Unlike other regenerative organs such as the liver, the adult mammalian heart does not regenerate tissue lost following injury such as myocardial infarction (MI). Instead a non-contractile fibrous scar develops that in the longer term leads to the development of heart failure (HF). In contrast to the adult, neonatal mammals, including mice and man, retain potent cardiac regenerative capacities and can replace myocardium lost following injury. Understanding the mechanisms underlying scar free repair in the neonate may help in development of new approaches to reduce the impact of myocardial injury in adults. In this thesis MI was induced by coronary artery ligation in mice at post-natal day 1 (P1). Novel electrocardiogram gated high resolution cardiac ultrasound was developed to permit non-invasive confirmation of injury 1 day later and regeneration 21 days later by loss, then restoration, of contractile function. Macrophages (MФ) play important roles in organ growth and homeostasis, and are required for scar-free regeneration of the neonatal mouse heart following MI. WNTs are secreted lipophilic proteins with multiple roles in development. MФ-derived WNTs are essential for scar free tissue regeneration following injury in the kidney, liver, and gut, but their role in the heart is unknown. The primary aim of this thesis was to investigate the role of MФ, and in particular MФ-derived WNTs in determining normal growth of the myocardium from neonate to adult and also in regeneration of the neonatal heart following injury. In wild-type neonatal mouse hearts, Csf1r-expressing cells density (mostly macrophages) was consistent across all time points studied. Three populations of resident cardiac mononuclear phagocytes were identified by flow cytometry: F4/80hi, CD11blo, Ly6C-ve - F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve. F4/80hi, CD11blo, Ly6C-ve cells were hypothesised to correspond to yolk-sac derived mononuclear phagocytes and F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve to foetal liver/bone marrow derived mononuclear phagocytes. Three phases of myocardial growth were identified by ultrasound and histological techniques: hyperplastic (P2-P8, with increased Ki67 and cardiac troponin immunopositive cells), hypertrophic/reorganisation (P8-P21, with increasing cardiomyocyte size and no change in left ventricle wall thickness), and finally hypertrophic solely (P21-P42, with increasing cardiomyocyte size and left ventricle wall thickness). Average coronary vessel size was shown to decrease between P2 and P8 whilst vessel density was increased. The number of α-smooth muscle actin (αSMA) coated vessels greatly increased between P8 and P42, indicating vessel maturation. Throughout all phases cardiac systolic function was maintained at steady state. Diastolic function was however shown to mature from a foetal to an adult pattern between P2 and P8, with reversal of the E:A wave ratio on Doppler ultrasound. In mice globally deficient in MФ due to a germline knock-out of the Csf1r gene (Csf1rnull mice), both body and heart weights were decreased from P7 onwards. The number of proliferating (Ki67+ve) cardiomyocytes at P1 and P7 was unchanged in Csf1r-null mice but there was a trend towards decreased cardiomyocyte size at P7, suggesting an influence on hypertrophic rather than hyperplastic growth of the myocardium. There was also a trend for slowed vascular network maturation, with a delay in the shift from large to smaller vessels in hearts from Csf1r-null mice. In mice with MФ-directed (Csf1r-icre mediated) depletion of Porcupine (Porcn), a gene encoding an enzyme required for WNT acylation and secretion cardiac growth, vascularisation, fibrosis and function were all similar in Cre-ve and Cre+ve animals until P41, when cardiomyocyte size and cardiac systolic function were both significantly increased in Cre+ve animals. However, the underlying mechanism is unknown. In the neonatal mice, Csf1r expressing cells, mostly MФ, were identified in association with regenerating myocardium after induction of MI at P1. Flow cytometry data showed that by P7 the putative resident yolk-sac derived population had mostly disappeared from the heart and was replaced by F4/80lo cells, similar to the pattern reported in the adult. In the regenerating myocardium, Axin2 expression was increased consistent with activation of canonical Wnt signalling. Expression of Wnt5b and Fzd2 receptor, both associated with fibrosis, was significantly increased relative to age matched uninjured hearts. MФ-directed depletion of Porcn did not influence either the functional decrease at day 1 or recovery at day 21 following induction of MI at P1. Coronary re-vascularisation was also unaffected by the genotype. However, retention of intra-myocardial fibrosis (picrosirius red staining) was significantly increased in hearts at day 21 post-MI from mice with MФ-directed depletion of Porcn. MФ-derived WNTs are therefore required for scar-free wound healing in the heart, as they are in the liver and the kidney where they regulate matrix metalloproteinase activity. In summary, novel ECG-gated high-resolution in vivo ultrasound developed in this project has allowed characterisation of cardiac structure and function during early post-natal growth and following injury and regeneration in neonatal mice. The resident MФ population of the heart is established pre-natally, and may play a role in determining maturation of the developing vascular network, although this does not involve MФ-derived Wnt signalling. Following MI, the MФ population may expand from bone marrow cells and MФ accumulate around the regenerating tissue. MФ derived WNTs are not required for regeneration of the neonatal myocardium but do have a role in ensuring scar free wound healing and this merits further investigation.

