Οι μηνισκικές ρήξεις διακρίνονται σε τραυματικές και εκφυλιστικές. Κλινικά δεδομένα υποδηλώνουν ότι η εκφύλιση των μηνίσκων συσχετίζεται με την οστεοαρθρίτιδα του γόνατος. Παρ’ όλα αυτά, τα μοριακά γεγονότα που καθορίζουν την παθογένεια της εκφύλισης των μηνίσκων σε ανθρώπους παραμένουν αδιευκρίνιστα. Στη μελέτη εξετάστηκε ανοσοϊστοχημικά η έκφραση της p38 MAPKινάσης, της ενεργού φωσφορυλιομένης μορφής της p-p38, του στόχου NF-kB (με τα διμερή p50-p65) καθώς και της COX-2 σε μηνισκικές ρήξεις, και διερευνήθηκε η συμμετοχή τους στην ανάπτυξη εκφύλισης. Τα ευρήματα απέδειξαν αυξημένη έκφραση του άξονα p38-NF-kB και της COX-2 στον αποδιοργανωμένο και εκφυλισμένο ινοχόνδρινο μηνισκικό ιστό, υποδεικνύοντας ένα ρόλο των μορίων αυτών στην παθοβιοχημεία της εκφύλισης και της επακόλουθης ρήξης. Η μελέτη είχε σκοπό να διερευνήσει και να χαρακτηρίσει την έκφραση και την ενεργοποίηση του σηματοδοτικού μονοπατιού της p38 MAPK-NF-kB και της COX-2 στα ινοχονδροκύτταρα των ανθρώπινων μηνίσκων με ρήξη. Επιπρόσθετα συσχετίσαμε τα επίπεδα έκφρασης των πρωτεϊνών αυτών με παθολογοανατομικές και κλινικές παραμέτρους, όπως η ύπαρξη εκφύλισης και η συνύπαρξη κλινικά εξακριβωμένης ΟΑ. Χρησιμοποιήθηκαν 57 ανθρώπινοι μηνίσκοι. 43 (75,4%) άνδρες και 14 (24,6%) γυναίκες, με μέσο όρο ηλικίας 32,6 έτη, με διάρκεια πόνου 17,36 μήνες. Σε 39 ασθενείς (68,4%) η ρήξη αποδόθηκε σε τραύμα και σε 18 (31,6%) σε προϋπάρχουσα κλινικά διαγνωσμένη ΟΑ. Η ιστοπαθολογοανατομική διευκρίνιση της μηνισκικής εκφύλισης βασίστηκε σε καθιερωμένα μικροσκοπικά κριτήρια. Εκφύλιση παρατηρήθηκε σε 34 μηνίσκους (59,4%). Χρησιμοποιήθηκαν τα αντισώματα, anti p38, anti p-p38 (μονοκλωνικά αντισώματα έναντι της ενεργοποιημένης μορφής), anti NF-kB, p50 πολυκλωνική, anti NFkBp65 πολυκλωνική και antiCOX-2. Η ένταση της χρώσης και η αναλογία των ανοσοθετικών ινοχονδροκυττάρων εκτιμήθηκε μικροσκοπικά και βαθμολογήθηκε σε κλίμακα 0-3 (0= χωρίς ανοσοδραστικότητα, 1= ήπια, 2= μέτρια, 3= ισχυρή). Στατιστική ανάλυση έγινε με τη δοκιμασία Mann-Whitney και η ισχύς της συσχέτισης των μεταβλητών με το Kendall’s T test, χρησιμοποιώντας το SPSS. 1.Η έκφραση της p38 ήταν στατιστικά σημαντικά υψηλότερη στους εκφυλισμένους συγκριτικά με μη εκφυλισμένους μηνίσκους. Μηνίσκοι σε ασθενείς με προϋπάρχουσα ΟΑ έδειξαν επίσης στατιστικά σημαντικά αυξημένη p38 σε σύγκριση με αυτούς με μη προϋπάρχουσα ΟΑ. 2.Η p-p38 είχε σημαντικά αυξημένη έκφραση σε εκφυλισμένους μηνίσκους έναντι μη εκφυλισμένων και σε ραγέντες μηνίσκους με ΟΑ σε σχέση με μη ΟΑ. 3.Οι υπομονάδες p50 και p65 ως τροποποιητές του άξονα p38-NFkB έδειξαν σημαντικά υψηλότερα επίπεδα στην εκφύλιση και την οστεοαρθρίτιδα. 4.Τα επίπεδα της COX-2 ήταν σημαντικά διαφορετικά μεταξύ εκφυλισμένων και μη εκφυλισμένων και σε οστεοαρθριτικές και μη αρθρώσεις, και συσχετίζονται απόλυτα με τη διακύμανση των προηγούμενων βημάτων του άξονα. 5.Η στατιστική ανάλυση αποκάλυψε σημαντική και θετική συσχέτιση μεταξύ COX-2 και p38-NF-kB και παράλληλη διακύμανσή της. / Meniscal tears are attributed to either trauma or degeneration processes. Clinical data suggest that meniscal degeneration (MD) is associated with knee osteoarthritis; however, the molecular events underpinning the pathogenesis of MD in humans remain elusive. Here we immunohistochemically examined the expression of p38 MAPK, its phosphorylated activated form p-p38, its target NF-kB (p50-p65 dimer), and COX-2 in ruptured menisci and investigated their involvement in MD development. Our findings demonstrate increased expression of the p38-NF-kB axis elements and COX-2 in disintegrated fibrocartilage suggesting a role of these molecules in the pathobiochemistry of MD and consequential rupture. We undertook this study to explore and characterize the expression and/or activation profile of the p38 MAPK-NF-kB signaling path away constituents and COX-2 in the fibrochondrocytes of human torn menisci. Furthermore we correlated the expression levels of the examined proteins with pathologic and clinical parameters, such as the presence of fibrocartilaginus degeneration and the coexistence of clinically identified OA. 57 human menisci were used for this study. Among the patients 43 (75,4%) were male and 14 (24,6%) female with mean age 32,6 years, with pain duration 17,36 months. In 39 patients (68,4%) meniscal tearing was attributed to trauma and in 18 (31,6%) to a background of clinically diagnosed OA. The histopathologic identification of meniscal degeneration (MD) was based on established microscopy criteria. MD was observed in 34 (59,4%) of the menisci. The following available antibodies were employed (anti-p 38), anti p-p38 (activated form monoclonal), anti NF-kB, p50 polyclonal, anti NFkBp65 polyclonal and anti COX-2. Strain intensity and proportion of immunopositive fibrochondrocytes was assessed by light microscopy and graded on a scale 0-3 (0= no immunoreachivity, 1= mild, 2= moderate, 3= strong). Statistical analysis was made with Mann-Whitney tests and the strength of association between the variables by Kendall’s T test, using SPSS for Windows. 