Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

Zebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.

Altas concentrações de ácidos graxos saturados desencadeiam resistência à insulina no músculo esquelético e o estresse de retículo endoplasmático (RE) é sugerido neste processo. Este estudo objetivou investigar, em células musculares L6, os efeitos do tratamento com palmitato sobre o estresse de RE e a ativação do NF-kB, relacionando-os com o prejuízo na expressão do GLUT4. Pelos resultados, observou-se que o tratamento com 0,75 mM de palmitato induziu redução no conteúdo de GLUT4 e Slc2a4. Os marcadores de estresse como GRP78, PERK/EIF2a e IRE1a/XBP-1/TRAF2 apresentaram pouca ativação. O fator transcricional NF-kB apresentou-se aumentado em seu conteúdo proteico e RNAm. A atividade de ligação do NF-kB à região promotora do Slc2a4 mostrou aumento independente do grau de fosforilação de IKK. Em suma, observou-se que o palmitato reprime a expressão do Slc2a4 e que induz pouco estresse de RE em células L6. Por outro lado, a participação do NF-kB parece ser importante no controle deste fenômeno reduzindo a expressão do Slc2a4 e prejudicando a homeostasia da glicose. / High concentrations of saturated fatty acids trigger insulin resistance in skeletal muscle and endoplasmic reticulum stress (ER) is suggested in this process. This study aimed to investigate, in L6 muscle cells, the effects of palmitate treatment on ER stress pathways and activation of NF-kB, relating them to the impaired expression of GLUT4. The results showed that treatment with 0.75 mM of palmitate induced reduction in the amount of GLUT4 and Slc2a4. Stress markers such as GRP78, PERK/EIF2a and IRE1a/XBP-1/TRAF2 showed little activation. The transcription factor NF-kB were increased in protein content and mRNA. The binding activity of NF-kB to the promoter region of Slc2a4 showed increased independent from the phosphorylation of IKK. Finally, it was observed that palmitate represses the expression of Slc2a4 and that this fatty acid induces little activation of reticulum stress pathways in L6 cells. On the other hand, the involvement of NF-kB appears to be important to control this phenomenon, reducing the expression of Slc2a4 and impairing glucose homeostasis.

A diabetes mellitus (DM) está entre as 10 principais causas de morte global e é considerada um dos principais fatores de risco para as doenças cardiovasculares. Dados da Federação Internacional de Diabetes indicam que 425 milhões de pessoas possuem esta doença e seu tratamento é considerado uma das prioridades de saúde. No coração, as alterações a nível celular causadas pela DM levam ao surgimento de anormalidades estruturais e funcionais que resultam na chamada cardiomiopatia diabética. A DM pode ser estudada a partir de modelos experimentais em animais, como a DM induzida pela estreptozotocina, uma substância citotóxica para as células pancreáticas que produzem insulina. Objetivos: O objetivo este trabalho foi avaliar a existência de alterações cardíacas precoces na contratilidade de miócitos isolados, na morfologia e função cardíaca por ecocardiograma de alta resolução e na atividade elétrica do coração. Metodologia: Foram utilizados ratos Wistar machos para indução da DM com estreptozotocina. Os cardiomiócitos foram isolados por dissociação enzimática utilizando-se a preparação de Langendorff e a contratilidade estudada através da medição da variação do comprimento da célula e dos sarcômeros de cardiomiócitos estimulados eletricamente. A avaliação da morfologia e da função cardíaca foi feita por ecocardiografia de alta resolução e o eletrocardiograma foi obtido nos animais anestesiados. Resultados: A DM resultou em alteração da contratilidade dos cardiomiócitos medida pela variação do comprimento da célula e pela variação do comprimento do sarcômero. Comparados aos controles, os animais diabéticos apresentaram aumento dos intervalos de tempo para atingir o pico de contração (0,049 vs 0,068 s, e 0,043 vs 0,063 s, P<0,001) aumento do relaxamento (0,028 vs 0,038 s e 0,025 vs 0,035 s, P<0,03) e do intervalo até a máxima velocidade de encurtamento (0,019 vs 0,024 s, P<0,001) e relaxamento (0,021 vs 0,032 s e 0,020 vs 0,025 