INTRODUÇÃO: As adaptações cardiovasculares decorrentes do treinamento físico aeróbio de natação são bem descritas na literatura, entre ela temos a angiogênese. O treinamento físico aeróbio é um dos estímulos que promove angiogênese. Os microRNAs (miRNAs) são uma classe de RNA não codificadora de proteínas de diferentes células em diversos tecidos e estão envolvidos em processos angiogênicos, mas o papel dos miRNAs na angiogênese cardíaca decorrente ao treinamento aeróbico ainda não foi esclarecido. OBJETIVO: Analisar os efeitos de diferentes volumes de treinamento físico de natação sobre a expressão de microRNAs envolvidos na angiogênese cardíaca de ratos. MATERIAIS E MÉTODOS: Ratas Wistar (n=21) foram divididas em grupos Sedentário (SC), Treinado 1 (P1): natação 60min/dia, 5x/sem/10sem, com 5% de sobrecarga, Treinado 2 (P2): mesmo protocolo P1 até a 8ªsem, 9ªsem 2x/dia, e na 10ªsem 3x/dia. Após o período de treinamento, os corações foram retirados e o RNA total foi isolado para a análise da expressão de miRNAs no coração por microarray de miRNA e os miR-126, -let-7f, -221 e -222 foram confirmados por RT-PCR em tempo real. Analisamos ainda os alvos do miR-126, Spred-1 e PI3KR2, e a expressão de proteínas que compõem as vias de sinalização em que esses alvos interferem por Western Blotting. Avaliamos também: Frequência cardíaca (FC) e pressão arterial (PA) por pletismografia caudal, VO2 pico, hipertrofia cárdica (HC) pelo peso do Ventrículo esquerdo/Peso corporal (mg/g), razão capilar/fibra (C/F) por histologia, expressão proteica de VEGF e seus receptores. RESULTADOS: O treinamento aeróbico diminuiu a FC sem alterar a PA, VO2 pico aumentou 11% e 15% em P1 e P2, a HC foi de 17% e 30% em P1 e P2, a razão C/F aumentou 57% e 100% em P1 e P2, acompanhada de um aumento da expressão de VEGF (P1 =42%, P2 =109%). A expressão do miR-let-7f foi aumentada em P2 (140%) comparado aos outros dois grupos (SC = 100%; P1 = 113%), o miR- 221 teve sua expressão diminuida em ambos os grupos treinados comparados xiii ao grupo SC (SC = 100%; P1 = 71%; P2 = 74%), o miR-222 não apresentou diferença na sua expressão entre os grupos (SC = 100%; P1 = 76%; P2 = 81%) e a expressão do miR-126 foi aumentada em P1 (126%) e P2 (142%) comparados ao grupo SC, a expressão de ambos os alvos desse miRNA foi diminuida nos grupos treinados (Spred-1 SC = 100 ± 12,4; P1 = 60 ± 5,6; P2 = 61 ± 8,4; PI3KR2 100 ± 12,3; P1 = 61 ± 12,3; P2 = 21 ± 7,1). Essa diminuição da expressão dos alvos desse miRNA favoreceu um aumento da expressão de proteínas pertencentes as vias de sinalização da PIK3 e MAPKs. CONCLUSÃO: O treinamento aeróbico foi eficaz em promover um aumento da angiogênese cardíaca comprovada por uma maior razão capilar/fibra no coração dos animais treinados e por maior expressão proteica de VEGF, sendo ainda mais evidente nos animais que realizaram um maior volume de treinamento. Os miRNAs relacionados à angiogênese parecem estar envolvidos na regulação desse processo. Além disso, o miR-126 parece ser um dos principais miRNAs envolvidos nesse processo / INTRODUCTION: The cardiovascular adaptations resulting from aerobic physical swimming training are well described in the literature, between these adaptations we have the angiogenesis. The aerobic physical training is one of the stimulus that promotes angiogenesis. The micro RNAs are a class of non coding protein RNAs of different cells in different tissues and are involved in angiogenic processes, but the role of micro RNAs in cardiac angiogenesis due to aerobic training is not clear. OBJECTIVE: To analyze the effects of different volumes of swimming physical training on the expression of micro RNAs involved in angiogenesis in heart of rats. MATERIALS AND METHODS: Wistar female rats (n=21) were divided into groups Sedentary (SC), Trained 1 (P1): 60min/day swimming, 5x/week/10weeks