As metaloproteinases da matriz (MMPs) são enzimas proteolíticas dependentes de zinco que degradam componentes da matriz extracelular, facilitando a remodelação tecidual e a migração celular. MMPs secretadas por osteoclastos exercem papel central na absorção óssea fisiológica e estão também associadas a processos de degradação patológica do osso. No entanto, o papel essencialmente degradador de osso das MMPs, particularmente da MMP-2, vem sendo questionado em anos recentes por estudos que evidenciam sua importância na diferenciação de células da linhagem osteoblástica e na formação de tecido ósseo em cultura. Neste sentido, é possível que a MMP-2 exerça um papel importante na formação de tecido ósseo em processo de reparação. O objetivo do presente trabalho foi investigar a pretensa ação osteo-estimulatória da rhMMP-2 ligada à monoleína (usada como carreador) implantada em defeito confeccionado na calvária de ratos. Foram confeccionados defeitos ósseos unilaterais de 4 mm de diâmetro na calvária de ratos adultos; nos animais controles o defeito ósseo foi mantido com o preenchimento natural de coágulo sanguíneo e nos animais implantados o defeito foi preenchido com monoleína ou com rhMMP-2 ligada à monoleína. Os animais foram eutanasiados após 2 e 4 semanas e a taxa de neoformação óssea foi estimada em cortes histológicos por um método histométrico de contagem diferencial de pontos. A taxa de neoformação óssea foi semelhante nos animais dos grupos controle e monoleína e significativamente maior nos animais do grupo MMP-2, em ambos os períodos analisados. Os resultados permitem concluir que a monoleína não interferiu com o processo reparacional e pareceu eficaz como carreador da rhMMP-2, e adicionam evidências á hipótese da importância da atividade da MMP-2 para a formação óssea, em um modelo experimental in vivo de reparo ósseo. / Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that degrade extracellular matrix components, facilitating cell migration and tissue remodeling. MMPs secreted by osteclasts are important in the physiological bone resorption as in pathological bone degradation. However, the essentially bone absorbing hole of MMPs, particularly of the MMP-2, has been questioned in recent years by studies that show its importance in osteoblastic cells differentiation and in vitro bone formation. Therefore, the MMP-2 may have also an important hole in reparational bone formation. The purpose of the present study was to investigate the pretense osteostimulatory effect of the rhMMP-2 linked to monoolein (used as a carrier) implanted into rat calvarial defects. Bone defects of 4mm in diameter were created unilaterally in rats calvaria and filled with natural blood clot (control), monoolein or rhMMP-2 linked to monoolein. The animals were killed 2 and 4 weeks postoperatively and the rate of new bone formation was estimated in histological sections by a histometric differential point-counting method. The rate of reparational bone formation was similar in the animals from control and monoolein groups and was significantly greater in the MMP-2 group, in both periods. From the results it may be concluded that monoolein did not interfere with the reparacional process and seemed effective as a rhMMP-2 carrier. In addition, the results add evidence to the importance of MMP-2 activity for bone formation, in an in vivo bone healing experimental model.

