O avanço tecnológico trouxe aumento na quantidade e na variedade de agentes eliminados na atmosfera, tendo relação direta com o aumento de partículas de poluição do ar e com a ocorrência de mortes por falência cardíaca, infarto agudo do miocárdio e arritmias. Muitos estudos já relataram que o aumento de material particulado (MP <10 nM) induz ao estresse oxidativo que, por sua vez, pode causar inflamação, aumentando a expressão de citocinas inflamatórias. Especificamente no miocárdio, quando agredido, ocorre necrose dos cardiomiócitos, apoptose, ativação do sistema complemento, acúmulo de células inflamatórias na área infartada e na área remota, tendo como mediadores dessa perda celular a inflamação e o estresse oxidativo. Essa cadeia complexa de eventos promove intenso remodelamento molecular e celular na região infartada e em regiões distantes a ela. Visto a poluição atuar tanto na inflamação quanto no estresse oxidativo, e esses serem mecanismos de lesão miocárdica, nossa hipótese é que a poluição poderia ser um amplificador dessa lesão. O objetivo deste trabalho foi avaliar o papel da poluição no remodelamento estrutural, geométrico e funcional do coração em modelo experimental de infarto do miocárdio. Para tal, foram analisados o acúmulo de colágeno intersticial no miocárdio, a inflamação, o estresse oxidativo e a apoptose. Foram estudados 75 ratos wistar divididos em 5 grupos: controle (CT), grupo controle exposto à poluição (CTP), grupo infartado (IAM), infartado exposto à poluição (IAM G1) e grupo exposto à poluição antes e após o infarto (IAM G2). Os métodos utilizados foram: histologia para análise morfométrica, RT-PCR real time para citocinas inflamatórias e apoptose, ecocardiograma para anatomia e função cardíaca e ELISA para avaliação do estresse oxidativo. Os resultados mostraram maior deposição de colágeno intersticial no ventrículo esquerdo nos grupos CTP, IAM, IAM G1 e IAM G2, quando comparados ao grupo controle (p <= 0,001). No VD, houve maior deposição de colágeno nos grupos CTP, IAM, IAM G1 e IAM G2 em relação ao controle (p <= 0,002). Ao final do estudo, o grupo CT apresentou o maior valor Resumo médio de fração de encurtamento, quando comparado aos outros grupos de estudo (p <= 0,03). Na análise do DSVE, verificamos que o grupo controle apresentou a menor média, quando comparado com os grupos infartados (p <= 0,003). Para a avaliação da apoptose, analisamos os genes p53 e Bcl-2 e verificamos maior expressão desses genes nos grupos IAM G1 e IAM G2 quando comparados com o CT, porém sem significância estatística. A poluição potencializou a expressão da citocina TGF-beta no grupo exposto previamente à poluição (IAM G2) em comparação ao grupo IAM (p = 0,004). Os grupos infartados também tiveram maior expressão do TGF-beta quando comparados com o grupo CT (p <= 0,04). O gene TNF-beta foi mais expresso apenas no grupo IAM G2, quando comparado com o grupo CTP, apresentando valor de p = 0,012. Ao analisarmos o gene IL-1beta, observamos maior expressão desse gene quando comparado o grupo CTP com o grupo IAM (p = 0,04) e com o grupo IAM G2 (p = 0,01). Na análise da expressão do gene INF-y, verificamos que houve maior expressão desse gene no grupo exposto previamente à poluição, em relação aos outros grupos do estudo, apresentando o valor de p <= 0,01. Ao analisarmos a expressão da citocina CCL-21, verificamos maior expressão desse gene no grupo IAM, quando comparado com os grupos controles CT e CTP, sendo p <= 0,035. O grupo IAM também apresentou maior expressão do gene CCR7 quando comparado ao grupo CTP (p = 0,03). A proteína glutationa total apresentou maior concentração nos grupos CTP (p = 0,034), IAM (p = 0,014), IAM G1 (p = 0,008), quando comparados ao grupo CT. Concluímos que a poluição estimulou a deposição de fibrose no miocárdio dos corações saudáveis, mas não amplificou essa resposta nos corações infartados nem aumentou a área de infarto. A poluição modulou a resposta inflamatória, sendo ainda maior no grupo exposto à poluição por tempo mais prolongado (IAM G2). A poluição também modulou o estresse oxidativo nos corações saudáveis, mas não amplificou essa resposta nos corações infartados / Technological improvement has brought an increased amount and range of agents disposed in the atmosphere. Moreover, there is a direct relationship with increased air pollution with the occurrence of deaths because heart failure, acute myocardial infarction and arrhythmias. Many studies have reported that the increase of particulate matter (<10 nM) induces oxidative stress which can cause inflammation increasing the expression of inflammatory cytokines. Specifically, in the myocardium affected by an ischemic process, cardiomyocytes necrosis, apoptosis, activation of the complement system, inflammatory cell accumulation in the infarcted area and in remote area occurs. The pathways for this cell loss also include inflammation and oxidative stress. This complex chain of events promotes intense molecular and cellular remodeling in the infarcted region and in regions distant to it. However, the available literature does not