1.Expression of p-38 was significantly higher in degenerated compared with non degenerated menisci. Menisci from patients with preexisting OA showed significantly increased p38 expression levels compared to those with no preexisting OA. 2.p-p38 expression was considerably elevated in degenerated compared with non degenerated and in OA compared with non OA ruptured menisci. 3.The downstream effectors of p38 NF-kB subunits p50 and p65, exhibited significantly higher levels in degenerated and OA fibrocartilage. 4.COX-2 levels were significantly different between degenerated and non degenerated menisci, as well as between OA and non OA joints. 5.Statistical analysis revealed significant and positive correlation between COX-2 and p-38 and NF-kB.

Orientador: Katashi Okoshi / Resumo: Introdução: A sobrecarga de pressão causada pela hipertensão arterial sistêmica (HAS) pode gerar mudança na arquitetura do colágeno, favorecer a fibrose, bem como o desbalanço entre a produção de espécies reativas de oxigênio (ERO) e a capacidade antioxidante. Aumento das ERO pode gerar ativação de vias sinalizadoras como a do fator nuclear kappa B (NF-kB) e das proteínas quinases ativadas por mitógenos (MAPK). Alterações dessas vias contribuem para o processo de remodelamento cardíaco causado pela HAS. O exercício físico desempenha importante papel na atenuação dos fatores de risco cardiovascular como a HAS. Dessa forma, o objetivo desse estudo foi avaliar a influência do treinamento físico sobre o remodelamento cardíaco de ratos espontaneamente hipertensos (SHR) na fase que antecede o desenvolvimento de insuficiência cardíaca. Métodos: Foram constituídos quatro grupos experimentais de ratos: normotensos Wistar (W) sedentários (W-SED, n=27); W exercitados (W-EX, n=31); SHR sedentários (SHR-SED, n=27); e SHR exercitados (SHR-EX, n=32). A partir de 13 meses de idade, os animais dos grupos exercitados foram submetidos a protocolo de exercício em esteira, cinco dias por semana, durante quatro meses. A avaliação estrutural e funcional in vivo do coração foi realizada por ecocardiograma. A função miocárdica in vitro foi avaliada em preparações de músculo papilar isolado do ventrículo esquerdo (VE). Amostras de tecido do VE foram obtidas para análises bioquímicas, histológicas e mo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The pressure overload caused by systemic arterial hypertension (SAH) may change the collagen architecture, induce fibrosis, as well as imbalance between the reactive oxygen species (ROS) production and antioxidant capacity. Increased ROS leads to activation of signaling pathways such as nuclear factor kappa B (NF-kB) and mitogen-activated protein kinases (MAPK). Alterations in these pathways contribute to cardiac remodeling process induced by SAH. Physical exercise plays an important role in mitigating cardiovascular risk factors such as hypertension. Therefore, the aim of this study was to evaluate the influence of physical training, started before clinical evidence of heart failure, on cardiac remodeling in spontaneously hypertensive rats (SHR). Methods: Four experimental groups were used: sedentary (W-SED n=27) and trained (W-EX, n=31) normotensive Wistar rats, and sedentary (SHR-SED, n=27) and exercised (SHR-EX, n=32) hypertensive rats. Rats of the exercise groups underwent a protocol of treadmill exercise five days a week, for four months; exercise started at 13 months of age. Echocardiogram was performed to evaluate in vivo cardiac structures and function. In vitro myocardial function was analyzed in left ventricular (LV) papillary muscle preparations. LV tissue samples were obtained for biochemical, histological, and molecular analysis. Total myocardial collagen was assessed by histology and hydroxyproline quantification. Cardiomyocyte size was measured i... (Complete abstract click electronic access below) / Doutor

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Submitted by Luana Urbano Pagan null (luanapagan@alunos.fmb.unesp.br) on 2018-03-13T18:51:41Z No. of bitstreams: 1 Tese Doutorado 1 fev 2018 - Luana Urbano Pagan.pdf: 2756763 bytes, checksum: 6b255d0ba5900dbacc830d74916982d2 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-03-16T19:35:20Z (GMT) No. of bitstreams: 1 pagan_lu_dr_bot.pdf: 2756763 bytes, checksum: 6b255d0ba5900dbacc830d74916982d2 (MD5) / Made available in DSpace on 2018-03-16T19:35:20Z (GMT). No. of bitstreams: 1 pagan_lu_dr_bot.pdf: 2756763 bytes, checksum: 6b255d0ba5900dbacc830d74916982d2 (MD5) Previous issue date: 2018-03-02 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: A sobrecarga de pressão causada pela hipertensão arterial sistêmica (HAS) pode gerar mudança na arquitetura do colágeno, favorecer a fibrose, bem como o desbalanço entre a produção de espécies reativas de oxigênio (ERO) e