O receptor NLRP12 é um receptor intracelular que está envolvido no reconhecimento de PAMPs e DAMPs durante uma infecção. Foi visto que durante a osteoclastogênese, há uma diminuição da transcrição de NLRP12, e que este receptor inibe a reabsorção óssea através da supressão da via alternativa de NF-κB, exercendo um papel protetor sobre o tecido ósseo. Além disso, foi observado que a deficiência de NLRP12 em monócitos sob o estímulo de RANKL levou a maior estabilização de NIK e maior translocação de RelB para o núcleo, aumentando a formação dos osteoclastos in vitro. Portanto, o objetivo deste estudo foi avaliar o papel de NLRP12 no desenvolvimento e progressão da lesão periapical induzida em camundongos. Para isso, foi induzida a lesão periapical dos primeiros molares inferiores dos camundongos fêmeas C57/Bl6 (WT) e camundongos deficientes para o receptor NLRP12 (Nlrp12-/-). Após 14 e 21 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos; expressão gênica de marcadores osteoclastogênicos por qPCR; contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); avaliação enzimática das MMPs por Zimografia. Os linfonodos cervicais foram submetidos à análise da expressão dos fatores de transcrição T-bet, RORγt, GATA-3 e Foxp-3 por qPCR. Aos 14 dias, na análise histomorfométrica os animais Nlrp12-/- apresentaram uma maior lesão periapical quando comparados aos animais WT, associado com o aumento da expressão de Trap, Catepsina K e MMP-9 em amostras de hemi-mandíbulas com lesão. Além disso, o número de células TRAP positivas foi significantemente maior em Nlrp12-/- com lesão quando comparado com seu controle, enquanto no grupo WT com e sem lesão foram semelhantes. Assim como foi observado o maior aumento dos osteoclastos presentes no local da lesão dos animais Nlrp12-/-, estes também se mostraram com maior atividade gelatinolítica de MMP-9 e MMP-2 em relação ao seu controle, diferente dos animais WT que não apresentaram diferença entre os grupos controle e com lesão. Ainda nesse período, foi observado nas amostras de linfonodos cervicais que, no grupo Nlrp12-/- houve uma tendência à maior expressão de RORγt, seguidos de menor expressão de T-bet quando comparados com o grupo WT. Aos 21 dias, os animais WT e Nlrp12-/- apresentaram lesões periapicais de tamanhos semelhantes. Além disso, somente o grupo Nlrp12-/- com lesão apresentou um aumento significativo da expressão de Trap em relação ao seu controle e a expressão de Catepsina K foi semelhante em ambos os grupos. Neste período houve um aumento na quantidade de células TRAP positivas em ambos os grupos com lesão quando comparados com seus respectivos controles, entretanto também não houve diferença entre os animais WT e Nlrp12-/-. A atividade de MMP-9 e MMP-2 foram semelhantes entre os animais WT e Nlrp12-/- e entre seus respectivos controles aos 21 dias. Nossos dados sugerem que a deficiência de NLRP12 levou a uma maior perda óssea aos 14 dias de lesão periapical e que isso ocorre via modulação da formação e atividade dos osteoclastos. Portanto, o NLRP12 inibe a osteoclastogênese e a atividade dos osteoclastos durante a fase inicial da lesão periapical, retardando o desenvolvimento da doença. / NLRP12 is an intracellular sensor that recongnizes PAMPS and DAMPs during infeccion. It was seen that NLRP12 transcription is down-regulated during osteoclastogenesis, and NLRP12 protect bone via suppression of alternative NF-κB-induced osteoclastogenesis. It has been shown that NLRP12 deficiency in monocytes under RANKL stimuli exhibited more stabilization of NIK and nuclear translocation of RelB, increasing osteoclasts formation in vitro. Therefore, the aim of this study was to evaluate the role of NLRP12 in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and NLRP12 knockout (KO) mice. Samples were collected at 14 and 21 days of the lesion for the analyses. Jaw samples with lesion and control area were subjected to periapical lesions area determination by histological sections; osteoclastogenic markers expression by q-PCR; count of osteoclasts submitted to the enzyme assay for TRAP; evaluation of MMPs activity by Zymography. The expression of T cells markers´ transcription factors was evaluated in lymph nodes by q-PCR. Fourteen days after periapical lesion induction, histological analysis revealed that NLRP12KO mice exhibited higher area of periapical lesion compared to WT group, which was associated with up-regulated mRNA expression of Trap, Cathepsin K and MMP-9 in the jaw samples. Moreover, the number of multinuclear TRAP-stained cells was significantly higher in the NLRP12KO with lesion group when compared to it control, whereas in the WT the number between the lesion and control groups were similar. Still, NLRP12KO showed higher MMP-9 and -2 gelatinolytic activity than it control, unlike WT mice that showed no difference between control and lesion group. In this period, NLRP12KO mice lymph nodes showed more RORγt expression than WT mice and less T-bet expression. At 21 days, WT and NLRP12KO presented periapical lesions of similar sizes. In addition, NLRP12KO group with lesion showed a significant increase in Trap expression when compared with their control, but the increase in Trap e Cathepsin K was similar in both groups. Additionally, there was an increase of multinuclear TRAP-stained cells in both lesion groups when compared with their respective controls; however, there was also no difference between WT and NLRP12KO mice. MMP-9 and -2 activity was similar between WT and NLRP12KO and with their respective controls at 21 days. Our results suggest that NLRP12 deficiency led to increased bone loss at 14 days of periapical lesion and it occurs due to increased osteoclasts formation and activity. Therefore, NLRP12 inhibits osteoclastogenesis and osteoclasts activity during the early stages of periapical lesion, slowing the development of the disease.

