Ainda que quando aplicada como primer antiproteolítico a clorexidina (CHX) minimize, embora não indefinidamente, a degradação de interfaces adesivas, parece não ser capaz de tornar os valores de resistência de união (RU) à dentina erodida equivalentes àqueles à dentina normal. Talvez seja possível, porém, que sua aplicação como agente para prevenção/controle do desgaste erosivo influencie o estabelecimento de tais interfaces, já que pode inibir completamente o processo. Este estudo in vitro avaliou, pois, o papel da aplicação da CHX como agente antiproteolítico para prevenção/controle do desgaste, ou seja, da condição do substrato dentinário no tocante à erosão, ou como parte do tratamento adesivo (primer antiproteolítico), na RU do conjunto sistema adesivo condicione e lave simplificado-resina composta à dentina. Para se determinar os diferentes níveis do fator substrato dentinário, a dentina superficial oclusal de terceiros molares (n=48) foi submetida exclusivamente à ação de uma lixa de SiC (#600; 1 min), configurando-se o que se denominou dentina normal - N, ou subsequentemente tratada com solução de digluconato de CHX a 2% (60 s) e erodida por um refrigerante à base de cola, determinando-se a dentina tratada com CHX para prevenção/controle do desgaste e então erodida - CHX.E, ou apenas erodida pelo refrigerante, estabelecendo-se a dentina erodida - E. Já para se determinar os diferentes níveis do fator CHX como parte do tratamento adesivo, a dentina dos grupos N, CHX.E e E foi, então, condicionada, lavada, seca e reidratada com 1,5 ?l, respectivamente, de água destilada (controle - Nc / CHX.Ec / Ec; n=8) ou de digluconato de CHX a 2% (primer antiproteolítico - Npa / CHX.Epa / Epa; n=8). O adesivo Adper Single Bond 2 foi aplicado em todos os espécimes e a porção coronária, reconstruída com a resina Filtek Z350. Após 24 h (água destilada/37?C), os espécimes foram seccionados em palitos e imediatamente testados (?TBS; 0,5 mm/min). As superfícies fraturadas de cada palito foram analisadas com o auxílio de um microscópio digital (50x de aumento). Um dente extra para cada grupo foi tratado exatamente como os outros, mas o corante fluorescente rodamina B foi previamente adicionado ao sistema adesivo para permitir a avaliação qualitativa da interface adesiva por meio de Microscopia Confocal de Varredura a Laser. Os valores de RU obtidos foram organizados de forma a se considerar cada dente como unidade experimental e, então, os testes de Análise de Variância a 2 critérios e de Tukey foram aplicados, com nível de significância de 5%. Apenas o substrato normal, quando reidratado, após condicionamento ácido, com água destilada (controle), é que apresentou valores de RU à dentina significativamente superiores aos demais. Quanto ao padrão de fratura dos palitos testados, as falhas adesivas e mistas foram predominantes em relação às coesivas, independentemente se em dentina ou em resina. Qualitativamente, a hibridização da dentina erodida pareceu menos satisfatória que a da dentina tratada com CHX como agente antiproteolítico para prevenção/controle do desgaste e então erodida, que, por sua vez, pareceu também menos satisfatória que a da dentina normal, particularmente se reidratadas com CHX a 2% como primer antiproteolítico. Ademais, a hibridização de cada substrato, individualmente, pareceu inferior quando da aplicação da CHX a 2% como primer antiproteolítico, se comparada com a da água destilada. Conclui-se, ora, que o uso da CHX como agente para prevenção/controle do desgaste erosivo parece, contrariamente ao que se observa para essa finalidade, não interessante de modo pleno quando, do ponto de vista da resistência de união, na sequência, há que se restaurar o substrato dentinário utilizando-se sistema adesivo do tipo condicione e lave simplificado em associação a resina composta. Quando aplicada como primer antiproteolítico, ainda, a CHX pode prejudicar a adesão à dentina normal, ainda que não o faça, nem o contrário, para a dentina tratada com CHX como agente antiproteolítico para prevenção/controle do desgaste e então erodida ou tão-somente erodida. / Even if when applied as a antiproteolytic primer the chlorhexidine (CHX) minimizes, although not indefinitely, degradation of adhesive interfaces, it seems not to be able to make bond strength (BS) values to eroded dentin equivalent to those to normal dentin. It may be possible, though, that its application as an agent for the prevention/control of the erosive wear influences the establishment of such interfaces, as it can completely inhibit the process. This in vitro study evaluated, thus, the role of CHX application as an antiproteolytic agent for preventing/controlling the wear, that is, the condition of the dentin substrate with respect to erosion,or as part of the adhesive treatment (antiproteolytic primer), on BS of a simplified etch-and-rinse adhesive plus a composite resin to dentin. In order to determine the different levels of the factor dentin substrate, superficial occlusal dentin of third molars (n=48) was only ground with a SiC paper (600-grit; 1 min), what was called normal dentin - N, or subsequently treated with 2% CHX digluconate (60 s) and eroded by a regular-cola soft-drink, determining the dentin treated with CHX to prevent/control the wear and then eroded - CHX.E, or only eroded by the soft-drink, establishing the eroded dentin - E. To determine the different levels of the factor CHX as part of the adhesive treatment, dentin of N, CHX.E and E groups was then acid-etched, washed, dried and rehydrated with 1.5 ?L, respectively, of distilled water (control - Nc / CHX.Ec / Ec; n=8) or of 2% CHX (antiproteolytic primer - Nap / CHX.Eap / Eap; n=8). Adper Single Bond 2 was applied in all specimens and resin composite buildups were constructed with Filtek Z350. After 24 h (distilled water/37?C), specimens were sectioned in beams and immediately tested (?TBS; 0.5 mm/min). Fractured surfaces of each beam were observed under a digital microscope (50X magnification). An extra tooth for each group was treated just like the others, but the fluorescent dye rhodamine B was previously added to the adhesive system to allow a qualitative evaluation of the adhesive interface by means of Laser Scanning Confocal Microscopy. Obtained BS values were organized in order to consider each tooth as an experimental unit and, then, two-way ANOVA and Tukey tests were applied, with a significance level of 5%. Only the normal substrate, when rehydrated, after acid etching, with distilled water (control), is the one that presented BS values to the dentin significantly higher to the others. As for the fracture pattern of the tested beams, adhesive and mixed failures were predominant in relation to the cohesive failures, regardless of whether in dentin or in resin. Qualitatively, the hybridization of eroded dentin appeared less satisfactory than that of the dentin treated with CHX to prevent/control the wear and then eroded, which, in turn, also appeared less satisfactory than that of normal dentin, particularly if rehydrated with 2% CHX as antiproteolytic primer. In addition, the hybridization of each substrate individually appeared to be inferior for the application of the 2% CHX as antiproteolytic primer, when compared to distilled water. It is concluded, therefore, that the use of CHX as an agent for preventing/controlling the erosive wear seems, contrary to what is observed for this purpose, not fully interesting when, from the point of view of the bond strength, in the sequence, the dentin substrate must be restored using a simplified etch-and-rinse adhesive system plus a composite resin. When applied as an antiproteolytic primer, moreover, CHX may impair bonding to normal dentin, even if not doing this, nor the opposite, for the dentin treated with CHX to prevent/control the wear and then eroded or for the only eroded dentin.