a capacidade antioxidante. Aumento das ERO pode gerar ativação de vias sinalizadoras como a do fator nuclear kappa B (NF-kB) e das proteínas quinases ativadas por mitógenos (MAPK). Alterações dessas vias contribuem para o processo de remodelamento cardíaco causado pela HAS. O exercício físico desempenha importante papel na atenuação dos fatores de risco cardiovascular como a HAS. Dessa forma, o objetivo desse estudo foi avaliar a influência do treinamento físico sobre o remodelamento cardíaco de ratos espontaneamente hipertensos (SHR) na fase que antecede o desenvolvimento de insuficiência cardíaca. Métodos: Foram constituídos quatro grupos experimentais de ratos: normotensos Wistar (W) sedentários (W-SED, n=27); W exercitados (W-EX, n=31); SHR sedentários (SHR-SED, n=27); e SHR exercitados (SHR-EX, n=32). A partir de 13 meses de idade, os animais dos grupos exercitados foram submetidos a protocolo de exercício em esteira, cinco dias por semana, durante quatro meses. A avaliação estrutural e funcional in vivo do coração foi realizada por ecocardiograma. A função miocárdica in vitro foi avaliada em preparações de músculo papilar isolado do ventrículo esquerdo (VE). Amostras de tecido do VE foram obtidas para análises bioquímicas, histológicas e moleculares. A avaliação do colágeno miocárdico total foi realizada pela histologia e por quantificação de hidroxiprolina. O tamanho dos miócitos foi medido em cortes histológicos do VE. A atividade das enzimas antioxidantes foi quantificada por espectrofotometria. A atividade da NADPH oxidase foi avaliada pela redução da lucigenina. A quantificação proteica dos colágenos I e III, lisil oxidase, vias MAPK e NF-kB, e inibidores teciduais 1 e 2 das metaloproteinases foi realizada por Western blot. A atividade das metaloproteinases foi realizada por zimografia. As comparações entre os grupos foram realizadas por análise de variância (ANOVA) complementada pelo teste de Bonferroni (distribuição normal), ou o teste de Kruskal-Wallis complementado pelo teste de Dunn (distribuição não normal). Resultados: A pressão arterial sistólica foi maior nos grupos SHR. Os grupos exercitados apresentaram maior capacidade física. Os sinais de insuficiência cardíaca foram maiores nos grupos hipertensos em relação aos controles, e o grupo SHR-EX apresentou menor prevalência de derrame pleural e taquipneia em comparação ao SHR-SED. O ecocardiograma mostrou reduções da espessura da parede do VE, espessura relativa do VE, diâmetro do átrio esquerdo e melhora do relaxamento no grupo SHR-EX vs. SHR-SED. O estudo da função miocárdica in vitro mostrou melhor performance no grupo SHR-EX (derivada positiva da tensão desenvolvida) vs. SHR-SED. O grupo SHR-EX mostrou maior atividade das enzimas antioxidantes em comparação SHR-SED. A produção de hidroperóxido de lipídeo, diâmetros dos miócitos, expressões proteicas da JNK fosforilada e da IkB total foram maiores nos grupos hipertensos. A quantificação de hidroxiprolina, malondialdeído, atividade da NADPH oxidase, expressões proteicas do colágeno III, lisil oxidase, TIMP-1, JNK total, p38 fosforilada, p65 fosforilada e total e IkB fosforilada não apresentaram diferença entre os grupos. A fração colágena intersticial, a atividade da MMP-2 e a expressão proteica da p38 total, ERK total e fosforilada foram maiores no SHR-SED em comparação com controle. O exercício causou redução da atividade da MMP-2 e da expressão da ERK fosforilada nos ratos hipertensos. Conclusão: O exercício físico em ratos espontaneamente hipertensos atenua o remodelamento cardíaco que está associado à melhora da tolerância ao esforço físico e redução da frequência de sinais de insuficiência cardíaca. Além disso, associa-se ao aumento da atividade das enzimas antioxidantes, diminuição da fosforilação da ERK e da atividade da MMP-2, e atenuação da expressão proteica da ERK total. / Introduction: The pressure overload caused by systemic arterial hypertension (SAH) may change the collagen architecture, induce fibrosis, as well as imbalance between the reactive oxygen species (ROS) production and antioxidant capacity. Increased ROS leads to activation of signaling pathways such as nuclear factor kappa B (NF-kB) and mitogen-activated protein kinases (MAPK). Alterations in these pathways contribute to cardiac remodeling process induced by SAH. Physical exercise plays an important role in mitigating cardiovascular risk factors such as hypertension. Therefore, the aim of this study was to evaluate the influence of physical training, started before clinical evidence of heart failure, on cardiac remodeling in spontaneously hypertensive rats (SHR). Methods: Four experimental groups were used: sedentary (W-SED n=27) and trained (W-EX, n=31) normotensive Wistar rats, and sedentary (SHR-SED, n=27) and exercised (SHR-EX, n=32) hypertensive rats. Rats of the exercise groups underwent a protocol of treadmill exercise five days a week, for