with 5% overload, Trained 2 (P2): same protocol of P1 until the 8th week, 9th week 2x/day, and at 10th week 3x/day. After the training period, the hearts were removed and the total RNA was isolated to analyze the miRNAs expression in the heart by microarray of miRNA and the miRs-126, -let-7f, -221 and -222 were confirmed by real time RT-PCR. We also analyzed the targets miR-126, Spred-1 and PI3KR2, and the protein expression that form the signaling pathways that affect those targets by Western Blotting. We evaluated: heart rate (HR) and blood pressure (BP) by tail plethysmography, peak VO2, cardiac hypertrophy (CH) by weight left ventricle/ corporal weight (mg/g), capillary/fiber (C/P) ratio for histology, VEGF protein expression and its receptors. RESULTS: The aerobic training decreased the HR without change the BP, peak VO2 increased 11% and 15% in P1 and P2, the HR was 17% and 30% in P1 and P2, the ratio C/F increased 57% and 100% in P1 and P2, followed by an increase of VEGF expression (P1=42%, P2=109%). The miR-let-7f had its expression increased in P2 (140%) compared to the other two groups (SC = 100%; P1 = 113%), o miR-221 had its expression decreased in both trained groups compared to SC group (SC = 100%; P1 = 71%; P2 = 74%), the miR-222 showed no difference on its expression between the groups (SC = 100%; P1 = 76%; P2 = 81%) and the miR-126 expression xv was higher in P1 (126%) and P2 (142%) compared to SC group, the expression of both targets of this miRNA was decreased on trained groups (Spred-1 SC = 100 ± 12,4; P1 = 60 ± 5,6; P2 = 61 ± 8,4; PI3KR2 100 ± 12,3; P1 = 61 ± 12,3; P2 = 21 ± 7,1). This decrease of expression of this miRNA targets favor an increase of protein expression belonging to the PIK3 and MAPKs signaling pathways. CONCLUSIONS: The aerobic training was effective on promoting an increased of cardiac angiogenesis comproved by a higher ratio capillary/ fiber on the heart of trained animals and by a higher VEGF protein expression, being even more evident in animals that realized a higher volume training. The miRNAs related to angiogenesis seem to be involved in the regulation of this process. Besides that, the miR-126 seems to be one of the principal miRNA involved on this process

Lai, Tsz Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-136). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Content / List of tables / List of figures / List of abbreviations / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Introduction of food market trends in Hong Kong and mushroom productivity in the world --- p.1 / Chapter 1.1.1 --- Agrocybe aegerita --- p.1 / Chapter 1.1.2 --- Pleurotus spp --- p.2 / Chapter 1.1.3 --- Pholiota nameko --- p.3 / Chapter 1.2 --- Objectives --- p.5 / Chapter Chapter 2: --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.6 / Chapter 2.1 --- Introduction --- p.6 / Chapter 2.1.1 --- Reactive oxygen species (ROS) --- p.6 / Chapter 2.1.1.1 --- Definition of ROS --- p.6 / Chapter 2.1.1.2 --- Sources of ROS --- p.6 / Chapter 2.1.1.2.1 --- Endogenous sources of ROS --- p.6 / Chapter 2.1.1.2.2 --- Exogenous sources of ROS --- p.8 / Chapter 2.1.1.3 --- Damaging effects of ROS --- p.8 / Chapter 2.1.2 --- Antioxidants --- p.10 / Chapter 2.1.2.1 --- Mechanism of action --- p.10 / Chapter 2.1.2.2 --- Sources of antioxidants --- p.11 / Chapter 2.1.2.2.1 --- Dietary antioxidants --- p.11 / Chapter 2.1.2.2.2 --- Antioxidants in edible mushrooms --- p.12 / Chapter 2.1.2.2.3 --- Phenolic compounds in mushrooms --- p.13 / Chapter 2.2 --- Materials and Methods --- p.16 / Chapter 2.2.1 --- Materials --- p.16 / Chapter 2.2.1.1 --- Mushroom fruiting bodies --- p.16 / Chapter 2.2.2 --- Principles of Methods and Experimental Protocols --- p.17 / Chapter 2.2.2.1 --- Sample preparation --- p.17 / Chapter 2.2.2.2 --- Evaluation of antioxidant capacity --- p.18 / Chapter 2.2.2.2.1 --- DPPH radical scavenging activity --- p.18 / Chapter 2.2.2.2.2 --- Superoxide anion scavenging activity --- p.19 / Chapter 2.2.2.2.3 --- Hydroxyl radical scavenging activity --- p.20 / Chapter 2.2.2.2.4 --- Hydrogen peroxide scavenging activity --- p.22 / Chapter 2.2.2.3 --- Determination of phenolic compounds --- p.24 / Chapter 2.2.2.3.1 --- Total phenolic content --- p.24 / Chapter 2.2.2.3.2 --- Identification of phenolic acids --- p.25 / Chapter 2.2.3 --- Statistical analysis --- p.27 / Chapter 2.3 --- Results and Discussion --- p.28 / Chapter 2.3.1 --- Extraction yield --- p.28 / Chapter 2.3.2 --- Evaluation of antioxidant capacity --- p.29 / Chapter 2.3.2.1 --- DPPH radical scavenging activity --- p.29 / Chapter 2.3.2.2 --- Superoxide anion scavenging activity --- p.31 / Chapter 2.3.2.3 --- Hydroxyl radical scavenging activity --- p.33 / Chapter 2.3.2.4 --- Hydrogen peroxide scavenging activity --- p.35 / Chapter 2.3.2.5 --- Comparison of the effective concentrations (EC50) of mushroom water extracts in different antioxidant assays --- p.37 / Chapter 2.3.3 --- Determination of phenolic compounds --- p.38 / Chapter 2.3.3.1 --- Total phenolic content --- p.38 / Chapter 2.3.3.2 --- Identification of phenolic acids --- p.39 / Chapter 2.4 --- Summary --- p.45 / Chapter Chapter 3: --- Anti-angiogenic properties of the Aa water extract --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.1.1 --- Angiogenesis --- p.46 / Chapter 3.1.1.1 --- Process of angiogenesis --- p.46 / Chapter 3.1.1.2 --- Regulations of angiogenesis --- p.47 / Chapter 3.1.1.2.1 --- Fibroblast growth factor (bFGF) --- p.47 / Chapter 3.1.1.2.2 --- Vascular endothelial growth factor (VEGF) --- p.48 / Chapter 3.1.2 --- Tumor angiogenesis --- p.49 / Chapter 3.1.2.1 --- ROS generation in tumor cells --- p.50 / Chapter 3.1.2.2 --- Hydrogen peroxide and VEGF --- p.51 / Chapter 3.1.2.3 --- Previous studies on tumor angiogenesis --- p.52 / Chapter 3.1.2.3.1 --- ROS and endothelial cells proliferation --- p.52 / Chapter 3.1.2.3.2 --- VEGF and endothelial cells functions --- p.53 / Chapter 3.1.3 --- Use of antioxidants in cancer treatment --- p.53 / Chapter 3.1.3.1 --- Antioxidant use of cancer therapy --- p.53 / Chapter 3.1.3.2 --- Antioxidant and endothelial cells functions --- p.54 / Chapter 3.1.3.3 --- Anti-angiogenic effects of polyphenols --- p.56 / Chapter 3.1.3.3.1 --- Phenolic acids --- p.56 / Chapter 3.1.3.3.2 --- Tea catechin --- p.57 / Chapter 3.1.3.3.3 --- Resveratrol --- p.57 / Chapter 3.1.3.3.4 --- Genistein --- p.58 / Chapter 3.2 --- Principles of Methods and Experimental Protocols --- p.60 / Chapter 3.2.1 --- Sample preparation --- p.60 / Chapter 3.2.2 --- Toxicity of the Aa water extract --- p.60 / Chapter 3.2.2.1 --- Limulus amebocyte lysate (LAL) test --- p.60 / Chapter 3.2.2.2 --- Toxicity towards normal cells --- p.61 / Chapter 3.2.2.2.1 --- Cell line and its subculture --- p.61 / Chapter 3.2.2.2.2 --- Colorimetric (MTT) assay --- p.62 / Chapter 3.2.3 --- Effect of the Aa water extract on cancer cells --- p.63 / Chapter 3.2.3.1 --- Cell line and its subculture --- p.63 / Chapter 3.2.3.2 --- Redox status --- p.63 / Chapter 3.2.3.3 --- VEGF secretion --- p.65 / Chapter 3.2.4 --- In vitro cell culture anti-angioenesis analysis --- p.66 / Chapter 3.2.4.1 --- Cell line and its subculture --- p.66 / Chapter 3.2.4.2 --- Endothelial cells proliferation --- p.67 / Chapter 3.2.4.3 --- Endothelial cells migration --- p.68 / Chapter 3.2.4.3.1 --- Wound healing assay --- p.68 / Chapter 3.2.4.3.2 --- Transwell culture insert assay --- p.69 / Chapter 3.2.4.4 --- Endothelial cells tubule formation --- p.71 / Chapter 3.2.5 --- In vitro organ culture anti-angiogenesis analysis --- p.72 / Chapter 3.2.5.1 --- Aortic ring assay --- p.72 / Chapter 3.2.6 --- Statistical analysis --- p.74 / Chapter 