s, P<0,03) em relação ao grupo controle medidos pelo comprimento da célula e pelo comprimento do sarcômero, respectivamente. O ecocardiograma evidenciou mudanças morfológicas com aumento do diâmetro diastólico (24,4 vs 28,0 mm/kg, P<0,01) e sistólico (11,0 vs 18,2 mm/kg, P<0,01), aumento da massa do ventrículo esquerdo (1,9 vs 2,5 mg/g, P<0,02) e manutenção da espessura relativa da parede posterior, caracterizando um quadro de hipertrofia excêntrica. Foram também observadas mudanças funcionais, como a diminuição significativa da fração de ejeção (73,4 vs 62,1%, P<0,01), da fração de encurtamento (44,2 vs 34,7%, P<0,01) e da onda S\' (46,9 vs 38,1 mm/s, P<0,01) avaliada pelo Doppler tecidual, indicando uma disfunção sistólica inicial nesse modelo experimental. No eletrocardiograma foi possível verificar um aumento na duração da onda P (9,15 vs 14,15 ms, P<0,005). Conclusão: Os resultados obtidos permitiram identificar alterações precoces nesse modelo de DM. Foi observada uma correlação entre as alterações da contratilidade observadas diretamente ao nível celular através da medição da variação do comprimento da célula e do sarcômero com as alterações na morfologia e função cardíaca em decorrência da DM. / Diabetes mellitus (DM) is among the top 10 causes of global death and is considered a major risk factor for cardiovascular disease. Data from the International Diabetes Federation indicate that 425 million people have this disease and its treatment is considered one of the health priorities. In the heart, changes at the cellular level caused by DM lead to the appearance of structural and functional abnormalities that result in so-called diabetic cardiomyopathy. DM can be studied from experimental models in animals including induction by streptozotocin, a cytotoxic substance for pancreatic cells that produce insulin. Objectives: The objective of this study was to evaluate the existence of early cardiac changes in the contractility of isolated myocytes, in the morphology and cardiac function by high resolution echocardiogram and in the heart electrical activity . Method: Male Wistar rats were used for induction of DM with streptozotocin. Cardiomyocytes were isolated by enzymatic dissociation using the Langendorff preparation and the contractility studied by measuring cell and sarcomere length variation in electrically stimulated cardiomyocytes. The evaluation of cardiac morphology and function was performed by high resolution echocardiogram and eletrocardiogram was was obtained in anesthetized animals. Results: DM resulted in alteration of cardiomyocyte contractility with measurement of cell length variation and with sarcomere length variation. Compared to controls, diabetic animals presented an increase in the time intervals to reach the peak of contraction (0.049 vs 0.068 s and 0.043 vs 0.063 s, P<0.001) increase in relaxation (0.028 vs 0.038 s and 0.025 vs 0.035 s, P<0.03) and the intervals until the maximum shortening velocity (0.019 vs 0.024 s, P<0.001) and relaxation (0.021 vs 0.032 s and 0.020 vs 0.025 s, P<0.03) in relation to the control group measured by length and by sarcomere, respectively. The echocardiogram evidenced morphological changes with an increase in the diastolic (24.4 vs 28.0 mm/kg, P<0.01) and systolic diameter (11.0 vs 18.2 mm/kg, P<0.01) increase in left ventricle mass (1.9 vs 2.5 mg/g, P<0.02) and maintenance of the relative thickness of the posterior wall in relation to the control group, characterizing eccentric hypertrophy in DM. Functional changes were also observed, such as a significant reduction in ejection fraction (73.4 vs 62.1 %, P<0.01), of the fractional shortening (44.2 vs 34.7 %, P<0.01) and the S\' wave (46.9 vs 38.1 mm/s, P<0.01) evaluated by tissue Doppler, indicating an initial systolic dysfunction in this experimental model. With the electrocardiogram it was possible to verify an increase in P wave duration (9.15 vs 14.15 ms, P<0.005). Conclusion: The results obtained allowed to identify early changes in this model of DM. A correlation was observed between contractility changes observed directly at the cellular level by measuring cell and sarcomere variation with changes in cardiac morphology and function as a result of DM.