four months; exercise started at 13 months of age. Echocardiogram was performed to evaluate in vivo cardiac structures and function. In vitro myocardial function was analyzed in left ventricular (LV) papillary muscle preparations. LV tissue samples were obtained for biochemical, histological, and molecular analysis. Total myocardial collagen was assessed by histology and hydroxyproline quantification. Cardiomyocyte size was measured in LV histological sections. Antioxidant enzymes activity was quantified by spectrophotometry. NADPH oxidase activity was analyzed by reduction of lucigenin. Protein expression of collagen I and III, lysyl oxidase, MAPK and NF-kB, and metalloproteinases tissue inhibitors 1 and 2 was quantified by Western blot. The activity of metalloproteinases was evaluated by zymography. Comparisons between groups were performed by two factors analysis of variance (ANOVA), complemented with the Bonferroni test (normal distribution), or Kruskal-Wallis complemented with Dunn test (non-normal distribution). Results: Systolic blood pressure was higher in the SHR groups. The exercised groups showed greater physical capacity. Prevalence of heart failure signs was higher in the hypertensive groups compared to controls, and the SHR-EX group showed lower prevalence of pleural effusion and tachypnea compared to SHR-SED. Echocardiogram showed lower LV wall thickness, LV relative wall thickness, left atrium diameter, and relaxation time in the SHR-EX group vs. SHR-SED. Myocardial functional study showed better performance in the SHR-EX group (positive derivative of the developed tension) vs. SHR-SED. The SHR-EX group showed higher antioxidant enzymes activity compared to SHR-SED. Lipid hydroperoxide production, myocyte diameters, and phosphorylated JNK and total IkB protein expression were higher in the hypertensive groups. Quantification of hydroxyproline, malondialdehyde, NADPH oxidase activity, and protein expression of collagen III, lysyl oxidase, TIMP-1, total JNK, phosphorylated p38, phosphorylated and total p65, and phosphorylated IkB did not differ between groups. The interstitial collagen fraction, MMP-2 activity, protein expression of total p38, and total and phosphorylated ERK were higher in the SHR-SED group compared to normotensive control. Physical exercise reduced the MMP-2 activity and the phosphorylated ERK expression in hypertensive rats. Conclusion: Physical exercise in spontaneously hypertensive rats attenuates cardiac remodeling associated with improved physical capacity and reduced prevalence of heart failure signs. In addition, it is associated with increased antioxidant enzymes activity, decreased ERK phosphorylation and MMP-2 activity, and attenuation of total ERK protein expression. / FAPESP: 2014/00747-1

L'avancée de la technologie des PRF a suscité l'intérêt de l'introduction de nouvelles fibres, comme la fibre de basalte, qui a un potentiel d'offrir une solution efficace, lorsqu’utilisée dans les structures en béton, soit sur la résistance à la corrosion, la durabilité et la rentabilité. En outre, les codes et les guides disponibles, ne fournissent pas de recommandations pour l'utilisation de barres en PRFB puisque les recherches passées dans ce domaine sont limitées. Donc, des travaux de recherche sont nécessaires pour caractériser et comprendre le comportement des barres de PRFB dans les éléments en béton armé. En conséquence, les objectifs principaux sont d'évaluer les caractéristiques à court et long terme des barres de PRFB nouvellement développées, ainsi que d'évaluer les performances structurales de ces nouvelles barres comme renforcement interne dans les poutres et les dalles de pont et d'introduire ce nouveau renforcement dans les codes et les guides de dimensionnement. Les tests expérimentaux ont été faits en trois parties. La première partie porte sur le développement de trois nouvelles barres et tendons en PRFB pour déterminer leurs propriétés physiques et mécaniques. Les performances à long terme et de durabilité ont été réalisées en conditionnant les barres de PRFB dans une solution alcaline simulant les conditions humides dans le béton pour déterminer la compatibilité comme renforcement interne dans les éléments en béton. Par la suite, les propriétés ont été déterminées et comparées avec des spécimens non conditionnés (référence). La seconde partie a porté sur sept dalles de pont en béton armé grandeur réelle avec les bords restreints, simulant les tabliers de pont les plus utilisés en Amérique du Nord, pour évaluer la performance des dalles renforcées de PRFB et d'acier. Les dalles mesurent 3000 mm de long × 2500 mm de large × 200 mm d'épaisseur. Les dalles ont été testées jusqu'à la rupture sous une charge concentrée au centre de celles-ci simulant l'empreinte d'une roue d'un camion. Les capacités en poinçonnement sont prédites en utilisant les exigences réglementaires disponibles, et sont