correlate the influence of exposure to pollution with remodeling, inflammation and oxidative stress specifically in the myocardium. The aim of this study was to evaluate the role of pollution in structural, geometric and functional remodeling in the heart using an experimental model of myocardial infarction. This study, analyzed the accumulation of interstitial collagen in the myocardium, as well as inflammation, oxidative stress and apoptosis 75 rats were divided into five groups: control group (CT), control group exposed to pollution (CTP), myocardial infarcted group (MI), infarcted group immediately exposed to pollution (MI G1) and infarcted previously exposed to pollution and kept on that after infarction (MI G2). The methods that was used: histology for morphometric analysis, real time RT-PCR for inflammatory cytokines and apoptosis, echocardiography for cardiac anatomy and function and ELISA to assess oxidative stress. The results showed greater deposition of interstitial collagen in the left ventricle in groups CTP, MI, MI G1 and MI G2 when compared to the control group (p <= 0.001). In the RV, there was a greater collagen deposition in groups CTP, MI, MI G1 and MI G2 Summary compared to control (p <= 0.002). At the end of the study, the CT group showed the higher mean shortening fraction than the other study groups (p <= 0.03). The analysis of LVSD found that the control group had lower average than infarcted groups (p <= 0.003). For the assessment of apoptosis, we analyzed the genes p53 and Bcl-2 with higher expression in groups MI G1 and MI G2 compared with CT, but without statistical significance. The pollution increased the expression of TGF-beta in the group previously exposed to pollution (MI G2) compared to MI group (p = 0.004). The infarcted groups had higher expression of TGF-beta compared to the CT group (p <= 0.04). The TNF-beta gene was over expressed only in the group MI G2 compared the CTP group (p = 0.012). By analyzing the gene IL-1y we observed higher expression this gene when compared with the group CTP with the group MI (p = 0.04) and the group MI G2 (p = 0.01). In the analysis of gene expression INF-y we observed that there was a higher expression of this gene in the group previously exposed to pollution (MI G2) compared to other groups (p <= 0.01). We also analyzed the expression of cytokines CCL-21 and CCR7. We observed significant expression this gene CCL-21 in the MI group compared with the control groups (CT and CTP) and the value (p <= 0,035). This group (MI) also showed higher expression this gene CCR-7 when compared CTP (p = 0.03). The protein showed higher glutathione concentration in the groups CTP (p = 0.03), MI (p = 0,014), MI G1 (p = 0,008) as compared to CT. We conclude that pollution has stimulated the deposition of fibrosis in the myocardium of healthy hearts, but did not amplify this response in infarcted hearts nor increased the infarcted area. The pollution modulated the inflammatory response and was even greater in the group exposed to pollution for longer periods (MI G2). The pollution has also modulated the oxidative stress in healthy hearts, but did not amplify this response in infarcted hearts

Introdução: No processo de remodelamento miocárdico ocorre hipertrofia de miócitos e deposição exacerbada de colágeno no interstício, promovendo alteração na geometria e na função do coração. A eritropoetina (EPO) tem sido amplamente estudada nesse cenário, pois exerce efeitos cardioprotetores. Objetivo: Avaliar o papel da EPO na atenuação do remodelamento estrutural, geométrico e funcional do coração, em modelo experimental de infarto do miocárdio. Materiais e Métodos: Estudados 60 ratos Wistar machos divididos em 4 grupos (Controle; Controle+EPO; Infartado; Infartado+EPO). A fração do volume de colágeno intersticial do ventrículo esquerdo (FVCI-VE) e do ventrículo direito (FVCI-VD) foi determinada em cortes histológicos, corados com picrosirus red utilizando-se o programa QWIN Image Processing and Analysis Software (Leica Microsystems Cambridge Ltd.). Essas mesmas lâminas e o software foram utilizados para a medida do tamanho da área de infarto. A análise anatômica e funcional foi realizada por ecocardiograma, avaliando-se a fração de encurtamento do VE (FE) e o diâmetro diastólico do VE (DDVE). Para o estresse oxidativo, dois kits comerciais foram utilizados na determinação da glutationa e do ADMA. A sobrecarga ventricular, apoptose e inflamação foram realizadas por PCR, em tempo real. Na avaliação da angiogênese, utilizamos a técnica de imunohistoquímica. A análise hematológica foi realizada por exames laboratoriais para dosagem de hemoglobina e hematócrito. Resultados: A FVCI-VE (%) foi maior nos grupos infartados em relação aos grupos controles (p < 0,001), e atenuada pela EPO (p < 0,001, IAM vs IAM+EPO) (CT = 0,76 ± 0,21; CT+EPO = 0,63 ± 0,15; IAM+EPO = 1,43 ± 0,92; IAM= 3,47 ± 2,5). A FVCI-VD (%) também foi maior nos infartados em relação aos grupos controles (CT = 0,60 ± 0,2; CT+EPO = 0,83 ± 0,3; IAM+EPO = 1,01 ± 0,55; IAM = 1,60 ± 1,15) (p < 0,001), mas sem diferença