Diabetes mellitus is a growing public health concern, presently affecting 25.8 million or 8.3% of the American population. While the availability of novel drugs, techniques, and surgical intervention has improved the survival rate of individuals with diabetes, the prevalence of diabetes is still rising. Type 2 diabetes (T2D) is a result of chronic insulin resistance and loss of -cell mass and function, and it is is always associated with the impairment in energy metabolism, causing increased intracellular fat content in skeletal muscle (SkM), liver, fat, as well as pancreatic islets. As such, the search for novel agents that simultaneously promotes insulin sensitivity and 𝜷-cell survival may provide a more effective strategy to prevent the onset and progression of this disease. Kaempferol is a flavonol that has been identified in many plants and used in traditional medicine. It has been shown to elicit various pharmacological activities in epidemiological and preclinical studies. However, to date, the studies regarding its effect on the pathogenesis of diabetes are very limited. In this dissertation, I explored the anti-diabetic potential of the dietary intake of kaempferol in diet-induced obese mice and insulin-deficient diabetic mice. For the first animal study, kaempferol was supplemented in the diet to determine whether it can prevent insulin resistance and hyperglycemia in high fat (HF) diet-induced obese mice or STZ-induced obese diabetic mice. For the second animal study, kaempferol was administrated once daily via oral gavage to diet-induced obese and insulin-resistant mice or lean STZ-induced diabetic mice to evaluate its efficacy for treating diabetes and further determining the underlying mechanism. The results demonstrated that dietary intake of kaempferol for 5 months (mo) improved insulin sensitivity and glucose tolerances, which were associated with increased Glut4 and AMPKα expression in muscle and adipose tissues in middle-aged mice fed a high-fat (HF) diet. In vitro, kaempferol increased lipolysis and restored chronic high fatty acid-impaired glucose uptake and glycogen synthesis in SkM cells, which were associated with improved AMPKα activity and Glut4 expression. In addition, dietary kaempferol treatment preserved functional pancreatic 𝜷-cell mass and prevented hyperglycemia and glucose intolerance in STZ-induced diabetic mice. Data from the second study show that oral administration of kaempferol significantly improved blood glucose control in obese mice, which was associated with reduced hepatic glucose production and improved whole body insulin sensitivity without altering body weight gain, food consumption, or the adiposity. In addition, kaempferol treatment increased Akt and hexokinase activity, but decreased pyruvate carboxylase and glucose-6 phosphatase activity in the liver homogenate without altering their protein expression. Consistently, kaempferol decreased pyruvate carboxylase activity and suppressed gluconeogenesis in HepG2 cells as well as primary hepatocytes isolated from the livers of obese mice. Kaempferol directly blunted the activity of purified pyruvate carboxylase. In the last study, we found that kaempferol stimulates basal glucose uptake in primary human SkM. In C2C12 mouse myotubes, kaempferol also increased insulin stimulated glycogen synthesis and preserved insulin dependent glycogen synthesis and glucose uptake in the presence of fatty acids. Kaempferol stimulated Akt phosphorylation in a similar time-dependent manner as insulin in human SkM cells. Consistent with this, kaempferol increased Akt and AMPK phosphorylation in isolated murine red SkM tissue. The effect of kaempferol on glucose uptake was blunted in the presence of chemical inhibitors of glucose transporter 4 (Glut4), phosphoinositide 3-kinase (PI3K), glucose transporter 1 (Glut1), and AMPK. The AMPK inhibitor also prevented kaempferol-stimulated Akt phosphorylation. Further, kaempferol improved the stability of insulin receptor substrate-1. Taken together, these studies suggest that the kaempferol is a naturally occurring compound that may be of use in the regulation of glucose homeostasis and diabetes by improving insulin sensitivity and glucose metabolism, as well as by preserving functional 𝜷-cell mass. / Ph. D.

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.

No description available.