comparées aux résultats expérimentaux. La troisième partie de cette étude portait sur les essais de 14 poutres en béton de 3100 mm de long × 200 mm de large × 300 mm de profond pour examiner le comportement en flexion et les performances en service des barres de PRFB avec deux états de surfaces: fini sablé et crénelé. Les poutres ont été testées en flexion en quatre points avec une portée libre de 2700 mm jusqu'à la rupture. Les résultats sont introduits et discutés en terme : du comportement de la fissuration, des flèches, de la capacité en flexion et des modes de ruptures. De plus, le coefficient d'adhérence (kb) des barres de PRFB est déterminé et comparé avec les recommandations des codes et guides actuels. Les résultats sont introduits et discutés en terme : du comportement de la fissuration, des flèches, de la capacité en flexion et des modes de ruptures. De plus, le coefficient d'adhérence des barres de PRFB est déterminé et comparé avec les recommandations des codes et guides actuels. Les résultats de l'étude concluent sur la viables pour la production des barres de PRFB pour respecter les exigences des codes actuelles. Également, les résultats d'essai indiquent que les barres de PRFB ont de bonnes propriétés mécaniques et peuvent être placées dans la même catégorie que les barres de PRFV, soit grade III. De plus, le comportement des poutres et des dalles de pont renforcées de PRFB est similaire que pour un renforcement en PRFV et PRFC et les exigences réglementaires sont applicables pour les barres de PRFB. / Abstract: The advances in fiber-reinforced-polymer (FRP) technology have spurred interest in introducing new fibers, such as basalt FRP (BFRP), which has the potential to offer an efficient solution when implemented in concrete structure, such as corrosion resistant, durable and cost-effective. Furthermore, the available design codes and guides do not provide any recommendations for the use of BFRP bars since fundamental studies and relevant applications are still limited. Therefore, investigations are needed to characterize and understand the behavior of BFRP bars in concrete members. Consequently, the main objectives of this experimental investigation are to evaluate the short- and long-term characteristics of newly developed BFRP bars, as well as evaluate the structural performance of these new bars as internal reinforcement for concrete beams and bridge-deck slabs to introduce these new reinforcing bars to the design codes and guides. The experimental tests were completed through three parts. The first part was conducted on three newly developed BFRP bars and tendons to investigate their physical and mechanical properties. Durability and long-term performance were assessed by conditioning the BFRP bars in an alkaline solution simulating the moist concrete environment to determine their suitability as internal reinforcement for concrete elements. Thereafter, the properties were assessed and compared with the unconditioned (reference) values. The second part of this study was conducted on seven full-scale edge-restrained concrete bridge-deck slabs simulating actual slab-on-girder bridge-deck that is commonly used in North America to evaluate the performance of concrete bridge-deck slabs reinforced with BFRP and steel bars. The deck slabs measured 3000 mm long × 2500 mm wide × 200 mm deep. The slabs were tested up to failure under single concentrated load acting on the center of each slab simulating the footprint of sustained truck wheel load. The punching shear capacities were predicted using the available provisions, and compared with the experimental results. The third part of this study included testing of fourteen concrete beams of 3100 mm long × 200 mm wide × 300 mm deep to investigate the flexural behavior and serviceability performance of sand-coated and ribbed BFRP bars in concrete beams. The beams were tested under four-point bending over a clear span of 2700 mm until failure. The results are introduced and discussed in terms of cracking behavior, deflection, flexure capacity, and failure modes. In addition, the bond-dependent coefficient (kb) of the BFRP bars was determined and compared with the recommendations of the current FRP design codes and guides. The findings of this study concluded the feasibility of producing BFRP bars meet the requirements of the current FRP standards. Also, the test results revealed that the BFRP bars had good mechanical behavior and could be placed in the same category as grade II and grade III GFRP bars. Moreover, the behavior of the concrete bridge-deck slabs and beams reinforced with BFRP bars was quite similar to the counterparts reinforced with glass- and carbon-FRP bars and the available FRP provisions are applicable for BFRP bars. The beam test results yielded an average bond-dependent coefficient (kb) of 0.76±0.03 and 0.83±0.03 for the sand-coated and ribbed BFRP bars, respectively.