Submitted by Programa de Pós-Graduação em Odontologia Saúde (mestrodo@ufba.br) on 2017-05-19T13:51:09Z No. of bitstreams: 1 Defesa de tese Gardênia.pdf: 37790525 bytes, checksum: 0ab1c619ff6642a89267aaee7ee90cee (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-07-03T14:58:28Z (GMT) No. of bitstreams: 1 Defesa de tese Gardênia.pdf: 37790525 bytes, checksum: 0ab1c619ff6642a89267aaee7ee90cee (MD5) / Made available in DSpace on 2017-07-03T14:58:28Z (GMT). No. of bitstreams: 1 Defesa de tese Gardênia.pdf: 37790525 bytes, checksum: 0ab1c619ff6642a89267aaee7ee90cee (MD5) / CAPES / Tumor Odontogênico Queratocístico (TOQ) e Ameloblastoma (AM) são tumores odontogênicos benignos comuns que apresentam comportamento localmente invasivo e altas taxas de recorrência. A via de sinalização Wnt desempenha papel no desenvolvimento e progressão destes tumores, mas os mecanismos moleculares envolvidos na gênese e comportamento tumoral ainda são pouco conhecidos. O objetivo deste estudo foi avaliar em TOQ e AM a expressão imuno-histoquímica de proteínas Wnt1, Wnt5a, β-catenina e Sufu, envolvidas na via de sinalização Wnt, na tentativa de contribuir com a melhor compreensão da patogênese destes tumores. Foram utilizados 37 amostras teciduais parafinizadas e fixadas em formalina de tumores odontogênicos, sendo 18 TOQs e 19 AMs, os quais foram submetidos à técnica imuno-histoquímica. A análise do perfil imuno-histoquímico demonstrou que ambos os tumores apresentaram similaridade na imunoexpressão de Wnt1 e Wnt5a (p=0,571 e p=0,157, respectivamente). Os TOQs apresentaram imunoexpressão de β-catenina significativamente maior que os AMs (p=0,000). Quanto ao Sufu, apesar de sua expressão ter sido maior nos TOQs, não houve diferença estatisticamente significante entre os tumores (p=0,216). Dentre os biomarcadores avaliados, a β- catenina (p=0,014) e o Wnt5a (p=0,014) apresentaram imunomarcação significativamente maior em TOQs, já nos AMs, a imunoexpressão foi maior principalmente para o Wnt5a (p=0,003). Houve predominância da imunomarcação nucleocitoplasmática do Wnt1 e citoplasmática da β-catenina e do Sufu em TOQs e AMs. Quanto à Wnt5a, os TOQs demonstraram maior imunomarcação nucleocitoplasmática, enquanto nos AMs houve predominância da marcação citoplasmática. Os altos índices de marcação da β-catenina em TOQs e da Wnt5a em TOQs e AMs sugere que estas proteínas tenham maior contribuição com o desenvolvimento, progressão e agressividade destes tumores, e que a Wnt5a atue em receptores diferentes nestes tumores. / Keratocystic Odontogenic Tumor (KCOT) and Ameloblastoma (AM) are common benign odontogenic tumors with locally invasive behavior and high recurrence rates. The Wnt signaling pathway plays a role in the development and progression of these tumors, but the molecular mechanisms involved in the genesis and tumor behavior are still unknown. The aim of this study was to evaluate the immunohistochemical expression of Wnt1, Wnt5a, β-catenin and Sufu proteins involved in the Wnt signaling pathway in KCOT and AM, in an attempt to contribute to a better understanding of the pathogenesis of these tumors. Thirty seven odontogenic tumor cases were paraffin embedded and fixed in formalin for proceeding immunohistochemical technique. Eighteen tumors were KCOT and 19 cases were AMs. Both tumors resembled the immunohistochemical profile for Wnt1 and Wnt5a (p= 0.571 and p=0.157, respectively). KCOTs presented significantly greater immunostaining of β- catenin than AMs (p=0.000). Although, KCOTs expressed more Sufu than AMs, there was no statistically significant difference between tumors (p=0.216). Among the evaluated biomarkers, β-catenin (p=0.014) and Wnt5a (p=0.014) had significantly higher immunostaining in all KCOT cases, meanwhile, the AMs expressed greater immunostaining for Wnt5a (p=0.003). Both tumors immunostaining of Wnt1 was predominantly nucleus/cytoplasmic, and of β-catenin and Sufu were cytoplasmic. KCOTs demonstrated greater immunostaining of Wnt5a in the nucleus/cytoplasm zone, while AMs showed greater predominance of this protein in the cytoplasm. The high intensity staining of β-catenin in KCOTs, and Wnt5a in KCOTs and AMs suggest that these proteins have greater contribution to the development, progression and aggressiveness of these tumors. Furthermore, Wnt5a acts on different receptors in these tumors.

