Introduction: Severe ischemia-reperfusion injury (IRI) after liver transplantation are correlated with lower early and late graft and patient survival, higher infection rates, appearance of acute and chronic rejections and other complications. In this field, it has been demonstrated that the prostaglandin E1 (PGE1) presents cytoprotective, anti-inflammatory and vasodilator effects. Therefore, the PGE1 assembles most of the requirements necessary to prevent the development of the ischemia-reperfusion injury, especially if it is administered through via portal vein at full doses in the moment of graft reperfusion.Materials and methods: Female Largewhite-Landrace pigs (body weight between 30-35 kg) were used. The orthotopic liver transplantation were performed in 18 pairs of pigs. The study was divided into three groups as follows: Control group, 4 experiments of orthotopic liver transplantation with a cold preservation time of 3 hours in Wisconsin solution; Groups A and B, 7 experiments with 24 hours of cold preservation time in each group. Concerning intraportal infusion of medication, the Group B was infused with prostaglandin E1 at a dose of 0.15 ug/kg/min (Alprostadil 500ug) for 90 minutes in the portal reperfusion of the graft; and Group A was infused with saline solution for 90 minutes in the same time period. In the recipient surgery, biochemical (GOT, GPT, LDH, B-Galactosidase, Hialuronic Acid), hemodynamic and histological studies were carried out to evaluate the ischemia-reperfusion injury (IRI).Results: Survival rate in control group was 100 % and it presented a lower level of sinusoidal-hepatocellular injury and leukocyte activation than the groups A and B. From the beginning of the surgery until the moment of the PGE1 administration, both the group A and the group B were similar in relation to the histology, hepatocellular, endothelial and inflammatory damage markers. After the infusion of the medication (PGE1), the group B developed an increase of the total hepatic flow and a lower alteration of all the parameters of hepatocyte-sinusoidal injury and hepatic function, with an statistical significance in the great majority of them. The portal PGE1 infusion at full doses did not generate hemodynamic instability. Conclusions: The liver transplantation in pigs with 24 hours of cold ischemia of the graft produced a severe IRI that caused the death of all the animals in the groups A and B due to primary graft nonfunction. Nevertheless, with the intraportal PGE1infusion, the group B presented an increase of the total hepatic flow and a lower alteration of all the parameters of hepatocellular injury, sinusoidal damage and hepatic function. The portal PGE1 perfusion at full doses was well tolerated at hemodynamic level.

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No. of bitstreams: 1 cuervo_jco_me_bot.pdf: 1620368 bytes, checksum: d662e09785a2bb73aaa15aaf92df4d19 (MD5) Previous issue date: 2016-09-29 / Em ruminantes a luteólise natural é caraterizada pela liberação de vários pulsos de prostaglandina F2alfa (PGF) produzidos pelo útero. A PGF é o hormônio luteolítico, enquanto a prostaglandina E1 (PGE1) é considerada um mediador luteoprotetor. Em estudos anteriores, infusões com doses baixas de PGF no útero com intervalos de 6 horas (h) resultou em regressão do corpo luteo (CL). A proposta deste experimento é desenvolver um modelo para avaliar o efeito de baixas doses de PGE1, também infundidas no lúmen uterino sobre a resposta luteal à PGF intrauterina (IU). Vacas no dia 10 do ciclo estral receberam infusões IU de salina (0,1 ml de salina + 0,1 ml de DMSO), PGE (2 mg de PGE1 em 0,1ml de DMSO) ou PGF (0,25 mg of PGF em 0,1 ml de salina) em intervalos de 6 h em um desenho experimental 2 X 2. Portanto os animais foram agrupados em quatro tratamentos: SALINA (4 infusões de salina; n=5), PGE (4 infusões de PGE1; n=5), PGF (4 infusões de PGF; n=5) e PGE+PGF (4 infusões de PGE1+PGF; n=5). As concentrações plasmáticas de progesterona (P4) foram dosadas por radioimunoensaio e o volume luteal foi determinado por ultrassonografia transretal. As concentrações circulantes de PGFM e PGEM foram dosadas antes e 10 minutos após as primeiras duas infusões. Biopsias luteais foram coletadas de cada vaca 30 minutos após cada infusão para determiner a expressão de genes em resposta a cada tratamento. As concentrações circulantes de PGFM 10 minutos após as infusões foram maiores em vacas que receberam tratamentos com PGF e PGE+PGF em comparação com as vacas tratadas com salina e PGE. Da mesma forma, as concentrações de PGEM 10 minutos após cada infusão foram maiores em vacas tratadas com PGE e PGE+PGF em comparação com vacas dos grupos salina e PGF. As concentrações de P4 diminuiram no grupo