Made available in DSpace on 2014-08-20T14:30:17Z (GMT). No. of bitstreams: 1 Dissertacao_Eliana_do_Nascimento_Torre.pdf: 1908566 bytes, checksum: 7f4e5292cab07c5cd5840870b92b0df1 (MD5) Previous issue date: 2011-07-19 / Adhesive fillings are satisfactory on short-term evaluation. However, when the evaluation period of longevity of these fillings is longer, problems with the stability of the polymer formed by the adhesive system and the degradation of collagen forming the hybrid layer provoke a large decrease in the durability of this type of filling. The extracellular matrix metalloproteinases (MMPs) are enzymes, which have been associated with the degradation of collagen present at the hybrid layer. Therefore, the possibility of inhibiting the activity of these enzymes has been considered an important strategy to maintain and increase the longevity of adhesive fillings. Hence, the aim of this work is to evaluate the inhibitory potential of MMPs through the addiction of the monomers with promising characteristics reported in previous studies. These monomers have molecules known as ―kidnappers‖ of bivalent cations. This characteristic is important because the catalytic place of MMPs has zinc and calcium in its constitution. Therefore, the coordination of molecules with the catalytic place of the enzyme would be able to inhibit the activity of the MMPs. Dentine from teeth recently pulled will be used to obtain and purify the MMPs of dentine.. The analysis of MMPs inhibition will be carried out through a zymography. In case of positive results for the inhibition of MMPs, cytotoxicity and genotoxicity tests will be carried out in cellular lineages of human pulp fibroblasts apart from an immortalized lineage of fibroblasts of 3T3/NIH mice. The MTT (bromide 3-(4.5-dimethylthiazol-2-ilo)-2.5-diphenyltetrazolium) colorimetric test will be used to measure the cytotoxicity of the products tested and the test of micronucleus will be used to measure genotoxicity. In all groups the 10mM concentration induced 100% cell death. There was no difference in sensitivity in both strains. Statistically, in relation to the control group/untreated, the monomers were more cytotoxic in HPFs of groups 4 and 5. Similar results were found for the 3T3 lineage, except for group 2, which also showed statistical significance for all concentrations. There was a higher number of micronucleated cells in the groups where the two monomers of intermediate chains longer (PEG200 and PEG400 DMA) were used when compared to control. The results suggest that these two monomers are more cytotoxic and genotoxic. Most of the methacrylate monomers showed considerable but not total inhibition of MMP-2 by zymography. The exception was the PEG200 DMA, which only the concentration of 5 mM inhibited the activity of this gelatinolitic MMP / Restaurações adesivas apresentam desempenho satisfatório em avaliações de curto prazo, porém quando o período de avaliação da longevidade dessas restaurações é maior, problemas com a estabilidade do polímero formado pelo sistema adesivo e com a degradação do colágeno formador da camada híbrida, provocam uma queda expressiva na durabilidade desse tipo de restauração. As metaloproteinases da matriz extracelular (MMPs) são enzimas que têm sido associadas com a degradação do colágeno formador da camada híbrida e, por isso, a possibilidade de inibir a atividade dessas enzimas tem sido considerada uma estratégia importante para a manutenção e aumento da longevidade das restaurações adesivas. O objetivo do presente estudo foi avaliar o potencial inibitório da MMP-2 por monômeros com características promissoras relatadas na literatura relacionadas