3.3 --- Results and Discussions --- p.75 / Chapter 3.3.1 --- Toxicity of the Aa water extract --- p.75 / Chapter 3.3.1.1 --- Limulus amebocyte lysate (LAL) test --- p.75 / Chapter 3.3.1.2 --- Toxicity towards normal cells --- p.75 / Chapter 3.3.2 --- Effect of the Aa water extract on cancer cells --- p.77 / Chapter 3.3.2.1 --- Redox status --- p.77 / Chapter 3.3.2.2 --- VEGF secretion --- p.79 / Chapter 3.3.2.3 --- Relationship between intracellular ROS and VEGF secretion detected --- p.80 / Chapter 3.3.3 --- Effect of the Aa water extract on angiogenesis --- p.82 / Chapter 3.3.3.1 --- Endothelial cells proliferation --- p.82 / Chapter 3.3.3.2 --- Endothelial cells migration --- p.84 / Chapter 3.3.3.2.1 --- Wound healing assay --- p.84 / Chapter 3.3.3.2.2 --- Transwell culture insert assay --- p.87 / Chapter 3.3.3.3 --- Endothelial cells tubule formation --- p.90 / Chapter 3.3.3.4 --- Aortic ring assay --- p.97 / Chapter 3.3.4 --- Effect of phenolic acids on endothelial cells --- p.101 / Chapter 3.3.4.1 --- Endothelial cells proliferation --- p.101 / Chapter 3.3.4.2 --- Endothelial cells migration --- p.102 / Chapter 3.3.4.2.1 --- Wound healing assay --- p.102 / Chapter 3.3.4.2.2 --- Transwell culture insert assay --- p.105 / Chapter 3.3.4.3 --- Endothelial cells tubule formation --- p.106 / Chapter 3.3.4.4 --- Aortic ring assay --- p.112 / Chapter 3.4 --- Summary --- p.116 / Chapter Chapter 4 --- Conclusions and future works --- p.118 / References --- p.121

Diss. (sammanfattning) Uppsala : Univ., 2005. / Härtill 4 uppsatser.

INTRODUÇÃO: As adaptações cardiovasculares decorrentes do treinamento físico aeróbio de natação são bem descritas na literatura, entre ela temos a angiogênese. O treinamento físico aeróbio é um dos estímulos que promove angiogênese. Os microRNAs (miRNAs) são uma classe de RNA não codificadora de proteínas de diferentes células em diversos tecidos e estão envolvidos em processos angiogênicos, mas o papel dos miRNAs na angiogênese cardíaca decorrente ao treinamento aeróbico ainda não foi esclarecido. OBJETIVO: Analisar os efeitos de diferentes volumes de treinamento físico de natação sobre a expressão de microRNAs envolvidos na angiogênese cardíaca de ratos. MATERIAIS E MÉTODOS: Ratas Wistar (n=21) foram divididas em grupos Sedentário (SC), Treinado 1 (P1): natação 60min/dia, 5x/sem/10sem, com 5% de sobrecarga, Treinado 2 (P2): mesmo protocolo P1 até a 8ªsem, 9ªsem 2x/dia, e na 10ªsem 3x/dia. Após o período de treinamento, os corações foram retirados e o RNA total foi isolado para a análise da expressão de miRNAs no coração por microarray de miRNA e os miR-126, -let-7f, -221 e -222 foram confirmados por RT-PCR em tempo real. Analisamos ainda os alvos do miR-126, Spred-1 e PI3KR2, e a expressão de proteínas que compõem as vias de sinalização em que esses alvos interferem por Western Blotting. Avaliamos também: Frequência cardíaca (FC) e pressão arterial (PA) por pletismografia caudal, VO2 pico, hipertrofia cárdica (HC) pelo peso do Ventrículo esquerdo/Peso corporal (mg/g), razão capilar/fibra (C/F) por histologia, expressão proteica de VEGF e seus receptores. RESULTADOS: O treinamento aeróbico diminuiu a FC sem alterar a PA, VO2 pico aumentou 11% e 15% em P1 e P2, a HC foi de 17% e 30% em P1 e P2, a razão C/F aumentou 57% e 100% em P1 e P2, acompanhada de um aumento da expressão de VEGF (P1 =42%, P2 =109%). A expressão do miR-let-7f foi aumentada em P2 (140%) comparado aos outros dois grupos (SC = 100%; P1 = 113%), o miR- 221 teve sua expressão diminuida em ambos os grupos treinados comparados xiii ao grupo SC (SC = 100%; P1 = 71%; P2 = 74%), o miR-222 não apresentou diferença na sua expressão entre os grupos (SC = 100%; P1 = 76%; P2 = 81%) e a expressão do miR-126 foi aumentada em P1 (126%) e P2 (142%) comparados ao grupo SC, a expressão de ambos os alvos desse miRNA