Le sarcome d’Ewing est une tumeur pédiatrique de l’os très agressive, généralement causée par une translocation chromosomique aboutissant à la protéine de fusion EWSR1-FLI1 qui dérégule l’expression d’un grand nombre de gènes participant à l’oncogenèse du sarcome d’Ewing. Au laboratoire, nous avons pu mettre en évidence une hétérogénéité intra-tumorale de l’expression d’EWSR1-FLI1 aboutissant à un nouveau modèle de la biologie du sarcome d’Ewing basé sur la coexistence de cellules EWSR1-FLI1high, participant à la prolifération de la tumeur primaire, et de cellules EWSR1-FLI1low, capables de dissémination métastatique. Au cours de ma thèse, je me suis intéressée en particulier aux mécanismes moléculaires sous-jacents aux capacités migratoires et invasives des cellules EWSR1-FLI1low. Nous avons ainsi pu mettre en évidence une diminution des jonctions serrées intercellulaires associée à une augmentation des interactions cellule-matrice chez les cellules EWSR1 FLI1low. Nous avons ensuite révélé le rôle clé des MMP dans l’invasion du sarcome d’Ewing, plus spécifiquement de la MMP2 et de la MT1-MMP qui sont toutes deux réprimées par EWSR1-FLI1 et sont apparues nécessaires à l’invasion des cellules EWSR1-FLI1low. L’un des défis majeurs actuels est de parvenir à visualiser cette sous-population très minoritaire et à suivre la plasticité de l’expression d’EWSR1-FLI1. Pour cela, l’identification de marqueurs des cellules EWSR1-FLI1low est cruciale. Ceci est également important pour déterminer si les chimiothérapies actuelles, qui ciblent plutôt les cellules prolifératives, n’entraînent pas une augmentation de la proportion de ces cellules au sein de la tumeur primaire. Enfin, nos essais infructueux d’utilisation de l’embryon de poulet comme nouveau modèle de la métastase du sarcome d’Ewing in vivo ont souligné la fragilité globale et les limitations de ce type de modèle, indiquant plutôt la nécessité d’améliorer les modèles murins existants. La mise au point de modèles in vivo apparaît en effet primordiale pour mieux disséquer les mécanismes moléculaires de la métastase et permettre le test de drogues susceptibles d’inhiber ce processus. / Ewing sarcoma is a very aggressive pediatric bone tumor generally caused by a chromosomal translocation that leads to the expression of the EWSR1-FLI1 fusion protein. This protein deregulates the expression of a number of genes involved in Ewing sarcoma oncogenesis. In the laboratory, we discovered an intra-tumor heterogeneity of EWSR1-FLI1 expression which lead us to propose a new model for Ewing sarcoma biology based on the coexistence of EWSR1-FLI1high cells, responsible for primary tumor growth, and EWSR1-FLI1low cells, with metastatic dissemination capacities. During my phD, I was especially interested in the molecular mechanisms underlying the migration and invasion capacities of EWSR1-FLI1low cells. We showed a decreased expression of intercellular junctions proteins associated with an increased expression of cell-matrix interaction proteins in EWSR1-FLI1low cells. We then revealed the key role of MMPs in Ewing sarcoma invasion, in particular of MMP2 and MT1-MMP, which are both repressed by EWSR1-FLI1 and were found necessary for invasion of EWSR1-FLI1low cells. One of the current major challenges is to visualize this very small sub-population and to track the plasticity of expression of EWSR1-FLI1. To this end, the identification of markers of EWSR1-FLI1low cell is crucial. This is also important in order to determine if current chemotherapies that primarily target highly proliferative cells do not increase the proportion of these EWSR1-FLI1low cells. Finally, our unsuccessful attempts to use the chick embryo as a new in vivo model of Ewing sarcoma metastasis indicate the need to improve the existing murine models. Indeed, the development of in vivo models appears essential for the dissection of the molecular mechanisms of Ewing sarcoma metastasis and the test of drugs susceptible to inhibit this process.