PGF em comparação com o grupo Salina no tempo 12-h (48,9% do controle) após a primeira infusão de PGF, no tempo 24-h (20,2% do controle), e em todos tempos subsequentes (P < 0,05). Não foram encontradas diferenças nas concentrações circulantes de P4 entre os grupos Salina, PGE e PGF+PGE. Houve também uma diminuição do volume luteal entre o grupo PGF e os outros três grupos que foi observada nos tempos 24-h (56,4% do controle), 48-h (30,6% do controle), e 72-h (20,4% do controle) após o tratamento com PGF (P<0.05). Não houve diferenças no volume luteal entre os tratamentos salina, PGE e PGE+PGF. Por tanto, infusões IU simultâneas de baixas doses de PGE1 bloquearam a ação luteolitica de pulsos IU de PGF em vacas, como observado nas mudanças circulantes de P4 e volume luteal. Análises da expressão génica nas biopsias luteais coletadas após o terceiro pulso de PGF, indicam o padrão tipico de expressão de genes em resposta ao tratamento com PGF (FGF2, EGR1, FOS e FAS aumentaram; PTGFR, VEGFA, NR5A1 e STAR diminuiram) e o tratamento PGE+PGF bloqueou completamente as mudanças na expressão destes genes. Infusões IU de PGF e PGE1 parecem ser um excelente modelo para determiner o padrão de expressão de genes envolvidos no efeito luteoprotetor da PGE1. / In ruminants, natural luteolysis is characterized by the release of several pulses of prostaglandin F2alpha (PGF) produced by the uterus. Prostaglandin F2alpha is the luteolytic hormone, whereas prostaglandin E1 (PGE1) is considered to be a luteoprotective mediator. In previous studies, low doses of PGF infused into the uterus at 6 hour (h) intervals resulted in regression of the corpus luteum (CL). This study was designed to develop a model to study the effect of low doses of PGE1, also infused into the uterine lumen, on the luteal responses to intrauterine (IU) PGF. Cows on day 10 of the estrous cycle received IU infusions of saline (0,1 ml of saline + 0,1 ml of DMSO), PGE (2 mg of PGE1 in 0,1ml of DMSO) or PGF (0,25 mg of PGF in 0,1 ml of saline) at 6-h intervals in a 2 X 2 experimental design. Thus, there were four treatment groups: SALINE (4 saline infusions; n=5), PGE (4 PGE1 infusions; n=5), PGF (4 PGF infusions; n=5), and PGE+PGF (4 PGE1+PGF infusions; n=5). Radioimmunoassay was used to measure circulating progesterone (P4) concentrations and luteal volume was determined by transrectal ultrasonography. Circulating concentrations of PGFM and PGEM were measured before and 10 minutes after the first two infusions. A luteal biopsy was collected from each cow at 30 minutes after each infusion for later determination of gene expression in response to each treatment. Circulating concentrations of PGFM 10 minutes after infusions were greater in cows receiving treatments with PGF and PGE+PGF than in Saline or PGE-treated cows. In the same way, concentrations of PGEM 10 minutes after infusions, were greater in cows that were treated with PGE and PGE+PGF than in saline and PGF-treated cows. Concentrations of P4 in the PGF group decreased compared to those in the saline group by 12-h (48.9% of control) after first infusion of PGF, at 24-h (20,2% of control), and all subsequent time points (P < 0,05). No differences in circulating P4 concentrations were found between Saline, PGE, and PGF+PGE. There was also a decrease of luteal volume between the PGF group and the other three groups that was detectable at 24 (56,4% of control), 48 (30,6% of control), and 72 (20,4% of control) h after PGF treatment (P<0.05). There were no differences in luteal volume between Saline, PGE, or PGE+PGF. Thus, simultaneous IU infusion of a low dose of PGE1 blocked the luteolytic actions of IU PGF pulses in cattle, as measured by changes in circulating P4 and luteal volume. Analyses of gene expression in the luteal biopsy taken after the third PGF pulse indicate a typical pattern of gene expression in response to the PGF treatments (FGF2, EGR1, FOS and FAS increased; PTGFR, VEGFA, NR5A1 and STAR decreased) and that simultaneous PGE1 treatment completely blocked these gene expression changes. Thus, IU infusion of PGF and PGE1 seems to provide an excellent model for determining the patterns of gene expression involved in the luteoprotective effect of PGE1.