No description available.

Les WNTs sont une famille de glycoprotéines qui, secrétées dans le milieu extracellulaire, jouent un rôle important dans l’embryogenèse. Chez l’adulte, leur dérégulation va entrainer diverses affections incluant des troubles du développement accompagnés ou non de malformations mais aussi des cancers. Au niveau de l’ovaire, le rôle des WNTs demeure peu défini même si des études chez l’humain et la souris prouvent l’implication de certains membres de cette famille dans le développement ovarien ainsi que dans les processus de maturation folliculaire et d’ovulation. Dans ce contexte, nous avons voulu évaluer l’expression de quelques membres de la famille des WNTs (-2, -2b, -4, -5a et -5b) durant l’expansion des cellules du cumulus et évaluer l’effet de certains d’entre eux sur le COC ainsi que la maturation de l’ovocyte chez la vache. Les COCs bovins étaient placés dans une solution de maturation in vitro pendant 0, 6, 12 et 22h et les niveaux d’ARNm mesurés par PCR en temps réel. L’abondance de l’ARNm pour WNT-2b était significativement plus élevée après 6h de maturation comparée aux COCs immatures (à 0h), alors que l’ARNm codant pour WNT-2, -4, -5a et -5b n’augmentait qu’en fin de culture. L’addition d’EGF provoquait l’expansion du COC et la progression de l’ovocyte vers la métaphase II (MII) comme nous l’espérions mais, à notre grande surprise, l’ajout de WNT-2b au milieu de maturation provoquait également l’expansion du COC (82% et 69% pour EGF et WNT-2b respectivement) et la progression de l’ovocyte vers le stade MII (62% et 56% EGF et WNT-2b respectivement). La combinaison d’EGF et WNT-2b n’a pas produit de meilleurs résultats. Notre étude met en lumière l’implication des WNTs dans la maturation du COC chez la vache. Leurs voies d’activation restent toutefois à déterminer. / The Wnts comprise a large family of secreted glycoproteins which, when secreted in the extracellular space, play a key role in embryonic development. In adults, Wnts play a role in homeostasis and their deregulation likely causes several problems including developmental abnormalities with or without deformities, and cancer. The role of Wnts in the ovaries is not clearly defined although studies in humans and mice show the involvement of some Wnts in ovarian development, follicular maturation and in the process of ovulation. In this study, we tested the hypothesis that members of the Wnt family are involved in oocyte maturation in cattle. The specific objectives were to measure the expression of key Wnts (Wnt-2, -2b, -4, -5a and -5b) in cumulus cells during expansion, and to assess the effect of select Wnt proteins on expansion of the cumulus oocyte complex (COC) and oocyte maturation. Bovine COCs were placed into IVM medium for 0, 6, 12 and 22 h and mRNA levels measured by real-time PCR. The abundance of Wnt-2b mRNA in the cumulus cells was significantly higher at 6 h of maturation compared to immature (time 0) COCs, whereas mRNAs encoding Wnt-2, -4, -5a and -5b did not increase until the end of culture. The addition of EGF induced COC expansion and progression of the oocyte to meiosis II (MII) as expected but, unexpectedly, addition of Wnt-2b also induced expansion and (82% and 69%, EGF and Wnt2b, respectively) and progression to MII (62% and 56%, EGF and Wnt2b, respectively); a combination of WNT-2b and EGF did not improve the rates over either alone. Our study provides new evidence for a role for Wnts in the maturation of the COC in cattle. The Wnt signalling pathways are still unknown and more studies are needed.