Canine Mammatumoren (CMT) sind eine der häufigsten Todesursachen bei Hündinnen (SIMON et al. 2001). Durch ihre periphere Lage sind zumeist nicht die Primärtumoren, sondern ihre Metastasen lebensbedrohlich, da diese häufig wichtige Organe wie Lunge Herz und Gehirn befallen (CLEMENTE et al. 2010). Aktuell angewandte Untersuchungsmethoden zur Erkennung von Metastasierung können entweder das invasive Wachstum beschreiben oder bereits bestehende Metastasen detektieren, jedoch fehlen bislang molekulare Marker, die frühzeitig und zuverlässig das metastatische Potential eines CMT anzeigen bevor Metastasen aufgetreten sind. Das Peptidhormon Relaxin ist in zahlreichen physiologischen und pathologischen Situationen beim Menschen als ein Induktor von Matrixmetalloproteinasen (MMP) bekannt (TOO et al. 1984; UNEMORI und AMENTO 1990; PALEJWALA et al. 2001; BINDER et al. 2002; KLONISCH et al. 2007; HENNEMAN et al. 2008). MMP sind Schlüsselenzyme des Bindegewebsabbaus, der jeder Metastasierung vorausgehen muss (WOODHOUSE et al. 1997). Bei der Frau ist im Blut messbares Relaxin ein Marker für metastatischen Brustkrebs (BINDER et al. 2004), für die Hündin scheint dagegen der Relaxinblutspiegel nicht aussagekräftig zu sein (SCHWEIZER 2010). Möglicherweise wird aber Relaxin lokal im caninen Mammagewebe exprimiert wie von GOLDSMITH et al. (1994) und SILVERTOWN et al. (2003) postuliert, so dass es wie in humanen Tumoren auto- oder parakrin Invasivität und Metastasierung (KLONISCH et al. 2007) fördern könnte. Daher sollte in der vorliegenden Arbeit die intratumorale Expression des Relaxins und seiner Rezeptoren sowie bekannter Faktoren des Bindegewebsabbaus untersucht und auf ihre prognostische Eignung überprüft werden. In zwei Studienabschnitten (LAMP et al. 2009; LAMP et al. 2011) wurden CMT-Gewebeproben von n=31 (LAMP et al. 2009) respektive n=59 Hündinnen (LAMP et al. 2011) mittels quantitativer Reverse-Transkriptase-Polymerasekettenreaktion (qRT-PCR) auf ihre Expression von Relaxin, seinen Rezeptoren, RXFP1 und RXFP2, sowie den Matrixmetalloproteinasen MMP-1, -2, -3, -9 und MMP-13, den Östradiolrezeptoren, ERα und ERβ, und dem Progesteronrezeptor (PR) analysiert. In beiden Studienabschnitten wurden die Plasmakonzentrationen der Hormone Relaxin, Östradiol und Progesteron auf mögliche Zusammenhänge mit der lokalen Genexpression überprüft. Im zweiten Studienabschnitt (LAMP et al. 2011) wurde darüber hinaus die Expression von Relaxin und RXFP1 auch immunhistologisch an n=9 CMT-Proben untersucht und in einer Multivarianzanalyse die prognostische Eignung aller untersuchten Parameter getestet. Die Expressionsanalyse konnte zeigen, dass CMT sowohl ein bisher unbekannter Ort der Relaxinexpression beim Hund sind als auch den Hauptrezeptor des Relaxins, RXFP1, exprimieren. Diese Ergebnisse der mRNA-Untersuchung ließen sich immunhistologisch bestätigen. Darüber hinaus ergab die immunhistologische Untersuchung, dass Relaxin vorwiegend im myoepithelialen Anteil der untersuchten CMT exprimiert wird. In den epithelialen CMT-Zellen fand sich die stärkste RXFP1-Reaktivität, so dass RXFP1 mit der von anderen Autoren beschriebenen MMP-2- und MMP-9-Expression in epithelialen Zellen kolokalisiert ist (PAPPARELLA et al. 1997; HIRAYAMA et al. 2002; PAPPARELLA et al. 2002). Die quantitativen Expressionsanalysen zeigten Korrelationen der Expressionsintensitäten von Relaxin, RXFP1 und MMP-2 auf. Die RXFP1 Expression war in dieser Studie sogar ein unabhängiger Marker für Metastasierung mit einem 15-fach höheren Risiko für Metastasierung für Patienten mit einer Expression oberhalb des studienspezifischen Cut-Offs. Alle untersuchten lokalen Genexpressionen waren von den systemischen Plasmakonzentrationen von Relaxin, Östradiol und Progesteron unabhängig. Die Resultate legen eine Bedeutung des intratumoral exprimierten Relaxins für eine auto- oder parakrine Steuerung der MMP-Expression, die für Invasivität und Metastasierung wichtig ist, nahe. Aufgrund des mRNA-Nachweises, der Kolokalisation der Proteine von RXFP1 und MMP-2 und -9 sowie der Korrelation der Genexpression von Ligand (Relaxin), Rezeptor (RXFP1) und Effektormolekül (MMP-2) ist es wahrscheinlich, dass CMT über das Relaxin eine autonome Steuerung ihrer Invasivität vornehmen können. RXFP1 scheint dabei eine Regulationsfunktion in der Relaxinsensibilität der CMT-Zelle zuzukommen, die in Zukunft durch die Messung der RXFP1-Expressionsintensität prognostisch nutzbar sein könnte. Zudem ist RXFP1 im CMT damit auch ein möglicher Ansatzpunkt für eine neue, auf Relaxinanaloga basierende, antimetastatische Therapie, die bereits an humanen Tumorzellen und in Mausmodellen erprobt wird (FENG et al. 2007; HOSSAIN et al. 2010). Durch den Nachweis von Relaxin und RXFP1 im CMT und ihre wahrscheinliche Relevanz für die Metastasierung ergeben sich somit neue Möglichkeiten für eine exaktere Prognose und verbesserte antimetastatische Therapie von CMT sowie die Chance, den Hund als Modell für die Erforschung Relaxin basierter Therapien des humanen Brustkrebses zu nutzen. / Canine mammary tumours (CMT) are one of the main reasons of death in female dogs (SIMON et al. 2001). Due to its peripheral location, it is normally not the primary tumour, but its metastases, which are life-threatening as they often impair the function of vital organs, such as lung, heart or brain (CLEMENTE et al. 2010). Currently used techniques for the detection of metastasis can either barely describe invasive growth patterns or detect already existing metastases. Molecular markers to determine the metastatic potential early and reliably, before metastatic spreading has occurred, are still lacking. The peptide hormone relaxin is well known as an inductor of matrix metalloproteinases (MMP) in numerous physiological as well as pathological situations in humans (TOO et al. 1984; UNEMORI und AMENTO 1990; PALEJWALA et al. 2001; BINDER et al. 2002; KLONISCH et al. 2007; HENNEMAN et