Early innate response of intestinal epithelial cells is the first line defense against enteric pathogens. Toxoplasma gondii infections acquired naturally via the peroral route, encounter intestinal epithelial cells early post-infection. Although the population structure of T. gondii is known to be highly clonal, clinical strains of T. gondii have been classified into three genotypes based on their virulence. In this study we investigated whether human intestinal epithelial cell immune response to T. gondii is virulence dependent. We demonstrated distinct virulence of the three T. gondii genotype strains evaluated in human intestinal epithelial cells by their capacity to replicate and induce host cell cytotoxicity. The early host innate mechanisms such as activation of signaling pathways and induction of innate effectors were likewise differentially elicited by the three T. gondii strains. Low levels of TLR dependent NF-kB activation and a failure to rapidly up-regulate innate cytokine and chemokine genes was observed after virulent Type I strain infection. In contrast, early innate response to the less virulent Type II strain was rapid, efficient and led to high levels of IL-8 and IL-6 secretion, whereas response to Type III parasites was intermediate. Early expression of b-defensin 2 gene was suppressed specifically by virulent Type I strain and its activation prior to infection in intestinal epithelial cells led to decreased parasite viability. These findings provide evidence for T. gondii strain virulence dependent down-modulation of early human intestinal epithelial cell innate responses and highlight the importance of these cells in host defense against this infection. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The zona-pellucida (zona, ZP) is an adhesion-refractory, acellular coat enclosing the rapidly growing, free-living mammalian preimplantation embryo which undergoes successive cleavage divisions to form the blastocyst, composed of ICM-cells surrounded by outer TE-cells. For further development, the blastocyst must ‘hatch’ or ‘escape’ out of the zona before it can implant into the endometrium for further development (Fig. 5.1A). Hence, the event of hatching or ‘zona escape’ assumes critical importance for the establishment of a successful pregnancy. The golden-hamster blastocyst offers a very unique paradigm to understand hatching, whereby upon attainment of a fully-expanded state, the blastocyst undergoes a dramatic (and molecularly unexplained) deflation event, followed by appearance of TE-derived dynamic cellular projections called TE-projections, whose appearance in an embryonic-stage and -time dependant manner suggest an intimate association with the hatching phenomenon (Fig. 5.1B). Thirdly, embryo-derived zonalytic proteases have been shown to bring about a focal-lysis of the ZP followed by global zona dissolution. Earlier work in the laboratory had demonstrated the intimate involvement of signaling molecules like LIF, HB-EGF, TGF-β and (ER)-α with hatching (Seshagiri et al., 2002, 2009). Investigations also revealed the involvement of cysteine-proteases of the cathepsin (cts) family, especially cts-L, -B and-P to be involved in zona lysis (Sireesha et al., 2008). In order to achieve a better understanding of mammalian preimplantation development, especially hatching, it was important to investigate the role and impact of other critical regulators of developmental and reproductive physiology. COX-2 is one such key signaling moiety and it was decided to investigate the role, if any, of COX-2 and its derived PGs in hamster peri-implantation events. COX-2 transcripts and immunoreactive COX-2 protein were detected in the different preimplantation stages, from 8-cell onwards. COX-2 protein was abundant in both the ICM and TE, but was especially enriched in the TE-cells of the late blastocyst. In order to investigate the function of this enzyme in preimplantation development and hatching, two very-specific inhibitors of COX-2 catalytic action, NS-398 and CAY-10404, were tested in identical concentrations of 25, 50 and 75 μM on in vitro cultured hamster blastocysts. In order to assess the impact of COX-2 inhibition on an embryo-stage and time-dependant manner, inhibitors were tested on freshly recovered 8-cell embryos or early blastocysts, continuously for 72 h or 48 h, respectively. COX-2-selective inhibitors inhibited hamster blastocyst hatching in a dose-dependant manner with maximum inhibition observed in the 75 μM dose. Surprisingly, there was a profound dose-dependent failure of deflation of late-blastocysts upon inhibitor treatment and embryos which hatched, did so in an inflated state and retained intact zonae in cultures. Moreover, embryos subjected to NS-398 treatment phenocopied those subjected to CAY-10404 treatment. Results demonstrate that the effect of inhibitors, and hence the need for COX-2 mediated signaling events is more pronounced in 8¬cell embryos than with early-blastocysts, indicating that COX-2 dependant molecular and cellular processes required for blastocyst morphogenesis and ZP-lysis may have been initiated prior to compaction and cavitation. The reversal of effects of COX-2 inhibition on hatching with exogenous addition of PGE2 and Iloprost (a stable PGI2 analogue) to inhibitor cultures, show that COX-2-derived eicosanoids could, in effect bring about hamster hatching, which is in agreement with previous reports (Davis et al., 1999) and augment peri-implantation development including hatching (Huang et al., 2003). Additionally, it has been successfully demonstrated that PGE2 was superior to PGI2 in augmenting blastocyst hatching in inhibitor-cultures. In this study, the modulation of critical cts-L, -B, -P proteases in COX-2 mediated hamster zona hatching has been verified by quantifying cts in transcripts in control and inhibitor-subjected embryonic samples which was further substantiated by the decreased intra-embryonal protein levels of cts-L and -P. These results demonstrate that COX-2 mediated signaling components directly and effectively modulate hamster preimplantation development, especially zona-hatching phenomenon by transcriptional-regulation of the critical zonalytic proteases. Another potential hatching-associated molecule i.e., NF-κB which is known to exert a great deal of influence on overall reproductive