ao seu potencial inibidor de MMPs, sendo estes, EGDMA (1), TEGDMA (2), T4GDMA (3), PEG200 DMA (4) e PEG400 DMA (5). Dentina humana derivada de dentes recentemente extraídos foi utilizada para a obtenção e purificação das MMPs. O ensaio de inibição da MMP-2 foi realizado por zimografia. Testes de citotoxicidade e genotoxicidade foram realizados com uma linhagem primária de fibroblastos pulpares humanos (FPH), além de uma linhagem imortalizada de fibroblastos de camundongos 3T3/NIH. O teste colorimétrico MTT (brometo de 3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazólio) foi usado para medir a citotoxicidade dos produtos testados, enquanto que o teste de formação de micronúcleos foi usado para avaliar a genotoxicidade. Em todos os grupos a concentração de 10mM induziu a 100% de morte celular. Não houve diferença de sensibilidade nas duas linhagens estudadas. Estatisticamente, em relação ao grupo controle/não tratado, os monômeros foram mais citotóxicos nos FPHs nos grupos 4 e 5. Resultados semelhantes foram encontrados na linhagem 3T3, à exceção do grupo 2, que apresentou diferença estatística em todas as suas concentrações, nesta célula. Houve maior número de células micronucleadas nos grupos onde foram utilizados os dois monômeros de cadeias intermediárias mais longas (PEG200 DMA e PEG400 DMA) quando comparados ao controle, sugerindo os resultados, que estes monômeros são mais citotóxicos e genotóxicos. A maioria dos monômeros metacrilatos apresentou considerável, mas não total inibição da MMP-2 em todas as concentrações através dos ensaios de zimografia. A exceção foi o PEG200DMA, o qual somente a concentração de 5mM inibiu a atividade gelatinolítica da referida MMP

La phase de l'épithélialisation de la réparation cutanée se déroule en impliquant plusieurs processus dynamiques et interactifs pendant lesquels les kératinocytes migrent, prolifèrent et se différentient afin de reconstruire la fonction de la barrière. La migration des kératinocytes est l'événement qui détermine l'efficacité du processus entier. Le comportement migratoire est contrôlé au même temps au niveau extracellulaire et intracellulaire et dépend d'interactions dynamiques entre les cellules et leur environnement extracellulaire, des facteurs de croissance et des cytokines. Parmi les protéines de la matrice extracellulaire, la laminine 332 est un substrat d'adhésion majeur des kératinocytes qui joue un rôle important au cours de la migration des kératinocytes, travers son domaine LG4/5 localisé à l'extrémité carboxy-terminale de sa chaine a. Des études récentes ont rapporté que l'induction de la migration des kératinocytes par LG4/5 est dépendante des Métalloprotéinases Matricielles pro-migratoires (MMP)-9 et -1 qui jouent des rôles essentiels au cours de la cicatrisation et surtout pendant la ré-épithélialisation. Etant donné que des travaux antérieurs du laboratoire ont montré que le domaine LG4/5 participe à la dynamique du cytosquelette et à la motilité cellulaire au travers de liaisons avec les récepteurs de type de protéoglycanes à heparane sulfate, syndécan-1 et -4 on a regardé l'implication potentielle de ces récepteurs au processus. Afin d'analyser la participation possible des syndecans dans ce processus, nous avons développé une approche de mutagénèse dirigée dans la protéine LG4/5 recombinante pour altérer les sites de liaison aux syndécan-1 ou -4. Notre analyse PCR et nos résultats de zymographie ont révélé une différence du profile d'activation des MMPs en fonction de la mutation produite et donc de la capacité de la protéine à recruter le syndécan-1 ou le syndécan-4, ainsi que le syndécan-1, et pas la syndécan-4, est