estatística quando comparados os grupos IAM vs IAM+EPO. A EPO não influenciou o tamanho do IAM. Os grupos infartados tiveram piora na fração de encurtamento em relação aos controles (CT = 45,65% ± 6,4; CT+EPO = 40,81% ± 4,44; IAM+EPO = 17,32% ± 6,01 e IAM = 20,11% ± 9,49) (p < 0,001), mas sem proteção da EPO. Os grupos infartados também tiveram maior dilatação do VE (p < 0,001) (CT = 0,73 ± 0,06; CT+EPO = 0,74 ± 0,05; IAM+EPO = 0,81 ± 0,09; IAM = 0,87 ± 0,11) sem atenuação da EPO. Os marcadores de estresse oxidativo, ADMA e glutationa, não evidenciaram ação da EPO nessa via. No que se refere à sobrecarga ventricular, o gene BNP apresentou maior expressão nos grupos infartados, em relação aos controles, porém não foi atenuado pela EPO (p = 0,103). Com relação à apoptose, os genes Bcl-2 e p53 mostraram-se mais expressos nos grupos infartados em comparação aos controles (p < 0,05), mas o Bcl-2 não foi ativado e nem o p53 inibido pela EPO. Os genes estudados na avaliação da inflamação foram TNF-alfa, TGF-beta1 e Ccr-5, também não demonstraram efeito anti-inflamatório da EPO. A semiquantificação da angiogênese pela marcação do VEGF também não apresentou diferença significativa entre os grupos (p = 0,95). A análise da hemoglobina e do hematócrito apresentou diferença significativa em relação aos grupos tratados ou não com EPO (p = 0,003 e p = 0,001), respectivamente. Conclusões: EPO atenuou significativamente o acúmulo de colágeno intersticial, mas não protegeu o coração quanto à dilatação, à disfunção e à sobrecarga ventricular neste modelo. Na fase crônica do infarto, avaliada neste estudo, a EPO não modulou as vias da apoptose, estresse oxidativo e inflamação / Introduction: The process of myocardial remodeling include inappropriate collagen deposition in the interstitium developing an overall process of structural and geometric remodeling of the heart. Erythropoietin (EPO) may have a cardioprotective effects including inflammatory and oxidative stress modulation. Objective: The aim of this study was to assess the role of EPO upon structural, geometric and functional remodeling at the heart. Materials and Methods: 60 Wistar rats were divided into 4 groups: Control, Control+EPO, Infarcted, Infarcted+EPO. Interstitial collagen volume fraction in the left (LV-ICVF) and right ventricle (RV-ICVF) was quantified by videomorphometry using a QWIN Image Processing and Analysis Software (Leica Microsystems Cambridge Ltd.). These same slides and software were also used to measure the size of the infarct area. The analyzed echocardiographic parameters were the left ventricle shortening fraction (LVFS) and diastolic diameter (LVDD). For oxidative stress, two commercial kits were used in to quantify ADMA and glutathione. RT-PCR was used to assess ventricular overload, apoptosis and inflammatory cytokines. For angiogenesis we used immunohistochemistry and hematological analysis was performed by laboratory tests for hemoglobin and hematocrit. Non parametric analysis was performed and p <=0.05 was considered significant. Results: LV-ICVF (%) was greater in the infarcted groups compared to controls (p < 0.001), and attenuated by EPO (p = 0.05, MI vs MI+EPO) (CT = 0.76 ± 0.20; CT+EPO = 0.62 ± 0.16; MI+EPO = 1.22 ± 0.86; MI = 3.80 ± 2.6). The RV-ICVF (%) was also greater in the infarcted groups compared to controls (CT = 0.60 ± 0.2; CT+EPO = 0.82 ± 0.28; MI+EPO = 1.02 ± 0.58; IAM = 1.62 ± 1.20) (p = 0.007) but without statistical difference between MI vs MI+EPO. Regarding infarct size we did not observe any difference. The infarcted groups had a worsening shortening fraction compared to controls (CT = 45.65% ± 6.4; CT+EPO = 40.81% ± 4.44; MI+EPO = 17.32% ± 6.01 and MI = 20.11% ± 9.41) (p < 0,001), but without EPO protection. The infarcted groups also showed increased LV dilation (p < 0.001) (CT = 0.73 ± 0.06; CT+EPO = 0.74 ± 0.05; MI+EPO = 0.81 ± 0.08; MI = 0.90 ± 0.11) without EPO attenuation. Oxidative stress markers ADMA and glutathione did not show EPO action in this pathway. The BNP that evaluate ventricular overload, presented increased expression in infarcted groups (p = 0.04), but not attenuated by EPO (p = 0.103). The genes of apoptosis Bcl-2 and p53 were more expressed in infarcted groups when compared to controls (p < 0.05), but Bcl-2 was not activated and p53 inhibited by EPO. For Inflammation just 3 genes exhibit expression with statistical differences between groups (TGF-beta1, TNF-alfa, and CCr-5), but it did not show the EPO anti-inflammatory effect. The semi-quantification of angiogenesis by VEGF expression also did not show statistically significant differences between groups (p = 0.95).The analysis of hemoglobin and hematocrit presented significant differences compared to groups treated or not with EPO (p = 0.003 and p = 0.001), respectively. Conclusions: EPO significantly attenuated the accumulation of interstitial collagen, but it did not reflected in the protection of the heart dilation or dysfunction and oxidative stress, ventricular overload, apoptosis and inflammation of gene expression in this model

No description available.

BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

La Nucléophosmine (NPM1) est l’une des protéines les plus fréquemment altérée dans les troubles hématopoïétiques. Sa mutation, au niveau de l’exon 12, est impliquée dans un tiers des leucémies myéloïdes aiguës et constitue l’un des premiers évènements dans la leucémogénèse. Elle est caractérisée par la séquestration aberrante de NPM1 au cytoplasme, on parle alors de NPMc+. NPM1 peut également subir plusieurs translocations menant à la synthèse de protéines de fusion dont NPM-MLF1. Cette dernière est impliquée dans les syndromes myéloprolifératifs et en plus faible proportion dans les leucémies myéloïdes aiguës. Cette translocation reste peu documentée. NPM1 est une protéine multifonctionnelle dont le rôle dans la régulation génique reste à être définie. La littérature fait état de quelques études rapportant que NPM1 influencerait l’expression génique mais aucun mécanisme n’a encore été clairement décrit. Nous proposons donc dans ce projet de doctorat, de définir comment NPM1 régule la transcription et nous déterminerons si la translocation NPM-MLF1 a un effet différent de celui de NPM1. Afin de déterminer de nouveaux partenaires d’interaction, nous avons purifié les complexes protéiques de NPM1 et NPM-MLF1 dans la lignée de cellules hématopoïétique K562. Dans une étude protéomique, nous avons mis en évidence par spectrométrie de masse puis par immunoprécipitation, que NPM1 et NPM-MLF1 interagissent avec les complexes de remodelage de la chromatine NuRD, P/BAF et ISWI. Par Ampli-Sequencing, nous avons identifié des gènes dérégulés par la baisse d’expression de NPM1 et anormalement exprimés chez les patients présentant une leucémie myéloïde aiguë avec mutation NPMc+. Nous avons déterminé par immunoprécipitation de la chromatine que NPM1 et NPM-MLF1 peuvent influencer l’expression de gènes cibles via le recrutement du complexe NuRD au niveau du site d’initiation de la transcription. Le profil des modifications d’histone est également perturbé. Nous montrons ici que NPM1 et NPM-MLF1 sont impliqués à la fois dans la répression et l’activation génique. Dans une seconde partie du projet, nous proposons de déterminer l’importance physiologique de la régulation, par NPM1-NuRD, de l’un des gènes cibles mis en évidence : SPARC. La méthode CRISPR-Cas9 a été utilisée pour générer des clones uniques K562 dont l’expression de SPARC est absente et celle de NPM1 est diminuée. Cette étude démontre que la diminution d’expression de NPM1 synergise avec la perte de SPARC afin d’induire la différenciation érythroïde et réduire l’invasion cellulaire. Nous montrons donc pour la première fois que NPM1 et la translocation NPM-MLF1 peuvent interagir avec des complexes de remodelage de la chromatine et peuvent influencer leur recrutement au niveau de gènes cibles afin de réguler la transcription. Nos résultats suggèrent que NPM1 est un facteur important dans le contrôle de la modification du transcriptome associée à la différenciation des cellules hématopoïétiques. Ce projet apporte donc de nouvel éclairage sur la compréhension des mécanismes associés à la mise en place des leucémies myéloïdes aiguës présentant une mutation NPMc+ ou la translocation NPM-MLF1. / Nucleophosmine (NPM1) is one of the most frequent altered proteins in hematopoietic disorders. NPM1 exon 12 mutation characterizes one third of all acute myeloid leukemia and can be fundamental to leukemogenesis. This mutation is characterized by the aberrant sequestration of NPM1 to the cytoplasm, the mutated protein is then called NPMc+. NPM1 can also undergo several translocations leading to the synthesis of fusion proteins including NPM-MLF1. The latter is involved in myeloproliferative disorders and in a lower proportion in acute myeloid leukemia. Little is known about this translocation. NPM1 is a multifunctional protein reported to influence gene regulation. The function(s) of NPM1 in gene regulation remains to be defined. The aim of this doctoral research project was to define how NPM1 regulates transcription and determine whether the NPM-MLF1 translocation could disrupt gene regulation imposed by NPM1. In order to determine new protein interaction partners, we purified NPM1 and NPM-MLF1 complexes in the K562 hematopoietic cell line. In a proteomic analysis performed by tandem immunoaffinity purifications followed by mass spectrometry and immunoprecipitation, we identified multiple nuclear protein partners of NPM1 and NPM-MLF1. Importantly, we found that NPM1 and NPM-MLF1 can interact with the chromatin remodeling complexes NuRD, P/BAF and ISWI. An analysis made by Ampli-Sequencing, indicated that multiple genes dysregulated by the decrease expression of NPM1 were also reported to be abnormally