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17993号 / 生博第296号 / 新制||生||39(附属図書館) / 80837 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 杉田 昌彦, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Ubiquitin is a 76 amino acid long polypeptide, which is present throughout eukaryotes in a highly conserved fashion. Ubiquitin can modify proteins by becoming covalently attached to them. Eukaryotic cells employ ubiquitin to maintain and regulate fundamental cellular processes like protein degradation, the immune response and transcriptional and translational regulation. Transfer of ubiquitin to the substrate is achieved by the catalysis of three classes of enzymes namely E1, E2 and E3. Together these enzymes form a pyramidal hierarchy, where E1 stands at the apex and E3 enzymes form the base of the pathway. The ubiquitin activating enzyme 1 (UBA1) plays a major role in ubiquitylation being the ubiquitin-dedicated E1 enzyme. In addition, it is the only enzyme in this pathway to use ATP as an energy source to catalyze two important reactions. The products of these reactions, ubiquitin adenylate and ubiquitin thioester, are the essential intermediate states of ubiquitin, for being conjugated to the target protein. With the help of X-ray crystallography and biochemical approaches, snapshots of multiple catalytic states of UBA1, where it is bound to Mg-ATP, ubiquitin and the E2 Ubc13 as substrates could be captured. With the help of these high-resolution crystal structures, deeper insights into the enzymatic mechanism of UBA1 could be attained. The resulting insights into the catalytic cycle were further validated by biochemical assays. It could be shown that ATP acts as a molecular switch to induce the enzyme’s open conformation. Ubiquitin-binding to the enzyme leads to domain rotations, which facilitate the recruitment of a cognate E2 enzyme. The interdomain communication as well as the cross-talk with the substrates and the products fuel the enzymatic cycle of UBA1. Due to the proven efficacy of proteasome inhibitors for cancer treatment, which block degradation of proteins labeled with ubiquitin, enzymes participating in the ubiquitylation cascade have been targeted by researchers for the development of novel anti-cancer therapeutics. UBA1 inhibition has been shown to preferentially induce cell death in malignant cells, and it can also be used as a strategy to overcome resistance against proteasome inhibitors. MLN7243, an adenosyl sulfamate inhibitor developed by Millenium Pharmaceutical to specifically target UBA1, is currently in Phase-I clinical trials for the treatment of solid tumors. UBA1 could be crystallized in complex with three adenosyl sulfamate inhibitors covalently linked to ubiquitin, which are promising drug candidates for cancer therapy. The inhibitors employed, MLN7243, MLN4924 and ABPA3, show distinct specificities towards different E1 enzymes. With the help of crystal structures the specificity determinants of these inhibitors could be deciphered, which were further confirmed by inhibition assays as well as molecular dynamics simulations. Together these crystal structures provide a starting point for developing E1-specific inhibitors, which, besides their potential for medicinal purposes, are important tools to better understand the function of the ubiquitin system as well as the action of ubiquitin-like proteins. / Ubiquitin ist ein 76 Aminosäuren langes Polypeptid, das in allen Eukaryoten vorkommt und hoch konserviert ist. Ubiquitin kann Proteine modifizieren indem es mittels einer kovalente Bindung an diese angeheftet wird. Eukaryotische Zellen nutzen Ubiquitin, um fundamentale zelluläre Prozesse wie den Proteinabbau, die Immunantwort sowie die Regulation der Transkription und Translation aufrecht zu erhalten. Der Transfer von Ubiquitin auf das Substrat wird durch die Katalyse von drei Enzymklassen E1, E2 und E3 erreicht. Zusammen bilden diese Enzyme eine pyramidale Hierarchie in der das E1 an der Spitze steht und die E3 Enzyme die Basis bilden. Das Ubiquitin-aktivierende Enzym 1 (UBA1) ist das E1 Enzym für Ubiquitin und spielt somit eine Hauptrolle in der Ubiquitinierung. Weiterhin ist es das einzige Enzym dieses Stoffwechselweges, das ATP als Energie nutzt, um zwei wichtige Reaktionen zu katalysieren. Die Produkte dieser Reaktionen, Ubiquitin-Adenylat und thioesterifiziertes Ubiquitin, sind die essentiellen Ubiquitinintermediate für die Konjugation an das Zielprotein. Mit Hilfe der Röntgenstrukturanalyse und biochemischer Ansätze konnten für UBA1 Momentaufnahmen multipler katalytischer Zustände erfasst werden, in denen es an die Substrate Mg-ATP, Ubiquitin und dem E2 Enzym Ubc13 gebunden vorliegt. Mit Hilfe dieser hochaufgelösten Kristallstrukturen konnten tiefere Einblicke in den enzymatischen Mechanismus des Enzyms erzielt werden. Die gesammelten Erkenntnisse zum katalytischen Zyklus wurden mittels biochemischer Methoden validiert. Es konnte gezeigt werden, dass ATP als molekularer Schalter fungiert, um das Enzym in seine offene Konformation zu überführen. Die Bindung von Bindung an das Enzym führt zur Rotation einzelner Domänen welche die Rekrutierung des E2 Enzymes erleichtern. Die Interdomänen-Interaktion sowie molekulare Wechselwirkungen mit den Substraten und