impliqué dans l'activation de la production de la MMP-9 par LG4/5. Nous avons ensuite confirmé ces résultats en réduisant l'expression du syndécan-1 dans des kératinocytes et on a pu aussi montrer que le traitement avec des cytokines telles que TNFalpha et IL-1beta, connues pour leur capacité d'induire l'activation de la MMP-9, a produit le même résultat dans ce systéme. L'addition de l'héparine dans nos experiences a inhibé l'activation de l'expression de MMP-9 suggerant que les heparanes sulfates dans syndecan-1 sont impliqué au mécanisme. Pour confirmer ces résultats des experiences avec des séries de syndecan-1 mutés sont en cours. Pour conclure, nos résultats montrent pour la première fois un rôle important de syndecan-1 à l'expression de MMP-9 suggérant que sa re-distribution au front des kératinocytes migratoires puisse éventuellement être liée au clivage ou à la dégradation des protéines de la matrice extracellulaire. En plus, nos résultats proposent que le domain LG4/5 de la laminin 332 libéré soit capable d'affecter la balance de l'expression de la MMP-9 lors de la migration des kératinocytes en leur permettant de traverser le caillot de fibrine / During skin repair, the epithelialization phase occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the barrier function. Keratinocyte migration determines the efficiency of the overall wound repair process. The migratory behaviour is governed at both the extracellular and intracellular levels and depends on the carefully balanced dynamic interactions of the cells with ECM components, growth factors and cytokines. Among extracellular matrix proteins, laminin 332, known as a major adhesion substrate for keratinocytes was shown to contribute to skin reepithelialization through its a3 chain C-terminal domains LG45. Recent studies have reported that LG45 induces keratinocyte migration, an event that relies on the involvement of the pro-migratory matrix metalloproteinases-1 and -9, two MMPs known to play a role in the reepithelialization phase of wound healing. As findings from our laboratory have reported that LG45 domains participate in cytoskeleton dynamic and cell movement through binding of the heparan sulphate proteoglycans syndecan-1 and -4, we analyzed the potential involvement of these receptors in this process. To that end, we have developed a site-directed mutagenesis approach within a recombinant LG45 protein to alter either the syndecan-1 or syndecan-4 binding site. Our PCR analysis and zymography results revealed that depending on the mutants, syndecan-1 or syndecan-4 recruitment induced different MMP activation profile and suggested that syndecan-1 plays a role in LG45 induced MMP-9 expression and activation. We confirmed these results by down regulating syndecans expression in keratinocytes and revealed that this phenomenon also occurred when cells were treated with TNFalpha or IL1beta, two cytokines known to up-regulate MMP-9 expression. Addition of heparin in these experiments abolished MMP-9 expression activation suggesting that syndecan-1 heparan sulfate moieties are involved in this mechanism. Confirming experiments using a series of mutated syndecan-1 in their ectodomain (lacking glycosaminoglycan chains) or in their cytoplasmic tail are ongoing in the lab. Taken together, our data demonstrate for the first time that syndecan-1 plays a pivotal role in MMP-9 expression, suggesting that its re-distribution at the front edge of migrating keratinocyte may have a role to play in the cleavage or degradation of extracellular matrix proteins. Our results further suggest that the released laminin 332 LG45 domain has the ability to impact the MMP9 expression balance during keratinocyte migration therefore facilitating their path through the fibrin clot