expressed in acute myeloid leukemia cells characterized by the NPMc + mutation. We demonstrate that not only NPM1 but also NPM-MLF1 can be recruited to NPM1 regulated genes and affect the chromatin recruitment of the NuRD complex. The variation in NuRD recruitment imposed by NPM1 knockdown or NPM-MLF1 expression, is associated to the abnormal transcriptional regulation of the target genes. We show here that NPM1 and NPM-MLF1 are involved in both gene repression and activation. In a second part of the project, we investigated the physiological importance of gene regulation imposed by NPM1-NuRD in these cells. Importance of the genetic link between NPM1 and the target gene SPARC was defined. Using the CRISPR-Cas9 methodology, we generated unique K562 clones whereby SPARC expression is abrogated and NPM1 expression is decreased. The decrease of NPM1 expression synergizes with the loss of SPARC expression to induce erythroid differentiation and reduce cell invasion. The results presented provide the first demonstration that NPM1 and NPM-MLF1 translocation can interact with chromatin remodeling complexes, influence their recruitment to target genes and thereby, influence the expression of specific genes. This project provides new information to understanding mechanisms affected in acute myeloid leukemia characterized by NPMc+ mutation or NPM-MLF1 translocation.

Les vésicules extracellulaires (VE), incluant exosomes et microparticules (MP), vecteurs d’information biologique, peuvent moduler la fonction de cellules cibles. Une élévation du taux de VE circulantes est observée dans les pathologies cardiovasculaires dont l’obésité est l’un des facteurs de risque majeur. Cependant, il existe peu de données concernant la production de VE adipocytaires et leur capacité à moduler le métabolisme des adipocytes. Tout d’abord, nous avons caractérisé de manière morphologique et biochimique les MP et les exosomes adipocytaires. Nous montrons une production accrue de ces VE dans un contexte d’obésité murine. L’analyse protéomique des VE adipocytaires révèle un enrichissement spécifique des MP et des exosomes en protéines clé du métabolisme énergétique et de l’inflammation, respectivement. Dans une seconde partie, nous avons étudié l’effet de MP lymphocytaires portant le morphogène Hedgehog (MPHh+) sur la différenciation adipocytaire. A l’instar d’une activation classique de la voie de signalisation Hh, les MPHh+ inhibent l’adipogenèse. Bien que dépendant du récepteur Smoothened (Smo), cet effet inhibiteur est indépendant des facteurs de transcription Gli. Nous montrons que les MPHh+ activent un axe anti-adipogénique Smo/Lkb1/Ampk pouvant être stimulé par un nouvel agoniste de Smo, le GSA-10. Nos résultats démontrent, d’une part, la capacité des adipocytes à sécréter des VE, et d’autre part, le potentiel fonctionnel des MPHh+ à inhiber l’adipogenèse par une voie de signalisation Hhnon-canonique. Les VE pourraient contribuer aux dysfonctions métaboliques associées à l’obésité en véhiculant des messages métaboliques à l’échelle de l’organisme. / Extracellular vesicles (EV), including microparticles (MP) and exosomes, are able to modulate target cell function through exchange or transfer of biological material. Although EV are present in the blood of healthy individuals, an elevated quantity of circulating EV is associated with cardiovascular diseases, which obesity is a major cardiovascular risk factor. Nevertheless, few studies have reported the ability of adipocytes to release EV and their implication in adipose tissue metabolism. First of all, we could determine morphological and biochemical features of adipocyte-derived exosomes and MP through a combination of methods. We were able to demonstrate an increase in adipocyte EV production in a murine model of obesity. Proteomic analysis of adipocyte EV further revealed a specific enrichment of proteins crucial for glucose and lipid metabolism and related to inflammation in MP and exosomes respectively. We then evaluated the ability of lymphocytes-derived MP harboring the Sonic Hedgehog morphogen to control adipocyte differentiation. Activation of the Hedgehog canonical pathway inhibited adipogenesis, as did MPHh+. Surprisingly, although Smo dependent, inhibitory potential of such MP did not involve the Gli transcription factors. We show that MPHh+ inhibit adipocyte differentiation through a Smo/Lkb1/Ampk axis as does a new agonist of Smo, GSA-10. Our results demonstrate, on one hand, the ability of adipocyte to release EV and on the other hand, the capacity of MPHh+ to control adipogenesis through a non-canonical Hh signaling pathway. In conclusion, EV might contribute to obesity related metabolic dysfunctions through systemic regulation of metabolic pathways.