A capacidade de sincronizar vias metabólicas a estímulos ambientais é um aspecto central da homeostase em mamíferos. Dentro desse contexto, o controle molecular da função mitocondrial representa um aspecto fundamental e defeitos na integridade desse sistema podem levar a severas perturbações à homeostase celular levando a um amplo espectro de doenças como a obesidade e o diabetes tipo 2. O controle transcricional do metabolismo energético é um processo dinâmico que depende da ação coordenada de fatores de transcrição, enzimas modificadoras de cromatina e coreguladores transcricionais. Co-reguladores podem agir como interruptores transcricionais ativando ou reprimindo a atividade de receptores nucleares. Neste estudo, demonstramos que o coativador PGC-1? e o co-repressor NCoR1 são importantes mediadores do metabolismo energético e da homeostase redox mitocondrial em células musculares. Nossos resultados sugerem que os efeitos desses co-reguladores são mediados pela transativação do elemento responsivo de PPAR (PPRE) em promotores de seletos grupos de genes. Ainda, a indução da capacidade oxidativa e da defesa antioxidante pelo silenciamento de NCoR1 ou pela expressão de PGC-1? atenua a produção de espécies reativas de oxigênio e a morte celular induzida por estresse metabólico. Essas evidências sugerem que o equilíbrio entre a ativação e a repressão transcricional em promotores contendo PPREs exerce um papel central na função mitocondrial em células musculares esqueléticas. Coletivamente, os resultados deste estudo indicam que o antagonismo entre os coreguladores PGC-1? e NCoR1 é um componente central no controle da função mitocondrial representando uma interface promissora para o desenvolvimento de novas abordagens terapêuticas para o tratamento e prevenção da disfunção metabólica. / The ability to synchronize metabolic pathways to environmental stimuli is a central aspect of mammalian homeostasis. Within this context, the molecular control of mitochondrial function represents a fundamental aspect and defects in the integrity of this system can lead to severe disturbances to cellular homeostasis causing a wide spectrum of pathologies such as obesity and type 2 diabetes. Transcriptional control of energy metabolism is a dynamic process that depends on the coordinated action of transcription factors, chromatin modifying enzymes, and transcriptional co-regulators. Co-regulators can act as transcriptional switches activating or repressing the activity of nuclear receptors. In this study, we demonstrated that the co-activator PGC-1? and NCoR1 co-repressor are essential mediators of energy metabolism and mitochondrial redox homeostasis in muscle cells. Our results suggest that the effects of these co-regulators are mediated by the transactivation of the PPAR responsive elements (PPREs) in promoters of selected gene groups. Furthermore, the oxidative capacity and antioxidant defense induction by either NCoR1 knockdown or PGC-1? overexpression attenuates the production of reactive oxygen species and cell death induced by metabolic stress. These evidence suggest that the balance between activation and transcriptional repression in promoters containing PPREs exert a central role in mitochondrial function in skeletal muscle cells. Collectively, the results of this study indicate that the antagonism between the co-regulators PGC-1? and NCoR1 is a central component of mitochondrial function representing a promising interface for the development of novel therapeutic approaches for the treatment and prevention of metabolic dysfunction.

Aumento nos níveis circulantes de alguns ácidos graxos (AGs) está relacionado com o quadro de resistência à insulina em músculo esquelético. Os mecanismos pelos quais os AGs diminuem a ação da insulina não estão elucidados, entretanto a participação destas biomoléculas no controle do NF-kB, SREBP-1c, HIF-1<font face=\"Symbol\">&#945;, LXR<font face=\"Symbol\">&#945; e PPARg considerados reguladores do Slc2a4, já foi sugerida. O objetivo deste estudo foi investigar a ação dos ácidos graxos, oléico (OFA) e linoléico (LFA), em células musculares L6, na regulação do Slc2a4. Redução no conteúdo protéico e de mRNA GLUT4 foi verificada na presença de ambos AGs. Esta redução foi relacionada com aumento na expressão e na atividade de ligação do NF-kB e diminuição na expressão e na atividade de ligação do SREBP-1 ao gene Slc2a4, na presença de OFA e LFA. Ambos AGs aumentaram a expressão do mRNA de LXR<font face=\"Symbol\">&#945;, PPARg and HIF-1<font face=\"Symbol\">&#945;, todavia apenas na presença de LFA foi detectada uma diminuição na ligação de PPARg ao Slc2a4. Ambos AGs reduzem a expressão do GLUT4 devido modulação da ligação do NF-kB e SREBP-1 ao gene Slc2a4. / High elevated levels of some free fatty acids (FFAs) are associated with insulin resistance in skeletal muscle. The mechanisms by which FFAs impair this hormone sensitivity need to be clarified; nevertheless, its effects in the modulation of NF-kB, SREBP-1c, HIF-1<font face=\"Symbol\">&#945;, LXR<font face=\"Symbol\">&#945; and PPARg which are related with Slc2a4 gene regulation have been suggested. The goal of this study was to investigate the action of oleic (OFA) and linoleic (LFA) fatty acids, in L6 muscle cells, in Slc2a4 regulation. The GLUT4 protein and mRNA expression decreased in the presence of OFA and LFA. The reduced GLUT4 expression was related to a significative enhancement of the NFkappaB mRNA expression and binding activity in presence of both FFAs and a decrease of SREBP-1 mRNA and binding activity in the Slc2a4. OFA and LFA increase LXR<font face=\"Symbol\">&#945;, PPARg and HIF-1<font face=\"Symbol\">&#945; mRNA expression, but only a reduction in PPARg binding activity was verified in presence of the LFA. A reduction in GLUT4 expression in the presence of OFA and LFA was detected and related with NF-kB and SREBP-1 binding activity in the Slc2a4 gene.