La dérégulation des tyrosine kinases (TK) joue un rôle majeur dans la tumorigénèse et la progression tumorale des cancers du sein. Dans ce contexte, mon travail de thèse s'est focalisé sur la caractérisation des mécanismes moléculaires régulés par les TK associés à l'invasion cellulaire à travers deux axes d'études distincts : la régulation de l'invasion cellulaire par la protéine TOM1L1 dans les cancers du sein ERBB2-positifs et le rôle des TK ABL/ARG dans l'invasion cellulaire des cancers du sein triple-négatifs (n'exprimant ni ERBB2, ni les récepteurs hormonaux) (TN). TOM1L1 est une protéine du trafic vésiculaire à domaine GAT initialement caractérisée au laboratoire comme un régulateur négatif de l'activité mitogénique de la TK Src. Cependant, nos travaux indiquent que les gènes codant pour TOM1L1 et le récepteur TK ERBB2, localisés au niveau du chromosome 17q, sont co-amplifiés dans environ 50% de ces cancers et associés à un mauvais pronostic suggérant une activité oncogénique non-anticipée de TOM1L1. Mon travail de thèse montre que le récepteur ERBB2 régule indirectement la phosphorylation de la sérine 321 de TOM1L1 afin de permettre son association avec la protéine du trafic vésiculaire TOLLIP au niveau d'endosomes Rab7/MT1-MMP-positifs. Ainsi localisée, TOM1L1 favorise le recrutement membranaire de la métalloprotéase MT1-MMP au niveau de structures d'invasion cellulaire spécifiques, les invadopodes, amplifiant ainsi la capacité invasive des cellules ERBB2+. Des données récentes indiquent que les TK ABL/ARG sont aussi impliquées dans la régulation des invadopodes des cellules des cancers TN. Grace à une approche préliminaire d'inhibition pharmacologique (Imatinib, Nilotinib), mes travaux de thèse ont permis de dévoiler un rôle anti-invasif non-anticipé de ABL dans certaines lignées de cancers du sein TN, dont nous recherchons actuellement le mécanisme. / Deregulation of tyrosine kinases (TK) plays crucial role in breast cancer tumorigenesis and metastatic progression. In this context, my work tried to decipher the molecular mechanisms of tyrosine kinases-induced cell invasion via two distinct research axes: the regulation of cell invasion by TOM1L1 in ERBB2-positive breast cancers and the role of ABL/ARG TK in triple-negative (TN) breast cancer cells (negative for ERBB2 and hormonal receptors expression). The GAT domain-containing vesicular trafficking protein TOM1L1 was first identified in the lab as a negative regulator of Src mitogenic activity. Here, we show that ERBB2 and TOM1L1, located on chromosome 17q, are co-amplified in 50% of breast tumor samples and define a subgroup of ERBB2-positive tumors with poor prognosis. We then studied TOM1L1 pro-oncogenic function and found that TOM1L1 regulates exclusively ERBB2-driven cell invasion. This effect requires indirect ERBB2-induced TOM1L1 phosphorylation on serine 321 and interaction with the endosomal sorting protein TOLLIP at Rab7/MT1-MMP-positive endosomes. Thus, TOM1L1 promotes membrane recruitment of MT1-MMP into specialized invasive structures called invadopodia and, as such, promotes cell invasion. Recent observations show that ABL/ARG TK are also involve in invadopodia regulation of TN breast cancers. With a preliminary pharmacological-inhibition strategy (Imatinib,Nilotinib), my work shows an unanticipated anti-invasive function of ABL in some TN cell-lines. We are currently looking for ABL kinases molecular mechanisms involved in this effect.