and developmental biology, was investigated in this study. Its specific effects on mammalian preimplantation development, especially hatching, remain totally uninvestigated. This formed the rationale to investigate the reach and impact of NF-κB signaling network in the modulation of peri-hatching events. Transcripts and immunoreactive NF-κB protein of crucial pathway-components like IKK, IκB-β and RelA were detected from 8-cell embryo to the zona-free blastocyst. In order to ascertain the impact of NF-κB signaling on peri-hatching events, two very-specific inhibitors of the NF-κB pathway, BAY-11-7082 and JSH-23 were employed which acted at two strategic signaling points. In order to assess the impact of NF-κB inhibition on an embryo-stage and time-dependant manner, inhibitors were tested on freshly recovered 8-cell embryos or early blastocysts, continuously for 72 h or 48 h, respectively. NF-κB-selective inhibitors inhibited blastocyst hatching in a dose-dependent manner. Interestingly, a profound dose-dependent failure of deflation of late-blastocysts upon inhibitor treatment was observed and embryos which hatched did so in an inflated state and also retained intact zonae in cultures. Moreover, embryos subjected to BAY-11-7082 treatment phenocopied those subjected to JSH-23 treatment, indicating specificity of inhibitor action. Time-course experiments demonstrated that the need for efficient NF-κB mediated signaling is distinctively more for 8-cell embryos than early-blastocysts, indicating that NF-κB dependant molecular and cellular processes required for blastocyst morphogenesis and ZP-lysis may have been initiated prior to compaction and cavitation. Moreover, modulation of zonalysins cts-L, -B and -P by NF-κB-signaling, during the event of zona lysis, both by real-time quantitation of its transcripts and intracellular protein levels has been demonstrated. These results demonstrate that NF¬κB mediated signaling components directly and effectively modulate hamster preimplantation development, especially zona-hatching phenomenon by transcriptional-regulation of the critical zonalytic proteases. The profound inhibition of hatching and effects on blastocyst morphogenesis observed by inhibition of COX-2 and NF-κB signaling systems demonstrate a fundamental need of the growing embryo for these critical signaling moieties. Moreover, the underlying similarity of consequences obtained upon inhibition of both signaling networks i.e., NF-κB and COX-2, perhaps indicate a linear mode of signaling between these principles. It remains to be tested, though, if it really is the case. A striking observation made in this study was the detection of immunoreactive signals for critical signaling moieties like ER-α, COX-2 and RelA onto TEPs of the deflated hamster blastocyst, in addition to earlier TEP-localisation of cathepsins. A B C (Figure) ENDOMETRIUM ENDOMETRIUM ENDOMETRIUM Fig 5.1. Schematic representation of the role of molecular and cellular factors in the regulation of concordant phenomena of mammalian blastocyst hatching and endometrial implantation. (A) Depicts a zona-intact well-formed blastocyst. Preimplantation embryo development and blastocyst formation involves close cooperation between several molecular principles (discussed in sections 1.3.1 to 1.3.3), (B) as the embryo prepares to hatch, prior to implantation, it initiates egression from the non-adhesive ZP coat by cathepsin (cts) protease-mediated lysis of zona (pink circles); there is concomitant appearance of cellular principles such as TEPs (undulating projections shown in green). Of interest is the intimate association of hatching-promoting molecules such as COX-2, NF-κB, ER-α, Cts etc. with the TEPs. (C) depicts a zona-free, TEP-rich blastocyst initiating implantation into the maternal endometrium. It is possible, that the embryonic TEPs with the associated hatching-regulatory molecules are also critical for implantation phenomena during the embryo-maternal recognition and implantation during the establishment of early pregnancy. Preliminary results indicate that TEPs could be the site of membrane lipid-rafts, focal points of membrane-based signaling. The definitive role of TEPs in peri-hatching events is yet to be confirmed, but it is presumed that these actin-based undulating structures, harboring several key molecules involved in peri-implantation events in the embryo as well as the maternal uterus could be instrumental in successfully bringing about the concomitant processes of hatching and implantation. Interestingly, during rodent implantation (hamster, guinea-pig, mouse and rat), the blastocyst orients in such a way that the ICM is oriented away from the endometrium and, at least in the hamster, the TEP-carrying abembryonic (mural) pole remains closest to the luminal epithelium (LE) (Gonzales et al., 1996b; Seshagiri et al., 2009; Fig. 5.1C). In contrast, in humans and other primates, the embryonic pole is closest to LE before implantation (Kirby, 1971; Lee and DeMayo, 2004). Although direct evidence is lacking, but these observations gives rise to a possibility that both hatching and implantation could be intimately related to the polar appearance of TEPs in the embryo. Several key signaling molecules like ER-α, LIF, HB-EGF and TGF-β have been already demonstrated to play crucial roles in mammalian hatching. In this thesis, we have exemplified the need for COX-2 mediated prostanoid signaling and the pleotropic NF-κB signaling system in bringing about mammalian blastocyst hatching. How exactly do these molecular entities communicate among themselves and with cellular principles like TEPs thereby effectively enabling peri-implantation development, remain to be understood. Taken together, these results demonstrate, for the first time, the involvement of embryo-derived signaling molecules, like COX-2 and NF-κB in an embryo stage-and time-dependant manner in mammalian peri-implantation events, especially blastocyst hatching. The association of TEPs with key molecules common to embryonic and maternal preparation for hatching and implantation, respectively, indicates towards a molecular and cellular continuity between the concomitant events. These fundamental findings on hamster blastocyst biology have profound clinical implications in the management of human infertility. (For figures pl see the abstract file).