The pulmonary vein (PV) constricts to hypoxia however little is known about the underlying mechanisms. Hypoxic PV constriction is proposed to recruit upstream capillary beds and optimise gas exchange in healthy humans and may play a role in high altitude pulmonary oedema. The PV is also intrinsic to disease states including pulmonary hypertension and pulmonary veno-occlusive disease. Blood vessel culture can be a powerful tool to enable assessment of the impact of environmental factors on vessel function and as a disease model. However culture conditions alone affect vessel contractility; the effect of culture conditions on PV function remained to be established. The aim of this project was to investigate hypoxic responses of porcine PVs including the impact of maintenance in culture. Maintenance of PVs in culture conditions for 24 hours increased contraction to hypoxia and inhibited hypoxic relaxation post-contraction. These changes to PV hypoxic responses were thought to result from endothelial dysfunction. However, the endothelial nitric oxide synthase inhibitor L-NAME inhibited PV hypoxic contraction and enhanced relaxation. The impact of K+ channel inhibitors on hypoxic contraction was also investigated. Penitrem A, 4AP, DPO-1, ZnCl2 and glyburide had no significant effect however TEA and BDM inhibited the hypoxic contraction. This suggested that TASK, KV1.5, BKCa and KATP do not play a role in the mechanism of hypoxic pulmonary venoconstriction however KV channels containing KV2.1 α subunits may modulate the response. Results with L-NAME suggested endothelial dysfunction may not fully account for the change in PV function after exposure to culture. Therefore the impact of PV maintenance in culture was further explored using an isolated PV smooth muscle cell (PVSMC) model. Maintenance of PVs in culture conditions had minimal impact on morphology and electrical properties of PVSMCs. Notably, resting membrane potential and hypoxia-induced depolarisation were not significantly different. Based on the findings of this study, the endothelium in PVs appears to a) play a major role in modulation of the hypoxic response b) be sensitive to short-term exposure to culture conditions. K+ channels appear to play a minor role in PV hypoxic contraction and SMCs isolated from PVs maintained in culture conditions have similar morphological and electrophysiological characteristics to freshly isolated PVSMCs. Taking all this into account, endothelial regulation of contractility should be a key focus for future PV research.

骨骼肌分化是由一个包括转录因子、表观遗传调控子和非编码RNA在内的复杂网络共同调控的。YY1能够通过募集PRC2抑制一系列肌肉结构基因的表达，进而抑制肌肉分化。miRNA是一组转录后调控基因表达的小片段非编码RNA，miRNA与转录因子的相互作用已经被广泛证实。在本次研究中，我们证实了一个YY1和肌肉特异性miRNA（miR-1,miR-133和miR-206）的调控回路。实验证实，YY1通过肌肉特异性miRNA增强子区域的YY1结合位点募集PRC2来抑制肌肉特异性miRNA的表达。YY1调控miR-1在体外和体内肌肉分化均被证实有重要意义。另外，我们还证实miR-1能够负反馈作用于YY1，抑制YY1的表达。 / 为了阐述YY1在基因组转录中的作用，我们做了肌肉中YY1的ChIP-seq。测序结果表明在C2C12肌肉母细胞中有1820个YY1结合位点，其中很大部分位于基因间的区域。进一步研究发现，基因间YY1的结合可能调控一些lincRNA，而这些lincRNA在肌肉发育的作用目前尚不清楚。进一步研究这些可能受YY1调节的lincRNA，我们证实了YY1能够正调控两个新的lincRNA，YAM-1和YAM-2。YAM-1在肌肉分化过程中逐渐下调，并且通过正调控他的临近基因miR-715，抑制肌肉分化，而YAM-2能够促进早期的肌肉分化。 / 总之，我们第一次在肌肉细胞中进行了YY1的ChIP-seq，并且证实在肌肉分化过程中转录因子和非编码RNA相互作用的重要性和普遍性。 / Skeletal muscle cell differentiation is a process orchestrated by a complex network of transcription factors, epigenetic regulators and non-coding RNAs. As a repressor of myogenesis, Yin Yang 1 (YY1) silences a number of muscle structural genes through recruiting Polycomb repressive complex2 (PRC2) in proliferating myoblasts. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidences support the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1). The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both in vitro and in vivo myogenesis. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. / To elucidate its role on genome-wide regulation of transcription, here in the second part of this study we performed ChIP-Seq for YY1 in muscle cells. Our results revealed 1820 YY1 binding peaks genome-wide in myoblasts, with a large portion residing in the intergenic region. A close analysis of the intergenic region bound by YY1 uncovered that YY1 may regulate a large number of lincRNAs (Long Intergenic non-coding RNAs), whose roles in skeletal myogenesis have not been explored yet. As further elucidation of the functional roles of YY1-lincRNA regulation, we identified two novel lincRNAs, YAM-1 and YAM-2 as positively regulated by YY1. YAM-1 was found to be down-regulated upon myogenic differentiation and acts as an inhibitor of myoblast differentiation. We further demonstrated that YAM-1 functions by its in cis regulation on a downstream gene, miR-715 which promotes differentiation. YAM-2, on the other hand, appears to promote myogenesis. / Together, our studies not only provide the first genome-wide picture of YY1 association in muscle cells but also uncovered novel regulatory circuits required for skeletal myogenesis and reinforce the idea that regulatory circuitry involving non-coding RNAs and TFs is essential components of myogenic regulatory network. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Leina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 144-167). / Abstract also in Chinese. / Abstract / 摘要 / Acknowledgement / Publications / List of figures / List of tables / Abbreviations / Table of content / Chapter Chapter 1: --- INTRODUCTION / Chapter 1.1 --- Skeletal Myogenesis --- p.1 / Chapter 1.2 --- Transcriptional Regulation of myogenic differentiation --- p.3 / Chapter 1.2.1 --- Transcriptional regulatory network in myogenic differentiation --- p.3 / Chapter 1.2.2 --- YY1 as a transcription factor in myogenic differentiation --- p.5 / Chapter 1.3 --- Epigenetic Regulation during skeletal muscle differentiation --- p.6 / Chapter 1.4 --- microRNA: Post-transcriptional regulation on myogenic differentiation --- p.11 / Chapter 1.4.1 --- Muscle specific miRNAs in skeletal myogenic differentiation --- p.15 / Chapter 1.4.2 --- Non-muscle specific miRNAs in skeletal myogenic differentiation --- p.20 / Chapter 1.4.3 --- miRNAs and skeletal muscle diseases --- p.23 / Chapter 1.5 --- Long Non-coding RNAs --- p.26 / Chapter 1.5.1 --- Long Non-coding RNAs and lincRNAs --- p.26 / Chapter 1.5.2 --- LincRNAs in muscles --- p.30 / Chapter Chapter 2: --- MATERIALS AND METHODS / Chapter 2.1 --- C2C12 cell line --- p.32 / Chapter 2.2 --- Primary Myoblast isolation and in vitro culture --- p.32 / Chapter 2.3 --- Animal studies --- p.33 / Chapter 2.4 --- RNA extraction --- p.34 / Chapter 2.5 --- RT-PCR and Real-Time RT-PCR --- p.35 / Chapter 2.6 --- Transfection and infection --- p.37 / Chapter 2.7 --- Oligonucleotides --- p.38 / Chapter 2.8 --- Dual-luciferase reporter assay --- p.43 / Chapter 2.9 --- Immunofluorencence staining --- p.44 / Chapter 2.10 --- Antibodies --- p.45 / Chapter 2.11 --- Protein extraction and Western blotting --- p.46 / Chapter 2.12 --- DNA constructs --- p.48 / Chapter 2.13 --- Mutagenesis --- p.49 / Chapter 2.14 --- RNA-Fluorescence In Situ Hybridization (RNA-FISH) --- p.51 / Chapter 2.15 --- C2C12 cells with YY1-stably knocked down --- p.52 / Chapter 2.16 --- Rapid Amplification of cDNA Ends (RACE) --- p.53 / Chapter 2.17 --- Chromatin Immunoprecipitation (ChIP) --- p.55 / Chapter 2.18 --- ChIP-PCR --- p.58 / Chapter 2.19 --- ChIP-sequencing --- p.58 / Chapter 2.20 --- Northern blotting --- p.59 / Chapter 2.21 --- Prediction of miRNA targets --- p.60 / Chapter 2.22 --- Statistical analysis --- p.60 / Chapter Chapter 3: --- Results / Chapter 3.1 --- YY1-miR-1/133 regulatory circuitry in skeletal myogenesis --- p.61 / Chapter 3.1.1 --- YY1 decreases miR-1/133 during skeletal muscle differentiation --- p.61 / Chapter 3.1.1.1 --- Negative correlation between YY1 and miR-1/133 during C2C12 differentiation --- p.61 / Chapter 3.1.1.2 --- Negative correlation between YY1 and miR-1/133 in primary cell differentiation --- p.63 / Chapter 3.1.1.3 --- Negative correlation between YY1 and miR-1/133 in postnatal muscle development and mdx mouse model --- p.65 / Chapter 3.1.1.4 --- Deletion of YY1 upregulates miR-1/133 both in C1C12 and primary myoblast --- p.68 / Chapter 3.1.1.5 --- Deletion of YY1 upregulates miR-1/133 at the transcriptional level --- p.70 / Chapter 3.1.2 --- YY1 represses miR-1/133 by binding to 4 enhancers --- p.72 / Chapter 3.1.2.1 --- Four enhancers of miR-1/133 with potential YY1 targeting sites --- p.72 / Chapter 