al. 2008). MMP are key-enzymes of connective tissue remodelling which is a prerequisite for metastasis (WOODHOUSE et al. 1997). In women, the plasma relaxin concentration is a marker for metastatic breast cancer (BINDER et al. 2004). However, in dogs, the concentration of circulating relaxin seems to have no diagnostic value (SCHWEIZER 2010). But, possibly relaxin is expressed locally in the canine mammary tissue as postulated by GOLDSMITH et al. (1994) and SILVERTOWN et al. (2003) and it could therefore act as a pro-invasive and pro-metastatic factor in an auto- or paracrine manner as it does in various human tumours (KLONISCH et al. 2007). Thus, the present study should examine the intratumoural expression of relaxin and its receptors as well as factors of connective tissue remodelling and evaluate their prognostic abilities. In two sections of the study (LAMP et al. 2009; LAMP et al. 2011), CMT-tissue samples from n=31 bitches (LAMP et al. 2009) and n=59 bitches (LAMP et al. 2011), respectively, were analysed for their expression of relaxin, its receptors RXFP1 and RXFP2, MMP-1, -2, -3, -9 and MMP-13 as well as the oestradiol receptors ERα and ERβ and the progesterone receptor (PR) using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Additionally, the plasma concentrations of the hormones relaxin, oestradiol and progesterone were tested for possible connections with the local gene expression. In the second section of the study, the expression of relaxin and RXFP1 was also examined immunohistologically in n=9 CMT tissue samples (LAMP et al. 2011) and the prognostic value of all parameters examined was assessed by a multivariate analysis. The expression analysis showed that CMTs are a novel site of expression of relaxin and its main receptor RXFP1 in the dog. These results were confirmed by the immunohistological examination. Moreover, the immunohistological analysis demonstrated that relaxin seems to be expressed mainly in myoepithelial cells. However, the strongest signals for RXFP1 were located in epithelial cells of the CMT, thus RXFP1 is colocalised with the expression of MMP-2 and MMP-9 reported in epithelial CMT-cells (PAPPARELLA et al. 1997; HIRAYAMA et al. 2002; PAPPARELLA et al. 2002). The quantitative expression analysis revealed correlations of expression intensities for relaxin, RXFP1 and MMP-2. The expression of RXFP1 presented as an independent marker for metastasis with a 15-fold risk increase for patients with an expression intensity above the study-specific cut-off. All local gene expressions examined where independent from systemic plasma concentrations of relaxin, oestradiol and progesterone. The results propose an important role for intratumourally expressed relaxin as an auto- or paracrine modulator of MMP expression, which is important for invasiveness and metastasis. Due to the mRNA detection, the protein colocalisation of RXFP1 with MMP-2 and MMP-9 as well as the correlation of gene expressions of the ligand (relaxin), the receptor (RXFP1) and the effector (MMP-2) it is highly probable that CMT can autonomously regulate their invasiveness via locally expressed relaxin. RXFP1 seems to have a regulatory function in the relaxin responsiveness of CMT cells, which may be of prognostical use in the future. In addition, RXFP1 is also a possible target for a novel antimetastatic therapy based on relaxin analoga which has been tested in human tumour cells and mice (FENG et al. 2007; HOSSAIN et al. 2010). The detection of relaxin and RXFP1 in the CMT and their probable relevance for metastasis could be a basis for a more precise prognosis of CMT, improved anti-metastatic therapies in the dog and the use of the dog as a model for relaxin-based therapies of human breast cancer.

Die Inzidenz des Diabetes mellitus (DM) nimmt weltweit jährlich zu und erlangt somit große Bedeutung für die Entwicklung der globalen Gesundheit. Die WHO rechnet bis zum Jahr 2030 mit ungefähr 366 Millionen erwachsenen Diabetikern. Es ist bekannt, dass Männer mit Diabetes mellitus Typ 1 (DMI) und 2 (DMII) häufiger an Subfertilität leiden, wobei dies möglicherweise auf erhöhte Apoptoseraten und vermehrte DNA-Fragmentierungen auf zellulärer Ebene zurückzuführen ist. Die Bedeutung der ROS als Regulatoren von physiologischen und pathologischen Signaltransduktionswegen ist bekannt. Demzufolge könnte die Aktivität der Stickstoffmonoxid-Synthetase (NOS) in diesem Zusammenhang eine Bedeutung haben. Das Ziel der vorliegenden Untersuchungen war es, die Auswirkungen von Apoptose und DNA-Fragmentierungen sowie die Bedeutung der NOS im Zusammenhang mit der Fertilitätsfähigkeit von Spermienzellen von DMI und DMII Patienten zu erfassen und damit erste Erklärungsansätze zur Pathophysiologie der diabetesassoziierten Subfertilität zu liefern. Samenproben von Normalspendern und Diabetikern wurden durch Dichtegradientenzentrifugation in Subpopulationen separiert und mittels fluoreszenzbasierten Assays zur Analyse von apoptoseassoziierten Parametern wie dem Zusammenbruch des mitochondrialen Membranpotentials (MMP), Aktivierung von Caspase-3 (CP3), DNA-Fragmentierungen und reaktiven Sauerstoffspezies (ROS) im Flowzytometer (FACS) untersucht. Die Ergebnisse zeigen eine signifikante Erhöhung von Apoptosemarkern (gestörtes MMP, aktivierte CP3), ROS und DNA-Fragmentationsraten in Spermien von DMI und DMII Patienten im Vergleich zu gesunden Normalspendern. Der Effekt ist bei DMII Patienten verstärkt ausgeprägt. Alle gemessenen Parameter korrelieren umgekehrt mit dem Fertilitätspotential der Spermien, gemessen anhand etablierter Spermiogramm-Analysen, womit ein möglicher Erklärungsmechanismus für die Subfertilität bei Diabetikern geliefert werden kann.

Cette thèse traite du rôle de la protéine FAK (Focal Adhésion Kinase) dans les mécanismes d'invasion cellulaire. / This thesis is about the role of the protein FAK (Focal Adhesion Kinase) in the cellular mechanisms of invasion.