Produkten treiben den enzymatischen Zyklus von UBA1 an. Die Einsatz von Proteasominhibitoren, die den Abbau von Ubiquitin-markierten Proteinen blockieren, in der Krebstherapie weckte das Interesse von Forschern Enzyme, die an der Ubiquitinierungs-Kaskade beteiligt sind, als neue therapeutische Ziele zur Bekämpfung von Krebserkrankungen zu erschließen. Es konnte gezeigt werden, dass die Inhibierung von UBA1 bevorzugt den Tod maligner Zellen induziert und als Strategie für die Überwindung der Resistenz von Proteasominhibitoren genutzt werden kann. MLN7243, ein Adenosyl-Sulfamat Inhibitor, der von Millenium Pharmaceuticals entwickelt wurde und spezifisch UBA1 angreift, befindet sich gegenwärtig in klinischen Studien der Phase I mit dem Ziel einer Behandlung von soliden Tumoren. UBA1 konnte im Komplex mit drei an Ubiquitin gekoppelten Adenosyl-Sulfamat Inhibitoren, die vielversprechende Wirkstoffe in der Krebstherapie sind, kristallisiert werden. Die Inhibitoren MLN7243, MLN4924 und ABPA3 besitzen unterschiedliche Spezifitäten für verschiedene E1 Enzyme. Mit Hilfe von Kristallstrukturen konnten Spezifitätsfaktoren dieser Inhibitoren entschlüsselt werden, die im Weiteren mittels Inhibierungstests und molekulardynamischer Simulationen bestätigt werden konnten. Diese Kristallstrukturen lieferten ein klareres Bild für die Entwicklung E1-spezifischer Inhibitoren mit deren Hilfe, neben ihrer potentiellen medizinischen Anwendung, ein besseres Verständnis des Ubiquitinsystems und Ubiquitin-ähnlicher kovalenter Verknüpfungen gewonnen werden kann.

Ruminococcus gnavus E1 appartient au groupe des Firmicutes, l’un des deux groupes dominants du microbiote intestinal humain. Les a-galactosidases sont des glycosides hydrolase (GH) actives sur des substrats contenant des galactoses liés en a. Elles sont très largement distribuées dans tous les domaines du vivant, bactéries, champignons, plantes et animaux, mais sont absentes du tractus digestif humain. Ces travaux portent sur les caractéristiques enzymatiques et la régulation de l’expression de deux &#61537;-galactosidase, Aga1 et Aga2, de R. gnavus E1. L’analyse bioinformatique de leur environnement génétique respectif indique une organisation simple pour Aga1 tandis qu’Aga2 est organisée en opéron. Elles ont été exprimées en système hétérologue chez E. coli, purifiées et leurs propriétés biochimiques ainsi que leurs spécificités de substrat ont été analysées. Le profil de croissance de la souche indique une préférence pour des substrats complexes (raffinose et mélbiose) faisant intervenir les a-galactosidase pour leurs utilisations ainsi que leur assimilation. / Ruminococcus gnavus E1 belongs to the Firmicutes, one of the two dominant groups in the human gut microbiota. a-galactosidases are glycoside hydrolases (GH) active on a-galactoside containing substrates. They are widely distributed through all the domains of life: bacteria, fungi, plants, and animals, but are absent from the human gastro-intestinal tract.Here we report the enzymatic characteristics and regulation of expression for two GH36 &#61537;-galactosidases, Aga1 and Aga2, from R. gnavus E1. Bioinformatics analysis of their respective genetic environment showed a different organisation, Aga1 having a simple organisation while Aga2 is organised as part of an operon. They were heterologously expressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. The growth pattern of the strain in minimum media demonstrates a preference for complex substrates (melibiose and raffinose) that require the expression of the a-galactosidases for their utilisation and assimilation

L'objectif du groupe Im3i est d'étudier les mécanismes moléculaires impliqués dans la relation symbiotique entre l'hôte et le microbiote intestinal afin de proposer des solutions pour le traitement de certaines pathologies humaines dans le contexte de la nutrition et de la sécurité alimentaire. Nous avons choisi une bactérie à Gram positif, Ruminococcus gnavus E1 , comme modèle d'étude. Cette bactérie, anaérobie stricte, a été isolée à partir du microbiote d'un adulte sain et son génome a été séquencé . L'étude in vivo a été menée sur des groupes de souris mono-colonisées par R. gnavus E1. Nos résultats montrent clairement une surexpression des gènes codant les fucosyltransférases et sialyltransférases intestinales au niveau du côlon. Les observations réalisées en microscopie confocale à l'aide de lectines ont confirmé ces résultats. L'étude in vitro nous a permis d'analyser l'impact des facteurs solubles présents dans le surnageant de R. gnavus E1 sur la glycosylation des cellules épithéliales intestinales. La caractérisation préliminaire de cette fraction soluble indique qu'elle est thermorésistante, de nature non lipidique et