<p>According to the Global Cancer Incidence, Mortality, and Prevention (GLOBOCAN) study for 2018, 2,089,000 women will have been diagnosed with breast cancer worldwide, with 627,000 breast cancer-related mortalities. It is estimated that between 15 – 20 % of breast cancer diagnoses are of the triple-negative subtype. Triple-negative breast cancers (TNBCs) do not express the receptors for estrogen, progesterone, and human epidermal growth factor 2, and hence cannot be treated using hormone receptor-targeted therapy. </p> <p>TNBCs are commonly of the basal-like phenotype, with high expression levels of proteins involved in epithelial-mesenchymal transition, extracellular-matrix (ECM) remodeling, cell cycle progression, survival and drug resistance, invasion, and metastasis. 5-year survival rates are significantly lower for TNBC patients, and the disease is characterized by poorer grade at the time of diagnosis as well as higher 5-year distant relapse rates, with a greater chance of lung and CNS metastases. Current treatments for TNBC take the form of aggressive cytotoxic chemotherapy regimens with multiple adverse side-effects. An important goal of on-going studies is to identify new compounds with significant TNBC-specificity, in order to improve patient survival outcomes while preserving a high quality of life during treatment.</p> <p> For several decades, compounds originally isolated from bioactive natural extracts, such as the taxanes and vinca<i> </i>alkaloids, have been at the forefront of chemotherapy. However, due to their non -specific mechanisms of action, treatment with these compounds eventually leads to significant toxicity to normal cells and tissues. Modern transcriptomics, metabolomics, and proteomics tools have greatly improved our understanding of the mechanisms governing cancer initiation and progression, and revealed the considerable heterogeneity of tumor cells. This has allowed for the identification of potential vulnerabilities in multiple cancers, including TNBCs. By leveraging these new technologies and insights with the tremendous diversity of bioactive compounds from organisms that remain unstudied, new classes of onco-drugs targeting pathways specific to TNBC cells could be identified in the near future.</p> <p>Here, we describe the cytotoxic effects of extracts from <i>Lippia origanoides </i>- a species of medicinal shrub native to Central and South America - on TNBC cells. We report that these extracts induce rapid, sustained, and irreversible apoptosis in TNBC cells <i>in vitro</i>, with significantly reduced cytotoxicity against normal mammary epithelial cells. The <i>L. origanoides </i>extracts LOE and L42 exploited two TNBC-specific characteristics to induce apoptosis in these cells: i) inhibiting the constitutively active survival and inflammatory NF-kB signaling pathway, and ii) significantly dysregulating the expression levels of mitochondrial enzymes required to maintain the TCA cycle and oxidative phosphorylation; metabolic pathways that are required for the maintenance of TNBC cell growth and proliferation.</p> <p>Finally, to lay the foundations for future studies on the abilities of these extracts to prevent tumor initiation and inhibit tumor growth <i>in vivo</i>, we also show that the <i>L. origanoides </i>extract, L42, is non-toxic<i> </i>to immunocompetent C57BL/6 mice, and have developed an <i>in vivo </i>model of human TNBC in athymic <i>nu/nu</i> mice. </p> <p>Collectively, our studies are the first to identify the anti-TNBC-specific properties of bioactive extracts from the <i>Lippia </i>species, and reveal that targeting NF-kB signaling and mitochondrial metabolism are potential avenues to new therapeutics against this subtype of breast cancer. Future work in our lab will focus on identifying the bioactive components (BACs) of the extract mediating its apoptotic effects, and shedding light on their protein binding partners within the cell.</p>

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京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18215号 / 薬博第805号 / 新制||薬||237(附属図書館) / 31073 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 橋田 充, 教授 髙倉 喜信, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM