce à leurs réponses AMPc inhibitrices, les cellules activées par les chimiokines seraient sélectionnées pour une réponse efficace en milieu inflammatoire. / CX3CL1 is a particular chemokine which, in contrast to other chemokines exists in two physiological forms: a soluble form is implicated in chemotaxis and cellular migration like all chemokines; whereas a membranous form confers to the CX3CL1/CX3CR1 couple an adhesive role, contributing in particular to the arrest of circulating leukocytes and their migration to the site of inflammation. The CX3CR1 belongs to the family of Receptor Coupled with Proteins Gi (RCPG), which inhibits the enzyme that syntheses cyclic AMP (cAMP), the adenylyl cyclase. The study of the kinetics and the role of the cAMP signal triggered downstream of CX3CR1, activated by soluble or membranous form of the CX3CL1 are the aims of this work of thesis. The response induced by the membranous form has the same amplitude as the one induced by the soluble form, but has a significantly slowed kinetic. This slowdown correlates with less internalization of CX3CR1, which seems to be retained on the surface by the membranous ligand. A slower recruitment of β-arrestin on the CX3CR1-activated by membranous CX3CL1 strengthens the hypothesis of a later internalization of CX3CR1 compared to activation by the soluble form. The physiological role of cAMP was also explored in the two principal functions of the couple CX3CL1 / CX3CR1: chemotaxis and adhesion. The inhibiting effect of cAMP in these two functions confirms an immunosuppressive action of this second messenger. These results indicate that through their inhibitory cAMP responses, cells activated by chemokines are selected for an effective response in the inflammatory conditions.

Le cancer épithélial de l'ovaire (CEO) est une cause majeure de mortalité parcancer gynécologique. Il est associé à un mauvais pronostic car il est souventdécouvert à un stade tardif. Mieux comprendre les causes et les mécanismesmoléculaires et cellulaires associés à la progression de ce cancer représente unenjeu majeur.Les deux chimiokines CXCL12/SDF-1 et fractalkine (FKN)/CX3CL1 ont étéimpliquées dans diverses tumeurs. La chimiokine SDF-1, a un effetimmunosuppresseur dans le CEO. Elle est aussi impliquée dans l'angiogenèsetumorale. L'effet de SDF-1 médié par CXCR4 est également impliqué dans larégulation de la prolifération, la survie, la migration et l'invasion des cellulescancéreuses. La FKN, a largement été mise en évidence dans les tissusépithéliaux et dans divers cancers où elle peut avoir soit un rôle anti-tumoral soitun rôle pro-tumoral. Jusqu'à présent la FKN n'a pas été étudié dans le CEO.Dans notre étude, nous avons démontré l'expression de SDF-1 et de la FKNdans l'épithélium de surface de l'ovaire sain et dans les tumeurs bénignes etmalignes. Ces résultats montrent que l'expression de SDF-1 et de la FKNpréexiste à la tumorigenèse. Nous avons démontré une expression hétérogènedes deux chimiokines dans les cellules du CEO. Les niveaux d'expression deSDF-1 dans les cellules tumorales sur une cohorte de 183 patientes n'ont aucunevaleur pronostique sur la survie globale et sur la survie sans progressiontumorale des patientes atteintes par le CEO. L'étude de la corrélation del'expression de la FKN avec les deux marqueurs de prolifération, Ki-67 etGILZ, sur une autre cohorte de 54 patientes, complétée par des expériences invitro, a montré que GILZ augmente l'expression de la FKN et d'autre part que laFKN elle-même augmente la prolifération. Cette étude contribue à élucider lerôle de SDF-1 et de la FKN dans le CEO.

The pathophysiology of atherosclerotic cardiovascular disease (CVD) is highlighted by vascular dysfunction and low-grade vascular inflammation. Furthermore, the site-specific distribution of atherosclerosis throughout the arterial vasculature is primarily determined by local hemodynamic force. Therefore, this dissertation outlines three experiments designed to investigate the role of acute mental and physical (i.e., aerobic exercise), and vascular wall shear stress (SS) on the inflammatory aspects of atherosclerosis. Chapter 2 examines the effect of acute laboratory-induced mental stress on intracellular pro-inflammatory signaling pathways in peripheral blood mononuclear cells. Chapter 3 investigates the impact of acute laboratory-induced mental stress and maximal aerobic exercise on the concentration of soluble VCAM-1 (sVCAM-1) and CX3CL1/fractalkine (sCX3CL1) in human serum. Lastly, Chapter 4 examines the role of short- (30 min) and long-term (24 hr) low-to-negative oscillating SS (LOSS) and high laminar SS (HLSS) on the expression and secretion (i.e., cleavage) of cell-membrane VCAM-1 and CX3CL1 by human umbilical vein endothelial cell cultures in vitro. Together, these experiments provide evidence that acute psychological stress, maximal aerobic exercise, and HLSS influence vascular inflammation and adhesive properties of the vessel wall. More specifically, the results from Chapter 2 provide evidence that acute mental stress promotes the immune-cell mediated synthesis of pro-inflammatory cytokines in circulation. In addition, Chapter 3 and Chapter 4 demonstrate that the elevations in blood flow and hemodynamic force associated with maximal aerobic exercise, and unidirectional high SS may have the capacity to alter the expression of endothelial-bound cellular adhesion molecules, in part by eliciting their release from the vessel wall.

La physiopathologie de la GVHa implique de nombreux mécanismes aboutissant à ces lésions tissulaires responsables d'une comorbidité majeure en allo-CSH. L'objectif principal de notre étude a été d'identifier des marqueurs précoces de GVHa et d'explorer leurs rôles dans la physiopathologie de ce phénomène. Quarante-deux cytokines ou chémokines, ont été étudiés à J0 dans le sérum de 109 patients allogreffés avec un conditionnement d'intensité réduite. Nous avons complété ce travail par une étude cinétique à différents temps de l'allo-CSH et une étude histologique de biopsies coliques de patients atteints de GVHa. A J0 de l'allo-CSH, seule la mesure de CX3CL1 J0 était significativement plus élevée chez les patients développant par la suite une GVHa en comparaison aux patients indemnes de GVHa (P=0 ,04). Cette observation persistait à J30 et J50 post allo-CSH mais pas à J100 (P=0,02, P=0,03 et P=0,12, respectivement). L'étude des dosages sériques avant le conditionnement (J-30) ne retrouvait pas de différence significative entre ces 2 groupes. L'analyse phénotypique des différents types cellulaires a mis en évidence une augmentation signification de la proportion de lymphocytes CD8+CX3CR1+ chez les patients présentant une GVHa (P=0,01). L'analyse histologique de biopsies coliques (n=12) montrait une nette augmentation de l'expression de CX3CL1 au niveau des cellules épithéliales de la muqueuse intestinale en cas de GVHa ainsi que la présence de cellules mononuclées CX3CR1+ aux contacts de ces cellules épithéliales. Les résultats de cette étude suggèrent fortement l'implication de CX3CL1 et de son récepteur CX3CR1 dans la physiopathologie de la GVHa. / This study investigated the role of cytokines and chemokines in acute graft-versus host disease (aGVHD) incidence and severity in 109 patients who underwent reduced-intensity conditioning allogeneic stem cell transplantation (HSCT). Among the 42 cytokines tested at day 0, only CX3CL1 levels at day 0 was significantly associated with grades II to IV aGVHD development (P=0.04). Increased levels of CX3CL1 at day 30 and day 50 post-HSCT were also significantly associated with aGVHD (P=0.02 and P=0.03, respectively). No such association was found before conditioning regimen or at day 100 post-HSCT. Because the receptor for CX3CL1 is CX3CR1, the number of CX3CR1+ cells was determined by flow cytometry. The CX3CR1+CD8+T cell proportion was significantly higher in patients with aGVHD than those without aGVHD (P=0.01). To investigate the distribution of the CX3CL1/CX3CR1 axis in the anatomic sites of aGVHD, CX3CL1 and CX3CR1 levels were studied using an in situ immunohistochemical analysis on gastro-intestinal biospsies of patients with intestinal aGVHD. CX3CL1 expression was significantly increased in the epithelial cells and mononuclear cells of the lamina propria. CX3CR1+ cells mononuclear cells were identified in close contact with epithelial cells. These findings strongly suggest the implication of the CX3CL1/CX3CR1 axis in the pathogenesis of aGVHD.