foi diminuida nos grupos treinados (Spred-1 SC = 100 ± 12,4; P1 = 60 ± 5,6; P2 = 61 ± 8,4; PI3KR2 100 ± 12,3; P1 = 61 ± 12,3; P2 = 21 ± 7,1). Essa diminuição da expressão dos alvos desse miRNA favoreceu um aumento da expressão de proteínas pertencentes as vias de sinalização da PIK3 e MAPKs. CONCLUSÃO: O treinamento aeróbico foi eficaz em promover um aumento da angiogênese cardíaca comprovada por uma maior razão capilar/fibra no coração dos animais treinados e por maior expressão proteica de VEGF, sendo ainda mais evidente nos animais que realizaram um maior volume de treinamento. Os miRNAs relacionados à angiogênese parecem estar envolvidos na regulação desse processo. Além disso, o miR-126 parece ser um dos principais miRNAs envolvidos nesse processo / INTRODUCTION: The cardiovascular adaptations resulting from aerobic physical swimming training are well described in the literature, between these adaptations we have the angiogenesis. The aerobic physical training is one of the stimulus that promotes angiogenesis. The micro RNAs are a class of non coding protein RNAs of different cells in different tissues and are involved in angiogenic processes, but the role of micro RNAs in cardiac angiogenesis due to aerobic training is not clear. OBJECTIVE: To analyze the effects of different volumes of swimming physical training on the expression of micro RNAs involved in angiogenesis in heart of rats. MATERIALS AND METHODS: Wistar female rats (n=21) were divided into groups Sedentary (SC), Trained 1 (P1): 60min/day swimming, 5x/week/10weeks with 5% overload, Trained 2 (P2): same protocol of P1 until the 8th week, 9th week 2x/day, and at 10th week 3x/day. After the training period, the hearts were removed and the total RNA was isolated to analyze the miRNAs expression in the heart by microarray of miRNA and the miRs-126, -let-7f, -221 and -222 were confirmed by real time RT-PCR. We also analyzed the targets miR-126, Spred-1 and PI3KR2, and the protein expression that form the signaling pathways that affect those targets by Western Blotting. We evaluated: heart rate (HR) and blood pressure (BP) by tail plethysmography, peak VO2, cardiac hypertrophy (CH) by weight left ventricle/ corporal weight (mg/g), capillary/fiber (C/P) ratio for histology, VEGF protein expression and its receptors. RESULTS: The aerobic training decreased the HR without change the BP, peak VO2 increased 11% and 15% in P1 and P2, the HR was 17% and 30% in P1 and P2, the ratio C/F increased 57% and 100% in P1 and P2, followed by an increase of VEGF expression (P1=42%, P2=109%). The miR-let-7f had its expression increased in P2 (140%) compared to the other two groups (SC = 100%; P1 = 113%), o miR-221 had its expression decreased in both trained groups compared to SC group (SC = 100%; P1 = 71%; P2 = 74%), the miR-222 showed no difference on its expression between the groups (SC = 100%; P1 = 76%; P2 = 81%) and the miR-126 expression xv was higher in P1 (126%) and P2 (142%) compared to SC group, the expression of both targets of this miRNA was decreased on trained groups (Spred-1 SC = 100 ± 12,4; P1 = 60 ± 5,6; P2 = 61 ± 8,4; PI3KR2 100 ± 12,3; P1 = 61 ± 12,3; P2 = 21 ± 7,1). This decrease of expression of this miRNA targets favor an increase of protein expression belonging to the PIK3 and MAPKs signaling pathways. CONCLUSIONS: The aerobic training was effective on promoting an increased of cardiac angiogenesis comproved by a higher ratio capillary/ fiber on the heart of trained animals and by a higher VEGF protein expression, being even more evident in animals that realized a higher volume training. The miRNAs related to angiogenesis seem to be involved in the regulation of this process. Besides that, the miR-126 seems to be one of the principal miRNA involved on this process

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.