Ainda que quando aplicada como primer antiproteolítico a clorexidina (CHX) minimize, embora não indefinidamente, a degradação de interfaces adesivas, parece não ser capaz de tornar os valores de resistência de união (RU) à dentina erodida equivalentes àqueles à dentina normal. Talvez seja possível, porém, que sua aplicação como agente para prevenção/controle do desgaste erosivo influencie o estabelecimento de tais interfaces, já que pode inibir completamente o processo. Este estudo in vitro avaliou, pois, o papel da aplicação da CHX como agente antiproteolítico para prevenção/controle do desgaste, ou seja, da condição do substrato dentinário no tocante à erosão, ou como parte do tratamento adesivo (primer antiproteolítico), na RU do conjunto sistema adesivo condicione e lave simplificado-resina composta à dentina. Para se determinar os diferentes níveis do fator substrato dentinário, a dentina superficial oclusal de terceiros molares (n=48) foi submetida exclusivamente à ação de uma lixa de SiC (#600; 1 min), configurando-se o que se denominou dentina normal - N, ou subsequentemente tratada com solução de digluconato de CHX a 2% (60 s) e erodida por um refrigerante à base de cola, determinando-se a dentina tratada com CHX para prevenção/controle do desgaste e então erodida - CHX.E, ou apenas erodida pelo refrigerante, estabelecendo-se a dentina erodida - E. Já para se determinar os diferentes níveis do fator CHX como parte do tratamento adesivo, a dentina dos grupos N, CHX.E e E foi, então, condicionada, lavada, seca e reidratada com 1,5 ?l, respectivamente, de água destilada (controle - Nc / CHX.Ec / Ec; n=8) ou de digluconato de CHX a 2% (primer antiproteolítico - Npa / CHX.Epa / Epa; n=8). O adesivo Adper Single Bond 2 foi aplicado em todos os espécimes e a porção coronária, reconstruída com a resina Filtek Z350. Após 24 h (água destilada/37?C), os espécimes foram seccionados em palitos e imediatamente testados (?TBS; 0,5 mm/min). As superfícies fraturadas de cada palito foram analisadas com o auxílio de um microscópio digital (50x de aumento). Um dente extra para cada grupo foi tratado exatamente como os outros, mas o corante fluorescente rodamina B foi previamente adicionado ao sistema adesivo para permitir a avaliação qualitativa da interface adesiva por meio de Microscopia Confocal de Varredura a Laser. Os valores de RU obtidos foram organizados de forma a se considerar cada dente como unidade experimental e, então, os testes de Análise de Variância a 2 critérios e de Tukey foram aplicados, com nível de significância de 5%. Apenas o substrato normal, quando reidratado, após condicionamento ácido, com água destilada (controle), é que apresentou valores de RU à dentina significativamente superiores aos demais. Quanto ao padrão de fratura dos palitos testados, as falhas adesivas e mistas foram predominantes em relação às coesivas, independentemente se em dentina ou em resina. Qualitativamente, a hibridização da dentina erodida pareceu menos satisfatória que a da dentina tratada com CHX como agente antiproteolítico para prevenção/controle do desgaste e então erodida, que, por sua vez, pareceu também menos satisfatória que a da dentina normal, particularmente se reidratadas com CHX a 2% como primer antiproteolítico. Ademais, a hibridização de cada substrato, individualmente, pareceu inferior quando da aplicação da CHX a 2% como primer antiproteolítico, se comparada com a da água destilada. Conclui-se, ora, que o uso da CHX como agente para prevenção/controle do desgaste erosivo parece, contrariamente ao que se observa para essa finalidade, não interessante de modo pleno quando, do ponto de vista da resistência de união, na sequência, há que se restaurar o substrato dentinário utilizando-se sistema adesivo do tipo condicione e lave simplificado em associação a resina composta. Quando aplicada como primer antiproteolítico, ainda, a CHX pode prejudicar a adesão à dentina normal, ainda que não o faça, nem o contrário, para a dentina tratada com CHX como agente antiproteolítico para prevenção/controle do desgaste e então erodida ou tão-somente erodida. / Even if when applied as a antiproteolytic primer the chlorhexidine (CHX) minimizes, although not indefinitely, degradation of adhesive interfaces, it seems not to be able to make bond strength (BS) values to eroded dentin equivalent to those to normal dentin. It may be possible, though, that its application as an agent for the prevention/control of the erosive wear influences the establishment of such interfaces, as it can completely inhibit the process. This in vitro study evaluated, thus, the role of CHX application as an antiproteolytic agent for preventing/controlling the wear, that is, the condition of the dentin substrate with respect to erosion,or as part of the adhesive treatment (antiproteolytic primer), on BS of a simplified etch-and-rinse adhesive plus a composite resin to dentin. In order to determine the different levels of the factor dentin substrate, superficial occlusal dentin of third molars (n=48) was only ground with a SiC paper (600-grit; 1 min), what was called normal dentin - N, or subsequently treated with 2% CHX digluconate (60 s) and eroded by a regular-cola soft-drink, determining the dentin treated with CHX to prevent/control the wear and then eroded - CHX.E, or only eroded by the soft-drink, establishing the eroded dentin - E. To determine the different levels of the factor CHX as part of the adhesive treatment, dentin of N, CHX.E and E groups was then acid-etched, washed, dried and rehydrated with 1.5 ?L, respectively, of distilled water (control - Nc / CHX.Ec / Ec; n=8) or of 2% CHX (antiproteolytic primer - Nap / CHX.Eap / Eap; n=8). Adper Single Bond 2 was applied in all specimens and resin composite buildups were constructed with Filtek Z350. After 24 h (distilled water/37?C), specimens were sectioned in beams and immediately tested (?TBS; 0.5 mm/min). Fractured surfaces of each beam were observed under a digital microscope (50X magnification). An extra tooth for each group was treated just like the others, but the fluorescent dye rhodamine B was previously added to the adhesive system to allow a qualitative evaluation of the adhesive interface by means of Laser Scanning Confocal Microscopy. Obtained BS values were organized in order to consider each tooth as an experimental unit and, then, two-way ANOVA and Tukey tests were applied, with a significance level of 5%. Only the normal substrate, when rehydrated, after acid etching, with distilled water (control), is the one that presented BS values to the dentin significantly higher to the others. As for the fracture pattern of the tested beams, adhesive and mixed failures were predominant in relation to the cohesive failures, regardless of whether in dentin or in resin. Qualitatively, the hybridization of eroded dentin appeared less satisfactory than that of the dentin treated with CHX to prevent/control the wear and then eroded, which, in turn, also appeared less satisfactory than that of normal dentin, particularly if rehydrated with 2% CHX as antiproteolytic primer. In addition, the hybridization of each substrate individually appeared to be inferior for the application of the 2% CHX as antiproteolytic primer, when compared to distilled water. It is concluded, therefore, that the use of CHX as an agent for preventing/controlling the erosive wear seems, contrary to what is observed for this purpose, not fully interesting when, from the point of view of the bond strength, in the sequence, the dentin substrate must be restored using a simplified etch-and-rinse adhesive system plus a composite resin. When applied as an antiproteolytic primer, moreover, CHX may impair bonding to normal dentin, even if not doing this, nor the opposite, for the dentin treated with CHX to prevent/control the wear and then eroded or for the only eroded dentin.