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Traumatic brain injury (TBI) produces axonal damage and deafferentation, triggering injury-induced synaptogenesis, a process influenced by matrix metalloproteinases (MMP) and their substrates. Here we report results of studies examining the expression and potential role of two membrane-bound MMPs, membrane-type 5-MMP (MT5-MMP) and a disintegrin and metalloproteinase-10 (ADAM-10), along with their common synaptic substrate N-cadherin, during the period of reactive synaptogenesis. Protein and mRNA expression of MT5-MMP, ADAM-10 and N-cadherin were compared in two TBI models, one exhibiting adaptive plasticity (unilateral entorhinal cortex lesion; UEC) and the other maladaptive plasticity (fluid percussion injury + bilateral EC lesions; TBI+BEC), targeting 2, 7, and 15d postinjury intervals. In adaptive UEC plasticity, membrane-bound MMP expression was elevated during synaptic degeneration (2d) and regeneration (7d), and normalized at 15d. By contrast, N-cadherin expression was significantly decreased at 2 and 7d after UEC, but increased during 15d synaptic stabilization. In maladaptive plasticity, 2d membrane-bound MMP expression was dampened compared to UEC, with persistent ADAM-10 elevation and reduced N-cadherin protein level at 15d. These results were supported by 7d microarray and qRT-PCR analyses, which showed transcript shifts in both hippocampus and dentate molecular layer (ML) for each model. Parallel immunohistochemistry revealed significant MT5-MMP, ADAM-10 and N-cadherin localization within ML reactive astrocytes, suggesting a glial synthetic or phagocytotic role for their processing during recovery. We also investigated the effect of MMP inhibition on molecular, electrophysiological, behavioral and structural outcome at 15d following TBI+BEC. MMP inhibitor GM6001 was administered at 6 and 7d postinjury, during elevated MT5-MMP/ADAM-10 expression and synapse regeneration. MMP inhibition showed: 1) reduced ADAM-10 and elevated N-cadherin protein expression, generating profiles similar to 15d post-UEC, 2) attenuation of deficits in the initiation phase of long-term potentiation, and 3) improved hippocampal dendritic and synaptic ultrastructure. Collectively, our results provide evidence that membrane-bound MMPs and N-cadherin influence both adaptive and maladaptive plasticity in a time and injury-dependent manner. Inhibition of membrane-bound MMPs during maladaptive plasticity produces more adaptive conditions, improving synaptic efficacy and structure. Thus, targeting MMP function and expression have potential to translate maladaptive plasticity into an adaptive process, facilitating improved recovery.

Malignant brain tumors are among the deadliest of human cancers. Despit recent advancements in conventional therapies, glioblastomas remain incurable, largel y due to their ability to invade surrounding tissue. Matrix metalloproteinases are thought to contribute to the invaseive phenotype of human gliomas. Absent in normal brain, matrix metalloproteinase-1 (MMP-1) has been shown to be present in gliomas, and in particular in glioblastoma multiforme (GBM). To begin to examine the role of MMP-1 in these tumore, two human glioma cell lines were stably transfected with MMP-1 cDNA. Confirmation of MMP-1 over-expression in these cells was achieved through real-time PCR and Western blot analysis. The functional consequences of MMP-1 over-expression were analyzed using a collagen type-I invasion assay along with clonogenic and ATP viability assays. Data presented demonstrate that MMP-1 over-expressing cells were more invasive in both cell types and interestingly more clonogenic in on of the glioma cell lines, supporting a possible role for MMP-1 in glioma growth and invasion.

La protéine C-réactive (CRP) est aujourd’hui considérée comme un biomarqueur indispensable dans la prédiction de maladies cardio-vasculaires et de complications aiguës associées, et ce, par des mécanismes qui ne sont pas encore totalement élucidés. Nous avons étudié les effets de la CRP dans divers aspects de la biologie des cellules mononucléaires du sang périphérique (PBMC) et des monocytes humains ex vivo. En effet, ces cellules peuvent être impliquées dans la physiopathologie de ces maladies. Nous avons aussi utilisé la lignée promonocytaire THP-1. Ces trois types cellulaires ont été incubés avec la CRP purifiée ou recombinante et l’expression génique de cytokines pro-inflammatoires, de chimiokines et de MMP-9 a été analysée par PCR semi-quantitative en temps réel et l’expression protéique par test immunométrique ou par test ELISA. La CRP ne semble agir ni sur la synthèse de ces cytokines ni sur celle de MMP?9. Une approche globale par puce à protéines avec les surnageants de culture de monocytes a démontré que la CRP augmentait la synthèse du VEGF-A. Ce résultat a été confirmé au niveau transcriptionnel par RT-PCR et au niveau protéique par test ELISA. Une étude complémentaire avec la lignée THP-1 a permis de montrer l’implication des voies PI3?Kinase et MEK mais pas celle de la p38MAPKinase dans cette augmentation. Ces travaux ont ainsi permis la mise en évidence de plusieurs mécanismes permettant d’associer la CRP aux pathologies cardiovasculaires dans une relation de cause à effet. Ces mécanismes pourraient représenter des cibles thérapeutiques potentielles des maladies cardiovasculaires. / C-reactive protein is now considered as an essential biomarker for predicting the occurrence of cardiovascular diseases and their acute complications through mechanisms that are not fully elucidated. We investigated CRP effects on several aspects of the biology of ex vivo human peripheral blood mononuclear cells (PBMC) and monocytes. In fact, these cells can be involved in the pathophysiology of these diseases. Also, we used the promonocytic line THP-1. These three cellular types were incubated with purified or recombinant CRP and gene expression of pro-inflammatory cytokines, chemokines and (pro)MMP-9 was analysed by real time semi-quantitative PCR and protein expression by immunometric or ELISA tests. CRP doesn’t seem to act upon neither the tested cytokines synthesis nor the (pro)MMP-9 expression. A global approach by protein array with the cultured monocytes supernatants showed that CRP induced VEGF-A protein synthesis. This result was confirmed at transcriptional level by RT-PCR and at protein level by ELISA. A complementary study with the monocytic cell line THP-1 demonstrated the activation of PI3-Kinase and MEK pathways but not of p38MAPKinase pathway in this regulation. These results provide insight into several mechanisms that could transform the statistical association between CRP and cardiovascular diseases into a cause-to-effect relationship. Some of these mechanisms could represent therapeutic targets for cardiovascular diseases.

Matrix metalloproteinases (MMPs) are required for tissue remodelling processes, including angiogenesis. MMP activity is generally proangiogenic but MMP-12 is suggested to be antiangiogenic and its precise role is still unclear. The work in this thesis describes the synthesis of an MMP-12 inhibitor and activity probe to address the hypothesis that MMP-12 inhibits angiogenesis. An inhibitor, synthesised in-house, selectively inhibited MMP-12 in in vitro recombinant enzyme assays. An activity probe, also synthesised in-house, was selective for MMP-12 in in vitro recombinant enzyme assays. The function of MMP-12 during angiogenesis was assessed using murine models of angiogenesis; the in vivo sponge implantation, and the ex vivo aortic ring assays. Angiogenesis and MMP activity were imaged in vivo in sponges in C57Bl6/J mice over 7 − 21 days (D) using commercial probes (MMPSense™ and AngioSense™). MMP-12 protein concentration and activity were higher in sponges during early angiogenesis (D 3 − 7) when gene expression of vascular endothelial growth factor (a proangiogenic marker) was also high. Gene expression for MMP-12 and platelet-derived growth factor receptor (a marker of vascular maturation) were both higher on D 21 as angiogenesis started to stabilise. The MMP-12 activity probe was unsuccessful in selectively detecting MMP-12 activity in sponge lysate mixtures from D 7 − 21. Administration of an MMP-12 inhibitor did not increase angiogenesis in the sponges in vivo. Additionally, sponges implanted in MMP-12-/- mice did not exhibit significant changes in angiogenesis or MMP activity when imaged in vivo using commercial probes (MMPSense™ and AngioSense™) on D 7. Supporting this, histological analysis of the sponges (removed on D 21) showed that deletion of MMP-12 also did not increase angiogenesis within the sponges.