qu'elle possède une faible masse moléculaire (< 3 kDa). Nous pouvons conclure que les variations d'expression des ARNm des différentes glycosyltransférases des cellules en gobelet et des entérocytes entrainant un programme de réinitialisation des glycoprotéines du mucus impacté par R. gnavus E1. Cela ouvre des perspectives considérables dans le dialogue moléculaire qui s'établit entre l'hôte et les bactéries endogènes. / The aim of IM3I group is study molecular mechanisms involved in the symbiotic relationship between the host and the intestinal microbiota, accounting for both partners, in order to propose solutions to human health problems, especially in the context of nutrition and food safety. We have chosen a Gram positive bacteria, Ruminococcus gnavus E1, as a model. This bacterium, in strict anaerobia, is isolated from the dominant microbiota of healthy human for which the genome is currently being sequenced. In vivo, using animals that are mono-associates with R. gnavus E1, our RT-qPCR studies clearly show an up regulation of the expression of genes coding for intestinal fucosyltranferases and sialyltransferases, in the colon. This is also confirmed by confocal microscopy using lectins. Thus, R. gnavus E1 is able to reestablish the glycosylation profile. In vitro, we have studied the impact of soluble factors from the R. gnavus E1 supernatant. We found that the soluble supernatant fraction of R. gnavus E1 cultures differentially modulates the intestinal glycosylation process in HT29-MTX, Caco-2, independently or coculture 75% Caco-2 and 25% HT29-MTX cell lines. Preliminary characterization of this soluble fraction indicates that it is heat resistant, is not affected by elimination of lipids, and has a low molecular weight form (< 3 kDa). We conclude that the variation of expression of mRNAs coding for different glycosyltransferases in goblet cells and enterocytes proves the re-initiating program of glycoproteins and mucus protection layer by the dominant bacteria R. gnavus E1 and opens the prospect to a new host-indigenous bacteria molecular crosstalk in the intestine.

Sabe-se que o flúor causa alterações químicas e celulares no osso, dependendo da dosagem e do tempo de exposição a esse elemento. Já foi demonstrado que alterações clínicas importantes, como a osteoporose, osteopetrose e esclerose óssea, podem ser geradas pela ingestão de flúor. Sabe-se que o flúor pode guiar os osteoblastos ao crescimento, sob doses e tempos de exposição específicos. A sensibilidade dos tecidos mineralizados ao fluoreto responde a mecanismos genéticos inerentes a cada população. Dentro de uma mesma espécie, diferentes linhagens apresentam um comportamento distinto frente a esse fármaco. Durante várias décadas, os estudos com fluoretos resumiram-se ao fluoreto de sódio, amplamente utilizado na prevenção das cáries dentárias. Recentemente, outras fontes de fluoretos como TiF4 e SnF2 têm sido buscadas, com intuito de potencializar as vantagens desse elemento. Porém não existem estudos ainda sobre o efeito do AlF3 sobre o tecido ósseo até a presente data. Desse modo, achamos oportuno investigar seu papel, quando administrado a células ósseas e para tal, investigamos a viabilidade e padrão de atividade das fosfatases desse sal (AlF3) quando administrado às células da linhagem pré-osteblástica MC3T3, em comparação ao NaF. Além disso, sabendo que diferentes células apresentam comportamento distinto ao tratamento com fluoretos, estudamos o papel do NaF quando administrado a duas linhagens celulares de camundongos com densidades ósseas distintas e com diferentes susceptibilidades ao fluoreto: C3H/HeJ e C57BL/6J (C3H e C57, alta e baixa densidades ósseas, respectivamente). As células também foram avaliadas quanto à viabilidade celular e ao perfil das fosfatases. Todos os resultados foram submetidos à Análise de Variância a três critérios e nos permitiu identificar que entre os sais de fluoreto, apenas o NaF promoveu alterações na viabilidade celular das células MC3T3. O AlF3 não exerceu influência na atividade da fosfatase alcalina quando consideramos as células pré-osteoblásticas, no entanto, o efeito desse fluoreto sobre a atividade das fosfatases ácidas totais e ácida resistente ao tartarato ocorre de modo dicotômico. Quando consideramos uma população celular heterogênea, coletada de calvária, como foi o caso das linhagens C3H e C57, verificamos que o NaF interferiu tanto sobre a proliferação, quanto a atividade das fosfatases de modo bastante distinto para ambas linhagens celulares. Assim concluimos que o NaF é capaz de exercer efeitos mais diversificados em diferentes tipos celulares, alterando viabilidade celular e atividade de enzimas; em contrapartida, o AlF3 produz efeitos mais especifícos