Contexte: La physiopathologie de la fibrillation auriculaire (FA) a été caractérisée par des changements de concentration cellulaire de Ca2+ et des processus connexes menant à l'apparition et au maintien de la maladie. Les récepteurs de trisphosphate d'inositol (IP3R) sont des canaux calciques ligand-dépendants pour lesquels la surexpression dans la FA a été liée à un remodelage cardiaque. Les microARN (miR, miARN), petits ARN non codants, sont d'une longueur d'environ 22 nucléotides et régulent l'expression des gènes par déstabilisation de l'ARN ou inhibition de sa traduction. De plus en plus de preuves ont été apportées sur le rôle des miARN dans la physiopathologie des troubles cardiaques, y compris le remodelage défavorable induit par la FA. Objectif: Notre laboratoire a montré que le niveau nucléaire IP3R1 est régulé à la hausse dans le modèle canin de FA, ce qui produit une augmentation de la charge nucléaire en calcium. Cette étude vise donc à étudier le rôle des miARN dans la régulation d'IP3R1 qui initie et/ou perpétue la FA dans les cardiomyocytes auriculaires du modèle de FA chez le chien. Méthodes: Nous avons utilisé un modèle canin de AF établi par méta-cardiographie auriculaire pendant 600 bpm × une semaine; des cœurs perfusés par Langendorff pour isoler les cardiomyocytes auriculaires pour des expériences moléculaires; le criblage des miRs qui ciblent le gène ITPR1, codant IP3R1, en utilisant des bases de données en ligne; RT-qPCR pour mesurer l'expression de l'ARNm de ITPR1 et confirmer le niveau d'expression des miARN criblés; l'analyse Western Blot pour évaluer le niveau de protéine d’IP3R1; le test de la double luciférase reporter, la surexpression et l'abattement des miARN en culture primaire de cardiomyocytes isolées ou de lignées cellulaires appropriées; et l'imagerie par fluorescence calcique Fluo-4 AM pour évaluer le rôle potentiel des miARN sur la manipulation du Ca2+. Pour les expériences de manipulation des miARN, les cellules ont été transfectées avec 1) un miARN non codant (miR-NC, groupe témoin), 2) un miARN mimétique et 3) un inhibiteur du miARN (AMO). La signification statistique est calculée avec le test t de Student ou l'analyse unidirectionnelle de variance (ANOVA) suivie par le test de Tukey à comparaisons multiples en utilisant le logiciel GraphPad Prism version 6.00. Résultats: Nos données indiquent une augmentation du niveau de la protéine IP3R1 sans changement apparent de l'expression du gène ITPR1 dans les cardiomyocytes de l'oreillette gauche par rapport à notre modèle canin de FA. Sur la base de l'analyse informatique, il a été prédit que miR-26a ciblerait l'ARNm de l'ITPR1. La FA a considérablement réduit la régulation du miR-26a dans les cardiomyocytes de l'oreillette gauche. Le dosage de la double luciférase reporté dans les cellules H9C2 a montré que le miR-26a agissait directement sur la région non traduite 3′ (3′UTR) de l'ARNm ITPR1. De plus, la surexpression de miR-26a a réduit le niveau de la protéine IP3R1 et a diminué le taux diastolique [Ca2+] dans le noyau et le cytosol des cardiomyocytes de chien, des transistors de Ca2+ stimulés électriquement; tandis que le knockdown de miR-26a a inversé ces effets. L'expression de l'ARNm de l'ITPR1 est restée inchangée dans les cardiomyocytes de chien isolées après la transfection avec l'imitateur et l'inhibiteur de l'ARNm. Conclusion: La régulation à la hausse d'IP3R1 dans la FA est due à l'inhibition de la traduction par le miR-26a, qui est régulé à la baisse dans les cardiomyocytes auriculaires du modèle canin de FA. Ce changement est associé à une altération de la manipulation du Ca2+, qui se traduit par une augmentation des taux de Ca2+ diastolique nucléaire. Nos résultats suggèrent que la régulation à la baisse de miR-26a augmente l'expression de l’IP3R1, contribuant au remodelage pro-arythmique dans la FA. / Background: The pathophysiology of atrial fibrillation (AF) has been characterized by changes in the cellular concentration of Ca2+ and related processes leading to the initiation and maintenance of the condition. Inositol trisphosphate-receptors (IP3Rs) are ligand-gated calcium channels for which overexpression in AF has been linked to cardiac remodeling. microRNA (miR, miRNA)s, small non-coding RNAs, are around 22 nucleotides in length and regulate gene expression by mRNA destabilization or inhibition of its translation. A growing body of evidence has emerged about miRNA's role in the pathophysiology of cardiac disorders, including AF-induced adverse remodeling. Objective: Our laboratory has shown that nuclear IP3R1 level is upregulated in the dog AF model, producing increased nuclear calcium loading. Hence, this study aims to investigate the role of miRNAs in the regulation of IP3R1 initiating and/or perpetuating AF in atrial cardiomyocytes of the dog AF model. Methods: We used AF dog model established by atrial-tachypacing for 600 bpm × one week; Langendorff-perfused hearts to isolate atrial cardiomyocytes for molecular experiments; screening miRs that target ITPR1 gene, encoding IP3R1, using online databases; RT-qPCR to measure ITPR1 mRNA expression and confirm the expression level of the screened miRNAs; western blot analysis to evaluate the protein level of IP3R1; dual-luciferase reporter assay, overexpression and knockdown of miRNAs in primary culture of isolated cardiomyocytes or appropriate cell lines; and Fluo-4 AM calcium fluorescence imaging to assess the potential role of the miRNA on Ca2+ handling. For miRNA manipulation experiments, cells were transfected with 1) non-coding miRNA (miR-NC, control group), 2) miRNA mimic, and 3) inhibitor of the miRNA (AMO). Statistical significance is calculated with Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test using GraphPad Prism software version 6.00. Results: Our data indicated a rise in IP3R1 protein level with no apparent change in ITPR1 gene expression in left atrial cardiomyocytes from our dog AF model. Based on the computational analysis, miR-26a was predicted to target the ITPR1 mRNA. AF significantly downregulated miR-26a in left atrial cardiomyocytes. The dual-luciferase reporter assay in H9C2 cells showed that miR-26a directly acted on the 3′ untranslated region (3′UTR) of ITPR1 mRNA. In addition, miR-26a overexpression reduced the IP3R1 protein level and decreased the diastolic [Ca2+] in both nucleus and cytosol of the electrically-stimulated Ca2+ -transients, dog cardiomyocytes, while miR-26a knockdown reversed these effects. ITPR1 mRNA expression remained unaltered in isolated dog cardiomyocytes after transfection with the miRNA mimic and inhibitor. Conclusion: IP3R1 upregulation in AF is due to translation inhibition by miR-26a, which is downregulated in the atrial cardiomyocytes of the dog AF model. This change is associated with altered Ca2+ handling, reflected as enhanced nuclear diastolic Ca2+ levels. Our results suggest that miR-26a downregulation enhances the IP3R1 expression, contributing to pro-arrhythmic remodeling in AF.

<div>Excessive deformation and stress of skin following reconstructive surgery plays a crucial role in wound healing, often leading to complications. Yet, despite of this concern, surgeries are still planned and executed based on each surgeon's training and experience rather than quantitative engineering tools. The limitations of current treatment planning and execution stem in part from the difficulty in predicting the mechanical behavior of skin, challenges in directly measuring stress in the operating room, and inability to predict the long term adaptation of skin following reconstructive surgery. Computational modeling of soft tissue mechanics has emerged as an ideal candidate to determine stress contours over sizable skin regions in realistic situations. Virtual surgeries with computational mechanics tools will help surgeons explore different surgeries preoperatively, make prediction of stress contours, and eventually aid the surgeon in planning for optimal wound healing. While there has been significant progress on computational modeling of both reconstructive surgery and skin mechanical and mechanobiological behavior, there remain major gaps preventing computational mechanics to be widely used in the clinical setting. At the preoperative stage, better calibration of skin mechanical properties for individual patients based on minimally invasive mechanical tests is still needed. One of the key challenges in this task is that skin is not stress-free in vivo. In many applications requiring large skin flaps, skin is further grown with the tissue expansion technique. Thus, better understanding of skin growth and the resulting stress-free state is required. The other most significant challenge is dealing with the inherent variability of mechanical properties and biological response of biological systems. Skin properties and adaptation to mechanical cues changes with patient demographic, anatomical location, and from one individual to another. Thus, the precise model parameters can never be known exactly, even if some measurements are available. Therefore, rather than expecting to know the exact model describing a patient, a probabilistic approach is needed. To bridge the gaps, this dissertation aims to advance skin biomechanics and computational mechanics tools in order to make virtual surgery for clinical use a reality in the near future. In this spirit, the dissertation constitutes three parts: skin growth and its incompatibility, acquisition of patient-specific geometry and skin mechanical properties, and uncertainty analysis of virtual surgery scenarios.</div><div>Skin growth induced by tissue expansion has been widely used to gain extra skin before reconstructive surgery. Within continuum mechanics, growth can be described with the split of the deformation gradient akin to plasticity. We propose a probabilistic framework to do uncertainty analysis of growth and remodeling of skin in tissue expansion. Our approach relies on surrogate modeling through multi-fidelity Gaussian process regression. This work is being used calibrate the computational model against animal model data. Details of the animal model and the type of data obtained are also covered in the thesis. One important aspect of the growth and remodeling process is that it leads to residual stress. It is understood that this stress arises due to the nonhomogeneous growth deformation. In this dissertation we characterize the geometry of incompatibility of the growth field borrowing concepts originally developed in the study of crystal plasticity. We show that growth produces unique incompatibility fields that increase our understanding of the development of residual stress and the stress-free configuration of tissues. We pay particular attention to the case of skin growth in tissue expansion.