Made available in DSpace on 2014-07-29T15:07:52Z (GMT). No. of bitstreams: 1 dissertacao leonardo ciencia animal.pdf: 381642 bytes, checksum: 916af2bb18068d3efe78bdc7c8582617 (MD5) Previous issue date: 2009-10-29 / Myxomatous mitral valve degeneration (MMVD) or mitral valve endocardiosis is an endocardial age-related disease characterized by the mucopolysaccharide (MPS) accumulation and by collagen degeneration in mitral valves of many species, especially in humans, dogs and swine. Metalloproteinases (MMP) are proteolytic enzymes responsible for the extracellular matrix (ECM) remodelling in regular and pathological tissue. It has been suggested that the changing of the valvular distribution of these enzymes might be responsible for MMVD genesis. Likewise, vasoactive substances like nitric oxide (NO) played a role in the endocardiosis development. The aim of this study was to evaluate the histological changes, the expression of nitric oxide synthase (NADPH-d) and the immunohistochemical localization of MMP-2 and MMP-9 in the anterior leaflet in dogs and swine with regular mitral valves and those with endocardiosis. For this purpose, two experiments were done. First, 12 mitral valves of dogs and 22 of swine were analyzed. Valves were fixed in a 4% paraformaldehyde, exposed to NADPH-d reaction, processed routinely and microscopically evaluated for the detection of MPS deposition, collagen degeneration and fibrosis. In dogs, very high intensity reaction to NADPH-d was associated with higher endocardiosis degree and with presence of MPS deposition as well as collagen degeneration. There was no alteration in colour during the swine valves reaction to NADPH-d. In conclusion, NO works in canine mitral valves, remodeling MEC and playing a role in dogs mitral endocardiosis disease. In swine, it is suggested that NO has restricted action in MEC or there are major differences on the structures of swine valves because there was no reaction to NADPH-d and absence of macroscopical endocardiosis lesions. For the second study, 25 mitral valves of dogs and 32 valves of swine were also analyzed. Valves were macroscopically evaluated for the occurrence or not of endocardiosis. They were fixed in a 4% paraformaldehyde, routinely processed and submitted to immunohistochemical reaction and microscopically evaluated for the intensity of antigen labelling and for the number of positive cells, as well as MPS deposition, collagen degeneration and fibrosis. In dogs, DMVM is characterized by MPS accumulation. This collagen deposition and degeneration are directly related to the endocardiosis level. The MMP-2 and MMP-9 enzymes are involved in dogs myxomatous mitral valve degeneration process. In swine, even with the lack of microscopical endocardiosis there was observed some MPS deposition changes, especially in females. In these animals valves there are constitutive expression of MMP-2 and MMP-9, what suggests the action of these enzymes in the normal MEC mitral valvular remodeling in young and old female pigs / A degeneração mixomatosa da válvula mitral (DMVM) ou endocardiose mitral é uma doença do endocárdio valvular relacionada com a idade e caracterizada por acúmulo de mucopolissacarídeos (MPS) e degeneração do colágeno na mitral de várias espécies mamíferas, sendo observada com maior frequência em humanos, cães e suínos. Metaloproteinases (MMP) são enzimas proteolíticas dependentes do zinco, responsáveis pelo remodelamento da matriz extracelular (MEC) em processos normais e patológicos de vários tecidos. A alteração na expressão e a distribuição valvular dessas enzimas estão envolvidas na gênese da DMVM. Da mesma forma, substâncias vasoativas como o óxido nítrico (NO) tem papel importante no desenvolvimento da endocardiose. Assim, o objetivo deste estudo foi avaliar as alterações histológicas, a expressão da sintase de óxido nítrico (NADPH-d), e o padrão de marcação imunoistoquímica de MMP-2 e MMP-9 no folheto anterior da válvula mitral normal e com endocardiose de cães, de suínos jovens e matrizes. Para isso, realizaram-se dois experimentos. No primeiro foram utilizadas 12 mitrais de cães adultos a idosos, 22 válvulas de suínos, 10 de animais com idade de sete e oito meses, e 12 mitrais de matrizes entre cinco e seis anos. As válvulas foram avaliadas macroscopicamente quanto ocorrência ou não de endocardiose, colhidas, fixadas em paraformaldeído a 4%, submetidas à reação de NADPH-d, incluídas em parafina e avaliadas microscopicamente quanto à deposição de mucopolissacarídeos (MPS), degeneração do colágeno, fibrose e grau de endocardiose. Nas válvulas caninas quanto maior a intensidade da reação de NADPH-d, maiores eram o grau de endocardiose, a deposição de MPS e a degeneração do colágeno. Nas válvulas suínas não houve coloração à reação de NADPH-d. Com isso, concluiu-se que o NO atua na mitral canina, remodelando a MEC e participando da patogenia da endocardiose mitral nessa espécie. Na mitral suína sugere-se a ação restrita do NO no remodelamento da MEC ou diferenças estruturais na válvula desta espécie, já que não houve reação ao NADPH-d e lesões macroscópicas de endocardiose. No segundo estudo foram utilizadas 25 mitrais de cães adultos e idosos e 32 válvulas de suínos, sendo 10 de animais com idade de sete e oito meses, e 22 mitrais de matrizes entre cinco e seis anos. As válvulas foram avaliadas macroscopicamente quanto ocorrência ou não de endocardiose, fixadas em paraformaldeído a 4%, incluídas em parafina, submetidas à reação de imunoistoquímica e avaliadas microscopicamente quanto à intensidade de marcação e número de células marcadas, quanto à deposição de MPS, degeneração do colágeno e fibrose. Nos cães, concluiu-se que a DMVM é caracterizada por acúmulo de MPS e degeneração do colágeno, que estão diretamente relacionadas ao grau de endocardiose e, consequentemente, à gravidade da doença valvular. Ainda, as enzimas MMP-2 e MMP-9 estão envolvidas na degeneração mixomatosa da válvula canina. Em suínos, mesmo na ausência macroscópica de endocardiose podem ser observadas alterações como deposição de MPS, degeneração do colágeno e fibrose, sendo esta última evidente em matrizes. Também, nesses animais há expressão constitutiva de MMP-2 e MMP-9, sugerindo a participação dessas enzimas no remodelamento da MEC valvular mitral normal em suínos jovens e matrizes

A endometriose é uma doença de origem multifatorial, caracterizada por presença de tecido endometrial fora da cavidade uterina, responsável por sintomas álgicos com grande impacto na qualidade de vida da paciente, além de ser um dos principais fatores de infertilidade. Muitos estudos já foram realizados no intuito de explicar a etiopatogenia da endometriose, assim como muito tem sido estudado para encontrar novas estratégias de tratamento. Várias linhas de medicamentos têm sido estudadas com este intuito, agindo em diferentes pontos da etiopatogênese da doença, uma delas na inibição de metaloproteinases da matriz extracelular, que tem papel no remodelamento do mesotélio do peritônio e angiogênese. O objetivo deste estudo foi avaliar a influência de uma droga (doxiciclina) de baixo custo, com ação conhecida na inibição das metaloproteinases, em endometriose peritoneal induzida em ratas. Para isso, foram usadas 30 ratas adultas Wistar com lesão induzida de endometriose, divididas em três grupos, um grupo controle (C, n=10) sem tratamento, um grupo onde foi administrado doxiciclina em baixa dose (BD, n=10) e um grupo onde foi realizado doxiciclina em alta dose (AD, n=10). Foi realizada avaliação da área das lesões de cada rata e estudo imunohistoquímico para positividade de anticorpo primário de metaloproteinase de matriz 9 (MMP9) e de inibidor de metaloproteinase de matriz 2 (TIMP2). A doxiciclina atuou reduzindo a área das lesões nos grupos BD e AD (p=0,0052) em relação ao grupo C e reduzindo a expressão do TIMP2 no grupo AD (p=0,0009) em relação aos grupos BD e C. Não houve resultado significativo na expressão da MMP9. / Endometriosis is a multifactorial origin disease, characterized by the presence of endometrial tissue outside the uterine cavity, responsible for painful symptoms with important impact on the life quality of the patient, besides being one of the main factors of infertility. Many studies have already been carried out to explain the etiopathogenesis of endometriosis, and much has been studied to find new treatment strategies. Several lines of drugs have been studied for this purpose, acting at different points in the etiopathogenesis of the disease, one of them in the inhibition of extracellular matrix metalloproteinases, which plays a role in the remodeling of the peritoneum mesothelium and angiogenesis. The purpose of this study was to evaluate the influence of a low-cost drug (doxycycline), with known action on the inhibition of metalloproteinases, in induced peritoneal endometriosis in rats. Thirty adult Wistar rats with endometriosis-induced lesions were divided into three groups: one untreated control group (C, n = 10), one group receiving low dose doxycycline (BD, n = 10) and a group where high dose doxycycline (AD, n = 10) was performed. An evaluation of the lesion area of each rat and immunohistochemical study for primary antibody to matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase inhibitor 2 (TIMP2) was performed. Doxycycline worked by reducing the area of lesions in the BD and AD groups (p = 0.0052) in relation to the C group and reducing the expression of TIMP2 in the AD group (p = 0.0009) in relation to the BD and C groups. There was no significant effect on MMP9 expression in the present study.

L’endothélium préserve l'homéostasie vasculaire et tissulaire qui contrôle plusieurs processus physiologiques dans les corps humain. Lors de certaines pathologies, l'intégrité vasculaire peut être perturbée par une diversité de médiateurs de perméabilité qui interrompent la fonction barrière de cet épithélium, et provoquer des lésions tissulaires au cours de la progression de la maladie. Par conséquent, l'intégrité de la barrière endothéliale est essentielle pour l'homéostasie vasculaire. Dans le cas de la fièvre hémorragique due à la dengue, de nombreuses voies de signalisation induisent une perméabilité vasculaire qui résulte d'une rupture de la barrière hémato-encéphalique qui, dans certains cas, peuvent permettre la pénétration virale dans le système nerveux central (SNC). La plupart de ces voies de signalisation (y compris le TNF-alpha) sur-activent les enzymes appelées métalloprotéinases matricielles (MMP), qui induisent la dégradation de la matrice extracellulaire (MEC) et des protéines intercellulaires régissant la perméabilité vasculaire. Parmi ces enzymes, les MMP gélatinolytiques (MMP-9 et MMP-2) sont considérées comme la classe la plus importante de MMP puisqu'elles sont capables de dégrader le collagène de type IV, constituant principal de la MEC, et les jonctions cellulaires PECAM- 1 et VE-cadhérine. Ainsi, dans cette étude, nous avons développé deux approches pour inhiber l'activité de MMP-9 et protéger la perméabilité vasculaire in vitro. Ces approches incluent (i) des antagonistes de MMP-9 conçus in silico, tels que le composée HA048 (ii) des composés lipophénoliques synthétiques dérivés du resvératrol. Ce dernier existe naturellement dans les tiges de raisin, les arachides et plusieurs autres plantes et qui est capable d'inhiber l'expression et l'activité de la MMP-9. Bien que le resvératrol ait plusieurs activités biologiques testées in vitro, son utilisation dans des modèles animaux est limitée en raison de sa faible biodisponibilité, de son métabolisme rapide et de son élimination. Par conséquent, cette étude a trois objectifs principaux. Premièrement, de trouver un nouveau composé capable d'inhiber l'activité de MMP-9 in vitro. Deuxièmement, de vérifier s’il peut protéger l'intégrité de la couche endothéliale dans les HUVEC. Enfin, de passer à des études précliniques sur le modèle de souris dengue afin de vérifier son activité, sa toxicité et sa biodisponibilité. Outre le resvératrol et le SB-3CT (inhibiteur commercial de la MMP-9), nous avons constaté que le resvératrol-acide linoléique oméga 6 (RES-LA), le resvératrol-docosanoïque (RES-C22) et le HA048 ont montré la plus forte activité anti-MMP-9 parmi d'autres composés. Cependant, RES-LA a été préférentiellement choisi par rapport à RES-C22 en raison de sa meilleure solubilité. Par conséquent, nous avons utilisé un nouveau concept de formulation de ces composés en les solubilisant dans un solvant compatible et moins toxique pour des expériences in vivo, un solvant eutectique profond naturel (NADES), composé de 1,2-propanediol:ChCl: eau (1:1:1) (NADES/PCW). Les résultats de la perméabilité vasculaire in vitro ont révélé que les inhibiteurs de la MMP-9 ont diminué la perméabilité endothéliale exacerbée induite par le TNF-α dans les HUVEC, mesurée à la fois en temps réel et en fluorescence. En outre, l'examen microscopique des jonctions cellulaires adhérentes a montré qu’il y avait plus de CD31/PECAM-1 entre les HUVEC traitées à la fois par le TNF-α et les inhibiteurs de la gélatinase par rapport au témoin positif (TNF-alpha).Le mécanisme d'action des lipophénols dérivés du resvératrol a été évalué. Nos résultats ont révélé que les lipophénols dérivés du resvératrol ont inhibé l'expression de la MMP-9 en atténuant la phosphorylation des kinases MAPK ERK1/2 et JNK1/2. / The endothelial barrier preserves the vascular and tissue homeostasis that controls several physiological processes inside the body. In pathologies, vascular integrity could be disrupted by a diverse of permeability mediators that interrupt the barrier function and cause tissue damage during disease progression. Therefore, endothelial barrier integrity is critical for vascular homeostasis. Although the mechanisms leading to vascular leakage have been studied over several past decades, recent approaches have pointed new therapeutic targets in pre-clinical studies. In the pathogenesis of dengue hemorrhagic fever (DHF), many signaling pathways induce vascular permeability that result of disruption of blood brain barrier, that in some cases could allow viral penetration into the central nervous system (CNS). Most of these signaling pathways (including TNF-alpha) activate the overproduction active enzymes called matrix metalloproteinases (MMPs) that induce degradation of the extracellular matrix (ECM) and intercellular proteins governing vascular permeability. Among these enzymes, gelatinolytic MMPs, (MMP-9 and MMP-2), which are considered the most important class of MMPs since they are capable of degrading collagen type-IV, the main constituent of ECM, and cell junctions (PECAM-1 and VE-cadherin). Thus, in this study, we have developed two approaches to inhibit MMP-9 activity and protect the vascular permeability in vitro. Those approaches include, (i) in silico designed MMP-9 antagonists, where we had determined a lead MMP-9 blocker (HA048). The second approach is to synthesize derived lipophenolic compounds from resveratrol, that naturally exists in grape stalks, peanuts, and several other plants, which is capable of inhibiting the expression and activity of active MMP-9 inside the cells. Despite that resveratrol has several biological activities tested in vitro, its use in animal models is limited due to its poor bioavailability, rapid metabolism, and elimination. Therefore, this study has three main goals. First, is to find a novel compound capable of inhibiting MMP-9 activity in vitro. Second, is to