e delimitados em pré-osteoblastos. / It is well known that fluoride causes chemical and cellular alteration on bone tissue, depending on the dosage and time of exposure to this element. Several studies have been conducted to show that some bone diseases, such as osteoporosis, osteopetrosis and esclerosis, can occur as a consequence of the excessive fluoride uptake. Fluoride can lead osteoblast to proliferate, under certain conditions as time and dosage. The same way, excessive dosages of fluoride can alter protein activity in osteoblasts, by changing the expression of retated genes. Sensitivity of mineralized tissues to fluoride depends on a genetic background inherent to each population. Within the same specie, different lineages present distint behavior to the ingestion of this íon. Over several decades, many researches focused on the study of NaF as the only source of fluoride, once it is wide spread used to the prevention of dental caries. More recently, different sources of fluoride have been assessed, like TiF4 e SnF2</sub, with the main aim to enhance biochemical and physical benefits of this element. Despite of this, none of these formulations have been tested to evaluate their effect on the bone tissue. Thus, we believe it is advisable to investigate viability and phosphatases activities of pre-osteoblast MC3T3 cells treated with Aluminium fluoride, compared to NaF treatments. Besides this, facing the knowledge that different cell lines have distinct behavior when treated with fluoride, we used two inbred strains of mice with distint mineral bone densities, C3H/HeJ (C3H, high bone density) and C57BL/6J (C57, low bone density), to assess the viability and phosphatases activities after the treatment with NaF. Overall results were tested by 3 way- Anova. AlF3 did not exibit influence over on alkaline phosphatase when pre-osteoblast cells are considered, but the effect of this fluoride salt on the activity of total and tartarate resistant acid phosphatases ocurrs on a dicotomic way. When we consider a mixed cell population, collected from calvária as for the C3H and C57 assays, NaF did interfered as with the proliferation rate as phosphatase activities on a very distinct fashion between the two lineages. Thus, it is possible to conclude that NaF is able to exert variable effects in different cell types and enzyme activities; on the other hand, AlF3 effects are more specific and surrounded in pre-osteblastic cells.

Sabe-se que o flúor causa alterações químicas e celulares no osso, dependendo da dosagem e do tempo de exposição a esse elemento. Já foi demonstrado que alterações clínicas importantes, como a osteoporose, osteopetrose e esclerose óssea, podem ser geradas pela ingestão de flúor. Sabe-se que o flúor pode guiar os osteoblastos ao crescimento, sob doses e tempos de exposição específicos. A sensibilidade dos tecidos mineralizados ao fluoreto responde a mecanismos genéticos inerentes a cada população. Dentro de uma mesma espécie, diferentes linhagens apresentam um comportamento distinto frente a esse fármaco. Durante várias décadas, os estudos com fluoretos resumiram-se ao fluoreto de sódio, amplamente utilizado na prevenção das cáries dentárias. Recentemente, outras fontes de fluoretos como TiF4 e SnF2 têm sido buscadas, com intuito de potencializar as vantagens desse elemento. Porém não existem estudos ainda sobre o efeito do AlF3 sobre o tecido ósseo até a presente data. Desse modo, achamos oportuno investigar seu papel, quando administrado a células ósseas e para tal, investigamos a viabilidade e padrão de atividade das fosfatases desse sal (AlF3) quando administrado às células da linhagem pré-osteblástica MC3T3, em comparação ao NaF. Além disso, sabendo que diferentes células apresentam comportamento distinto ao tratamento com fluoretos, estudamos o papel do NaF quando administrado a duas linhagens celulares de camundongos com densidades ósseas distintas e com diferentes susceptibilidades ao fluoreto: C3H/HeJ e C57BL/6J (C3H e C57, alta e baixa densidades ósseas, respectivamente). As células também foram avaliadas quanto à viabilidade celular e ao perfil das fosfatases. Todos os resultados foram submetidos à Análise de Variância a três critérios e nos permitiu identificar que entre os sais de fluoreto, apenas o NaF promoveu alterações na viabilidade celular das células MC3T3. O AlF3 não exerceu influência na atividade da fosfatase alcalina quando consideramos as células pré-osteoblásticas, no entanto, o efeito desse fluoreto sobre a atividade das fosfatases ácidas totais e ácida resistente ao tartarato ocorre de modo dicotômico. Quando consideramos uma população celular heterogênea, coletada de calvária, como foi o caso das linhagens C3H e C57, verificamos que o NaF