3.1.2.2 --- YY1 represses the four enhancers’ activities --- p.75 / Chapter 3.1.2.3 --- Depletion of YY1 up-regulates the four enhancers’ activities --- p.77 / Chapter 3.1.2.4 --- YY1 directly binds to the putative binding sites and mediates the repression on miR-1/133 --- p.79 / Chapter 3.1.2.5 --- YY1 recruits Ezh2 to the enhancers which subsequently causes histone modification --- p.82 / Chapter 3.1.3 --- YY1 repressing miR-1/133 is functionally significant in myogenesis --- p.84 / Chapter 3.1.3.1 --- Negative correlation between YY1 and miR-1/133 in CTX induced muscle regeneration model --- p.84 / Chapter 3.1.3.2 --- Depletion of YY1 in CTX induced muscle regeneration model promotes miR-1/133 expression --- p.87 / Chapter 3.1.3.3 --- Depletion of YY1 in CTX induced muscle regeneration model promotes muscle differentiation --- p.89 / Chapter 3.1.4 --- miR-1 can target YY1 forming a feedback loop --- p.92 / Chapter 3.1.5 --- miR-1 can repress Pax7 by targeting two binding sites on 3’UTR --- p.95 / Chapter 3.1.5.1 --- miR-1 targets Pax7 by binding to two target sites --- p.95 / Chapter 3.1.5.2 --- miR-1 represses Pax7 forming an YY1-miR-1-Pax7 regulating circuitry in skeletal myogenesis --- p.98 / Chapter 3.1.6 --- Conclusion: YY1-miR-1-Pax7 regulatory circuitry in skeletal myogenesis --- p.100 / Chapter 3.2 --- ChIP-seq reveals YY1-lincRNA regulation in skeletal myogenesis --- p.102 / Chapter 3.2.1 --- ChIP-seq uncovered a large number of genes under YY1 regulation --- p.102 / Chapter 3.2.2 --- ChIP-seq reveals that YY1 associates with lincRNA loci --- p.105 / Chapter 3.2.2.1 --- YY1 associates with lincRNA-YAM loci --- p.105 / Chapter 3.2.2.2 --- YY1 positively regulates YAM-1 and YAM-2 both in vitro and in vivo --- p.107 / Chapter 3.2.3 --- YY1-YAM-1-miR-715 regulatory pathway in muscle differentiation --- p.109 / Chapter 3.2.3.1 --- Genomic organization and cellular localization of YAM-1 --- p.109 / Chapter 3.2.3.2 --- Expression of YAM-1 decreases during myogenic differentiation --- p.112 / Chapter 3.2.3.3 --- YAM-1 represses myogenic differentiation both in vitro and in vivo --- p.115 / Chapter 3.2.3.3.1 --- YAM-1 inhibits C2C12 differentiation --- p.115 / Chapter 3.2.3.3.2 --- YAM-1 inhibits muscle differentiation in vivo --- p.117 / Chapter 3.2.3.4 --- A functional YY1-YAM-1-miR-715 regulatory axis in skeletal myogenic differentiation --- p.119 / Chapter 3.2.3.4.1 --- miR-715 is down-regulated during muscle differentiation --- p.119 / Chapter 3.2.3.4.2 --- miR-715 is under the regulation of YY1-YAM-1 --- p.122 / Chapter 3.2.3.4.3 --- miR-715 represses muscle differentiation forming a YAM-1-miR-715 regulatory axis during muscle differentiation --- p.124 / Chapter 3.2.4 --- YAM-2 promotes early myogenic differentiation --- p.126 / Chapter 3.2.4.1 --- Genomic organization and cellular localization of YAM-2 --- p.126 / Chapter 3.2.4.2 --- YAM-2 is regulated during myogenic differentiation --- p.129 / Chapter 3.2.4.3 --- YAM-2 promotes early myogenic differentiation --- p.131 / Chapter Chapter 4: --- DISCUSSION / Chapter 4.1. --- YY1-miRNA regulatory circuit in skeletal myogenesis --- p.133 / Chapter 4.2 --- YY1 mediates epigenetic modification in skeletal myogenesis --- p.135 / Chapter 4.3 --- miRNAs in skeletal myogenesis --- p.136 / Chapter 4.4 --- YY1 regulates long intergenic non-coding RNAs in skeletal myogenesis --- p.138 / Chapter Chapter --- 5: SUMMARY AND FUTURE WORK --- p.142 / REFERENCE --- p.144

Os efeitos da obstrução uretral parcial na musculatura da bexiga urinária de coelhos foram investigadas usando as ferramentas estereológicas. Foram utilizadas 12 fêmeas de coelhos da raça Norfolk, com três meses de idade e peso corporal variando de 2,5-3,0 kg. O procedimento cirúrgico consistiu de celiotomia mediana retro-umbilical para exposição da bexiga urinária. A parede dorsal da uretra foi divulsionada de sua íntima associação com o útero e vagina, o suficiente para a passagem de fio nylon 2-0. Um pino de Steinmann (3 mm de diâmetro) foi interposto temporariamente entre a uretra e o fio para determinar indiretamente o grau de obstrução uretral. Após três, sete e doze semanas os animais foram ortotanasiados e comparados com o grupo de animais controle (não obstruídos). Os fragmentos da bexiga foram preparados para microscopia de luz. Cortes seriados foram realizados para o estudo morfométrico e estereológico. Os três eixos: crânio-caudal (CC), dorso-ventral (DV) e latero-lateral (LL) aumentaram em todos os grupos analisados: controle, 3, 7 e 12 semanas. Os valores para CC foram estatisticamente similares para 3, 7 e 12 semanas. O mesmo foi observado no eixo DV. Os valores para o eixo LL foram similares para os grupos de 7 e 12 semanas. O estudo morfométrico baseou-se em determinar o tamanho da fibra (área seccional) e comprimento da fibra muscular. Nos animais do grupo de 3, 7 e 12 semanas foi observado um aumento de 4,63x, 4,32x e 7,10x no tamanho celular e um decréscimo de 2,55x, 1,94x e 4,04x no comprimento da fibra muscular quando comparados ao grupo controle. O estudo estereológico baseou-se em estimar o volume referência (Vref), a densidade numérica (Nv), o número total de fibras musculares (N), a densidade de volume (Vv) e o volume da fibra muscular (Vn). O Vref apresentou um aumento de 11,07x, 7,98x e 31,7x quando comparado com o grupo controle. A densidade numérica (Nv) aumentou 0,06x e 0,05x para os grupos de 3 e 7 semanas, respectivamente, em relação ao grupo controle. O grupo de 12 semanas, no entanto, apresentou um decréscimo de 0,01x em comparação com o grupo controle. Os grupos de 3, 7 e 12 semanas apresentaram, respectivamente, um aumento de 0,81x, 12,56x e 38,43x em número total de células. A densidade de volume (Vv) para os grupos de 3, 7 e 12 semanas apresentou um aumento de 0,97x, 0,56x e 0,86x em relação ao grupo controle. E finalmente, o volume médio da fibra muscular apresentou um aumento de 0,62x, 0,81x e 0,82x, respectivamente para os animais de 3, 7 e 12 semanas. Os dois mecanismos: hipertrofia e hiperplasia ocorrem na bexiga urinária de coelhos, porém não sabemos a seqüência exata em que aparecem. / The effects of partial urethral obstruction on rabbit´s urinary bladder musculature were investigated using stereological designed methods. A total of 12 female Norfolk rabbits weighing from 2.5 to 3 kg were used. A retro-umbilical celiotomy was made to expose the urinary bladder. The urethra´s dorsal wall was isolated from its association with the uterus. A 3mm-Steinmann-pin was positioned on the urethra to produce a standard degree of obstruction and a ligature was tied up around it, using a 2-0 nylon silk. Three, seven and twelve weeks after the surgery procedures the rabbits were euthanised. Bladder fragments were prepared for light microscopy. Serial sections were performed to morphometric and stereological study. In relation to the bladder axis: cranio-caudal (CC), dorso-ventral (DV) and latero-lateral (LL) increased in all groups analysed: control, 3, 7 and 12 week-obstructed animals. Values for CC were statistically similar for 3, 7 and 12-week-obstructed groups. The same was observed for DV axis. The LL axis showed values statistically similar for 7 and 12-week-obstructed groups. The morphometric study was based on the muscle fibre size (sectional area) and the muscle fibre length. In 3, 7 and 12-week-obstructed animals, it was observed a 4.63, 4.32 and 7.10-fold cell size increase and a 2.55, 1.94 and 4.04-fold decrease in length, respectively, when compared to control group. As for the stereological study. Vref presented a 11.07, 7.98 and 31.7-fold increase when compared to control subjects. Numerical density (Nv) increased by 0.06 and 0.05 in 3 and 7-week-obstructed groups, respectively, in relation to control group. Twelve week-obstructed group. Presented however a 0.01x-decrease compared to control animals. Three, seven and twelve-week-obstructed groups presented, respectively, 0.81, 12.56 and 38.43-fold increase in total number of cells (N). Volume density presented a 0.97, 0.56 and 0.86-fold increase in 3, 7 and 12-week-obstructed groups, respectively. And finally, mean muscle cell volume (Vn) presented a 0.62, 0.81 and 0.82-fold in 3, 7 and 12-week obstructed groups, respectively. Both mechanisms: hypertrophy and hyperplasia happened to occur on rabbit´s urinary bladder, thought we do not know the exact sequence in which they appear altogether.

Une des propriétés majeures de la thrombine est le caractère pléiotropique de ses effets physiologiques et pathologiques à la fois dans le compartiment sanguin et tissulaire de la paroi. La voie de signalisation RhoA est activée par la fixation de la thrombine aux récepteurs PARs et cette voie est un régulateur principal de la mécanotransduction et de la plasticité cellulaire. Le facteur d’échange de RhoA, Arhgef1, est impliqué dans le développement de l’hypertension dépendante de l’angiotensine II et dans l’athérothrombose. Notre hypothèse est que le contrôle de la signalisation intracellulaire de RhoA par Arhgef1 est un élément régulateur de la coagulation plasmatique et pourrait participer aux modifications phénotypiques des plaquettes et des cellules vasculaires et ainsi contribuer à l’augmentation de la génération de thrombine tissulaire. Les objectifs ont été de caractériser la génération de thrombine et la fonction plaquettaire depuis leur activation jusqu’à leurs implications dans un modèle de thrombose tissulaire et d’étudier le rôle prothrombotique des cellules musculaires lisses vasculaires (CMLVs) chez des souris Arhgef1 -/-. Résultats : Les souris Arhgef1-/- ont une numération plaquettaire normale mais présentent une diminution significative de l’activation plaquettaire, de la génération de thrombine en sang total et en présence de plaquettes (mais pas en plasma pauvre en plaquettes) et de l’adhérence plaquettaire par rapport aux souris contrôles. Ces modifications se traduisent, in vivo, par un plus grand nombre d’arrêts transitoires de l’écoulement sanguin dans le modèle de saignement à la queue et un allongement du temps de survenue du thrombus occlusif carotidien en réponse au FeCl3 chez les souris Arhgef1 -/- comparées aux contrôles. Les CMLVs des souris Arhgef1 -/- génèrent moins de thrombine à leur surface et ont une prolifération diminuée par rapport aux CMLVs des souris contrôles. En conclusion, les résultats démontrent le rôle d’Arhgef1 dans les fonctions plaquettaires et dans la régulation du phénotype des CMLVs. Le mécanisme principal fait intervenir la Rho GTPase dans l’adhésion plaquettaire et la génération de thrombine à la surface CMLVs qui contrôlent la formation du thrombus. Ces résultats suggèrent que ce facteur d’échange est capable d’amplifier la thrombose artérielle et pourrait être impliqué via les récepteurs à la thrombine dans le couplage thrombine tissulaire-rigidité cellulaire via les plaquettes et les CMLVs dans les pathologies vasculaires. / One of the major properties of thrombin is the pleiotropic character of its physiological and pathological effects in both the blood and the tissue compartment of the vessel wall. The RhoA signaling pathway is activated by the binding of thrombin to the PARs receptors and this pathway is a major regulator of mechanotransduction and cellular plasticity. The RhoA exchange factor, Arhgef1, is involved in the development of angiotensin II-dependent hypertension and in atherothrombosis. Our hypothesis is that the control of intracellular RhoA signaling by Arhgef1 is a regulatory element of plasma coagulation and could participate in phenotypic modifications of platelets and vascular cells and thus contribute to the increase of tissue thrombin generation. The objectives were to characterize thrombin generation and platelet function from their activation to their implications in a model of tissue thrombosis, and to study the prothrombotic role of vascular smooth muscle cells (VSMCs) in Arhgef1 -/- mice. Results: Arhgef1 -/- mice had a normal platelet count but showed a significant decrease in platelet activation, thrombin generation in whole blood and in the presence of platelets (but not in platelet poor plasma) and platelet adhesion compared to control mice. These modifications result, in vivo, by a greater number of transitory stopping of the blood flow in the tail bleeding model and an increase in the time of occurrence of the carotid occlusive thrombus in response to FeCl3 in Arhgef1 -/- mice compared to controls. The VSMCs of Arhgef1 -/- mice generate less thrombin at their surface and have decreased proliferation compared to VSMCs of the control mice. In conclusion, the results demonstrate the role of Arhgef1 in platelet function and in the regulating of the phenotype of VSMCs. The main mechanism involves Rho GTPase in platelet adhesion and in thrombin generation at the VSMC surface that control thrombus formation. These results suggest that this exchange factor is able to amplify aterial thrombosis and could be involved via thrombin receptors in tissue thrombin-cell stiffness coupling via platelets and VSMCs in vascular pathologies.