</div><div>Patient-specific geometry and material properties are the focus on the second part of the thesis. Minimally invasive mechanical tests based on suction have been developed which can be used in vivo, but these tests offer only limited characterization of an individual's skin mechanics. Current methods have the following limitations: only isotropic behavior can be measured, the calibration problem is done with inverse finite element methods or simple analytical calculations which are inaccurate, the calibration yields a single deterministic set of parameters, and the process ignores any previous information about the mechanical properties that can be expected for a patient. To overcome these limitations, we recast the calibration problem in a Bayesian framework. To sample from the posterior distribution of the parameters for a patient given a suction test, the method relies on an inexpensive Gaussian process surrogate. For the patient-specific geometry, techniques such as magnetic resonance imaging or computer tomography scans can be used. Such approaches, however, require specialized equipment and set up and are not affordable in many scenarios. We propose to use multi-view stereo (MVS) to capture patient-specific geometry.</div><div>The last part of the dissertation focuses on uncertainty analysis of the reconstructive procedure itself. To achieve uncertainty analysis in the clinical setting we propose to create surrogate and reduced order models, especially principal component analysis and Gaussian process regression. We first show the characterization of stress profiles under uncertainty for the three most common flap designs. For these examples we deal with idealized geometries. The probabilistic surrogates enable not only tasks such as fast prediction and uncertainty quantification, but also optimization. Based on a global sensitivity analysis we show that the direction of anisotropy of skin with respect to the flap geometry is the most important parameter controlled by the surgeon, and we show hot to optimize the flap in this idealized setting. We conclude with the application of the probabilistic surrogates to perform uncertainty analysis in patient-specific geometries. In summary, this dissertation focuses on some of the fundamental challenges that needed to be addressed to make virtual surgery models ready for clinical use. We anticipate that our results will continue to shape the way computational models continue to be incorporated in reconstructive surgery plans.</div>

Introduction: The inflammation plays the essential role in the bone loss in juvenile idiopathic arthritis (JIA). Proinflammatory cytokines and also glucocorticoids (GCs) may activate bone resorption by osteoclasts. Simultaneously, bone formation can be attenuated, especially by inhibitors of proteins, which control the osteoblast differentiation. The aim was to verify the hypothesis that in patients with highly active JIA, reduction of bone formation via Wingless (Wnt) proteins inhibitors - Dickkopf 1 (Dkk-1) and sclerostin could be found. Except the densitometry measurements of bone and lean mass, we assessed markers of disease activity, bone metabolism and remodeling in young adult patients with JIA before and during 2 years of anti TNFα (tumour necrosis factor α) treatment, which decreases disease activity. Results: In patients with JIA before antiTNFα treatment, bone mineral density (BMD, g/cmš) was significantly reduced compared to controls. Values of BMD and body composition in JIA significantly depended on disease duration and GCs treatment. Serum concentration of sclerostin was significantly elevated in JIA compared to values in healthy controls. Values of the other monitored markers did not differ between JIA and controls. In patients with JIA, Dkk-1 correlated positively with C-reactive...

Development and evaluation of new cementless revision acetabular components for total hip arthroplasty / type TC / Objective: The aim of the clinical part of the study was to introduce a new revision oval cup type TC, and evaluate its reliability and utility in revision endoprosthesis. Due to the different structure of the implant, we aimed to objectively demonstrate the remodeling of bone tissue in its vicinity. In the experimental part of the work we verified the hypothesis that the use of locking screws has an effect on the bond strength of the implant with bone tissue, and therefore on the primary stability of the acetabular component. Method: We evaluated 31 patients that underwent revision hip surgery between 2004 and 2008. The mean follow-up was 7.1 years (range 5.3 to 9.3 years, minimum 5 years after surgery). Osteointegration of the implant and remodeling of bone tissue around the implant and its ribs were evaluated by digital radiography and computed tomography, and clinical results according to Harris. The experiment was based on the execution of pull-out tests after the implantation of TC cups in cadaveric bovine pelves. We evaluated the dependence of tensile forces in the axis of the implant on extraction of the metal cup from the bone bed. Four tests were conducted with the cup fixed...