verify whether it can preserve the endothelial monolayer integrity. Finally, is to move to the preclinical studies in dengue mouse model to verify its activity in vivo, toxicity, and bioavailability.Besides resveratrol and SB-3CT (commercial MMP-9 inhibitor), we found that resveratrol-linoleic acid, omega-6 (RES-LA), resveratrol-Docosanoic acid (RES-C22) and HA048 (in silico designed inhibitor) have demonstrated the highest MMP-9 inhibitory activity among dozens of synthesized compounds. However, RES-LA was preferably selected over RES-C22 due to for solubility reasons. Moreover, we formulated these compounds in novel solvents that could be compatible and less toxic for in vivo experiments, which is known as natural deep eutectic solvent (NADES) based on 1,2-propanediol:ChCl:water (1:1:1) (NADES/PCW).Results of in vitro vascular permeability revealed that MMP-9 inhibitors have decreased TNF-α induced-exacerbated endothelial permeability in HUVEC, measured in both real-time impedance sensing and fluorescence count. In addition, microscopic examination of cell junction proteins showed that CD31/PECAM-1 were more adherent and attached between HUVEC treated with both TNF-α and gelatinase inhibitors comparing to the positive control (TNF-alpha).The mechanisms of action of resveratrol derived lipophenols were assessed. Our findings revealed that resveratrol derived lipophenols have inhibited MMP-9 expression by attenuating the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinases (JNK) mitogen-activated protein kinases (MAPK).

Les mécanismes de reconnaissance moléculaire sont à la base de nombreuses fonction biologiques essentielles (transduction du signal, régulation de l’expression génique, stimulation du système immunitaire,...). La compréhension des phénomènes physiques et chimiques à la base de ces phénomènes est fondamentale pour de nombreuses applications telles que la conception de médicaments, le développement d’outils diagnostiques ou tout autre procédé biotechnologique. Dans cette étude, nous avons étudié de manière extensive l’interaction entre la métalloélastase du macrophage (MMP-12) et le RXP470.1, un inhibiteur puissant et sélectif. En combinant des approches de cristallographie, de microcalorimétrie (ITC) et des tests enzymatiques, nous avons pu quantifier l’importance énergétique du transfert d’un seul proton suite à la liaison du RXP470.1, et mettre en lumière l’importance des contributions entropiques. Ainsi, la protonation du Glu219, un résidu catalytique, permet de compenser une enthalpie de liaison intrinsèque défavorable. Cette protonation est rendue possible par le large shift de pKa que subit le Glu219 en réponse à la liaison du RXP470.1 (pKalibre = 5.7 ± 0.1 / pKalié = 10 ± 0.04). Enfin, cette étude est la première, à notre connaissance, ayant combiné données d’affinité, thermodynamiques et structurales pour aborder le rôle du groupe chélatant. Nous avons ainsi étudié deux analogues du RXP470.1 variant seulement par la nature de leur pince à zinc. Ces modifications se traduisent par un effet marqué sur les profils d’affinité et de sélectivité ainsi que sur la signature énergétique des composés étudiés. L’étude des facteurs B, associés à l’analyse des structures cristallographiques, de ces inhibiteurs en complexe avec la MMP-12, suggère que des différences mineures de structures peuvent engendrer des variations de mobilité importantes au niveau des résidus impliqués dans l’interaction inhibiteur - enzyme. Ces différences trouvent leur origine dans un positionnement très légèrement différent du groupe chélatant par rapport au zinc.Pris dans leur ensemble, ces résultats pointent la nécessité de combiner un ensemble d’approches expérimentales pour décrire la complexité des interactions protéine/ligand. Ces associations doivent permettre d’évaluer le potentiel de méthodes théoriques capables de décrire des systèmes complexes. / Protein-ligand recognition mechanisms are essential to many fundamental biological functions such as signal transduction, gene regulation or stimulation of the immune system. Understanding the physical and chemical phenomenon upon protein-ligand binding is essential for many practical applications such as drug design, ligand based diagnostic tools and any other study based on biotechnology. In this study, we extensively explored the interaction between human macrophage metallo elastase MMP-12 and RXP470.1, a potent and selective inhibitor. By combining high resolution X-ray crystallography, FRET based enzyme assays and Isothermal Titration Calorimetry, we were able to highlight the importance of entropic contributions and to quantify the importance of a single proton transfer upon RXP470.1 binding. We show, here, how the protonation of Glu219 upon RXP470.1 binding rescues an otherwise unfavourable binding enthalpy. This protonation is made possible by the large pKa shift Glu219 undergoes as RXP470 enters MMP12h To our knowledge, this study is also the first to address the zinc binding group effect from affinity, thermodynamlc and structural data. We tested two RXP470 analogues, which only differ by their zinc-binding group. We show that this, apparently minor, change has great consequences regarding their affinity profiles and thermodynamic signatures. In addition, the analysis of the b factors, associated to the X-ray structures of these compounds in complex with MMP-12, suggests that small modifications of the zinc binding group might imply important mobility variations of the residues involved in the protein-ligand interaction. These modifications are initiated by a small shift of the zinc binding groups positioning in the active site.Taken together, these results point toward the necessity to combine several experimental approaches to describe the complexity of protein-ligand interactions. These associations should allow the evaluation of new theoretical methods able to describe complex systems.