interferiu tanto sobre a proliferação, quanto a atividade das fosfatases de modo bastante distinto para ambas linhagens celulares. Assim concluimos que o NaF é capaz de exercer efeitos mais diversificados em diferentes tipos celulares, alterando viabilidade celular e atividade de enzimas; em contrapartida, o AlF3 produz efeitos mais especifícos e delimitados em pré-osteoblastos. / It is well known that fluoride causes chemical and cellular alteration on bone tissue, depending on the dosage and time of exposure to this element. Several studies have been conducted to show that some bone diseases, such as osteoporosis, osteopetrosis and esclerosis, can occur as a consequence of the excessive fluoride uptake. Fluoride can lead osteoblast to proliferate, under certain conditions as time and dosage. The same way, excessive dosages of fluoride can alter protein activity in osteoblasts, by changing the expression of retated genes. Sensitivity of mineralized tissues to fluoride depends on a genetic background inherent to each population. Within the same specie, different lineages present distint behavior to the ingestion of this íon. Over several decades, many researches focused on the study of NaF as the only source of fluoride, once it is wide spread used to the prevention of dental caries. More recently, different sources of fluoride have been assessed, like TiF4 e SnF2</sub, with the main aim to enhance biochemical and physical benefits of this element. Despite of this, none of these formulations have been tested to evaluate their effect on the bone tissue. Thus, we believe it is advisable to investigate viability and phosphatases activities of pre-osteoblast MC3T3 cells treated with Aluminium fluoride, compared to NaF treatments. Besides this, facing the knowledge that different cell lines have distinct behavior when treated with fluoride, we used two inbred strains of mice with distint mineral bone densities, C3H/HeJ (C3H, high bone density) and C57BL/6J (C57, low bone density), to assess the viability and phosphatases activities after the treatment with NaF. Overall results were tested by 3 way- Anova. AlF3 did not exibit influence over on alkaline phosphatase when pre-osteoblast cells are considered, but the effect of this fluoride salt on the activity of total and tartarate resistant acid phosphatases ocurrs on a dicotomic way. When we consider a mixed cell population, collected from calvária as for the C3H and C57 assays, NaF did interfered as with the proliferation rate as phosphatase activities on a very distinct fashion between the two lineages. Thus, it is possible to conclude that NaF is able to exert variable effects in different cell types and enzyme activities; on the other hand, AlF3 effects are more specific and surrounded in pre-osteblastic cells.

Du fait de leur présence à la surface de la particule virale, les glycoprotéines d’enveloppe E1 et E2 du virus HCV jouent un rôle essentiel dans sa morphogenèse ainsi que lors de son entrée dans la cellule hôte. Jusqu’à récemment, les travaux de recherche sur les glycoprotéines d’enveloppe du virus HCV se sont essentiellement focalisés sur E2 car elle est la protéine d’attachement du virus. De plus, elle est la cible majeure des anticorps neutralisants et il a été longtemps postulé qu’elle était la protéine de fusion du virus. Cependant, les récentes publications de la structure de E2 ne mettent pas en évidence la présence d’un peptide de fusion et sa structure ne correspond pas aux critères attendus pour une protéine de fusion, suggérant que la glycoprotéine E1 seule ou en association avec E2 pourrait être responsable de l’étape de fusion. La structure de la région N-terminale de E1 (acides aminés 192 à 270) a récemment été résolue et a mis en évidence la présence d’une épingle à cheveux formée par 2 feuillets beta (β1 et β2) suivie par un segment de 16 acides aminés qui forme une hélice alpha (α1) flanquant 3 feuillets beta antiparallèles (β3, β4 et β5). En plus de la caractérisation de ces structures secondaires de E1, une région qui se situe au milieu de la protéine (approximativement entre les résidus 274 et 292) a été proposée avoir un rôle actif au cours du processus de fusion et elle pourrait correspondre à un peptide de fusion.Nous nous sommes basés sur ces travaux récents pour investiguer le rôle fonctionnel de la glycoprotéine E1 par une approche de mutagenèse dirigée des résidus conservés dans la région N-terminale et dans la région du potentiel peptide de fusion, dans le contexte d’un clone infectieux du HCV. Comme attendu, nos résultats indiquent que ces mutations introduites dans E1 n’ont aucun effet sur la réplication virale. Cependant, vingt-et-un parmi les vingt-huit mutants produits conduisent à une atténuation ou une perte de l’infectiosité virale. D’une manière très intéressante, deux mutants atténués, le T213A et le I262A, se sont montrés moins dépendants au co-récepteur claudine-1. D’autre part, nous avons montré que ces mutants utilisent un autre récepteur de la famille des claudines (claudine-6) pour l’entrée virale, indiquant ainsi un changement de dépendance à son co-récepteur claudine-1. A l’opposé, deux autres mutants, le L286A et le E303A, se sont révélés avoir une plus grande dépendance au co-récepteur claudin-1 pour l’entrée dans les cellules d’hépatome. Au cours de ce travail, nous avons également identifié une mutation intéressante à proximité du potentiel peptide de fusion. Cette mutation, G311A, conduit à la sécrétion de particules virales entières mais non infectieuses, suggérant un défaut d’entrée cellulaire pour ce virus. De façon très surprenante, nous avons également identifié une mutation (D263A) qui conduit à la sécrétion de particules virales dépourvues d’ARN génomique. Une caractérisation plus poussée de ce mutant a de plus révélé une modification dans la co-localisation subcellulaire entre l'ARN viral et la glycoprotéine E1, mettant en évidence pour la première fois un dialogue croisé entre E1 et l'ARN génomique du HCV lors de la morphogenèse du virus.En conclusion, nos observations permettent d’identifier précisément les régions spécifiques de la protéine E1 qui jouent un rôle dans l’assemblage et l’entrée du virus dans la cellule, mettant en évidence le rôle majeur de la glycoprotéine E1 au niveau des différentes étapes du cycle infectieux du HCV. / Being part of the viral particle, HCV envelope glycoproteins E1 and E2 play an essential role in virion morphogenesis as well as in HCV entry into liver cells. These glycoproteins form a non-covalent heterodimer, and until recently, research on HCV envelope glycoproteins has been mainly focused on E2. Indeed, this glycoprotein is the receptor-binding protein, it is also the major target of neutralizing antibodies and it was postulated to be the fusion protein. However, the recent publications of the structure of E2 do not show the presence of a fusion peptide and its structure does not fit with what one would expect for a fusion protein, suggesting that E1 alone or in association with E2 might be responsible for the fusion step. Concerning E1, only the crystal structure of the two-fifth N-terminal region, comprising amino acids 192 to 270, has been reported. This partial structure reveals a complex network of covalently linked, intertwined homodimers. The overall fold of the N-terminal E1 monomer consists of a beta-hairpin (β1 and β2) followed by a segment composed of a 16 amino-acid long alpha-helix (α1) flanking a three-strand antiparallel beta-sheet (β3, β4 and β5). In addition to the characterization of secondary structures within E1, a region located in the middle of the polypeptide (approximately between aa 274 and 292) has been suggested to play an active role during the fusion process and might potentially act as a fusion peptide. We took advantage of these recently published data to further investigate the functional role of HCV glycoprotein E1 by using a site-directed mutagenesis approach targeting conserved amino acids in the N-terminal region as well as in the region postulated to contain the fusion peptide in the context of an infectious clone. As expected, our results indicate that these mutations have no effect on virus replication. However, twenty-one out of twenty-eight mutations led to attenuation or inactivation of infectivity. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on tight junction protein claudin-1, a co-receptor for HCV. Instead, these mutant viruses relied on another claudin (claudin-6) for cellular entry, indicating a shift in receptor dependence. In contrast, two other mutants, L286 and E303, were more dependent on claudin-1 for cellular entry into hepatoma cells cells. We also identified an interesting mutation downstream of the putative fusion peptide, G311A, which leads to the release of non-infectious particles having a defect in cellular entry. Finally, an unexpected phenotype was also observed for D263A mutant, which was no longer infectious but led to the secretion of viral particles devoid of genomic RNA. Further characterization of the D263A mutant revealed a change in subcellular co-localization between HCV RNA and E1, highlighting for the first time a crosstalk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.In conclusion, our observations allowed for the identification of specific regions in the E1 glycoprotein that play a role in virion assembly and entry, highlighting the major role played by this protein at different steps of the HCV infectious cycle.