Le syndrome d'apnée obstructive du sommeil (SAOS), est une maladie chronique qui se caractérise par des interruptions répétées de la respiration durant le sommeil en raison de la fermeture temporaire des voies aériennes supérieures. L'hypoxie intermittente chronique (HI) résultante de cette fermeture transitoire des voies aériennes supérieures, constitue l’une des conséquences majeures du SAOS, et elle est la responsable de la plupart des complications liées à cette pathologie, dont nous citons: l’hypertension artérielle, l’infarctus de myocarde et plus généralement le remodelage cardiovasculaire.D’autre part, l’entrainement physique intensif(EI)est bien connu d’avoir des bénéfices sur le système cardiovasculaire, d’où nous avons poser l’hypothèse que l’EI peut inverser les effets délétères de l’HI sur la réactivité et le remodelage vasculaire ainsi que sur la signalisation calcique intracellulaire dans les cellules musculaires.Pour répondre à cette question, nous avons choisi le rat comme modèle animal, pour étudier l’effet potentiel de l'EI dans la prévention et l’inversion des effets délétères de (HI) en termes de réactivité et signalisation calcique dans les tissues musculaires.Des rats ont été exposés durant 21 jours à l’hypoxie intermittente dans des cages spécialement équipées pour maintenir un flux d’air alternant entre 21% et 5% de PO2 dans les cages contenant les rats hypoxique et a 21% de PO2 dans les cages contenant les rats contrôles. Durant les deux dernières semaines d’exposition à l’HI, un groupe des rats hypoxiques et un des rats normoxiques ont subi des sessions d'EI en courant sur un tapis roulant avec une vitesse allant de 16m/min jusqu'à 30 m/min.Les paramètres physiologiques ont été mesurés (Pression artérielle, fréquence cardiaque, hématocrites), l’aorte a été prélevé pour étudier la réactivité vasculaire, les cellules musculaires lisses de l’aorte ont été ensuite prélevés et cultivées pour étudier la signalisation calcique par microscopie à EPIfluorescence. Finalement les gènes codant pour les médiateurs de la signalisation calcique : RyR1, RyR2 RyR3, (ryanodine receptors), TRPV4 (transient receptor potential channel), SERCA1, SERCA2 (Sarco/Endoplasmic Reticulum Ca2+ -ATPase) et IP3R1 (Inositol 1,4,5-Trisphosphate Receptor) dans différentes tissues vasculaires et squelettiques ont été étudiés au niveau moléculaire par Q-PCR et Western Blot.Nos résultats montrent que l'HI induit une augmentation significative de pression artérielle et de l’hématocrite et une diminution dans la relaxation de l'aorte induite par l'acétylcholine pré contractée par la phénylnephrine. Ceci est conforme à notre observation selon laquelle HI augmente le niveau de calcium intracellulaire dans le muscle lisse aortique cultivé. D'autre part, l'EI induit une diminution significative de l’hématocrite et de la vasoconstriction aortique induite par la phénylnephrine et l'endothélie-1, conformément à l'observation que l'EI réduit la différence HI-N dans la réponse calcique. A l’échelle moléculaire, HI induit une augmentation significative de l'expression de RyR1, RyR2, RyR3, SERCA1, SERCA2, TRPV4 et IP3R1 au niveau de l'ARNm dans les tissus de tous les groupes, avec une plus grande quantité de RyR1,RyR2,et RyR3 dans les tissus HI des muscles lisses (principalement dans l'aorte thoracique et abdominale) et le SERCA1 (9 fois plus haut dans les tissus IH) et le SERCA2 (10 fois plus élevé dans les tissus HI) dans les muscles squelette (Gastrocnemius, plantaris et soléus). De plus, HI induit une augmentation significative de RYR1, RYR2 et TRPV4 au niveau protéique dans l'aorte thoracique et abdominale; et l'EI réduit la différence d'expression entre les animaux N et IH.Nos résultats suggèrent que l'EI représente un traitement prometteur non pharmacologique ou complémentaire pour limiter les complications cardio-vasculaires induites par l’HI et le remodelage musculaire chez les patients atteints de SAOS. / Obstructive sleep apnea syndrome (OSAS) is a chronic disease characterized by repeated interruptions of breathing during sleep due to the temporary closure of the upper airway. Its prevalence increases with the increasing in prevalence of obesity, especially in developed countries.Chronic intermittent hypoxia (IH) resulting from this transient closure of the upper airway is one of the major consequences of OSAS and is responsible of most of the complications related to this pathology, including hypertension, myocardial infarction, atherosclerosis and more generally cardiovascular remodeling.On the other hand, intensive physical training(IT) is well known to have benefits on cardiovascular system, thus we hypothesize that physical training can reverse the deleterious effects of IH on reactivity and vascular remodeling as well as intracellular calcium signaling in muscle cells.To answer this question, we chose the rat as an animal model to study the potential effect of IT in the prevention and reversal of deleterious (IH) effects in terms of reactivity and calcium signaling in muscle tissue.Rats were exposed for 21 days to intermittent hypoxia and housed in cages specially equipped to maintain an airflow alternating between 21% and 5% PO2 in cages containing hypoxic rats and 21% PO2 in cages containing the control rats. During the last two weeks of exposure to IH, a group of hypoxic rats and one of the normoxic rats underwent IT sessions on a treadmill at a speed of 16m / min to 30m / min.Physiological parameters were measured (blood pressure, heart rate, hematocrit), the aorta was removed to study the vascular reactivity, then vascular smooth muscle cells were removed and cultured to study calcium signaling by EPIfluorescence microscopy. Finally, the genes coding for the key mediators of the calcium signaling: RyR1, RyR2 RyR3, (ryanodine receptors), TRPV4 (transient receptor potential channel), SERCA1, SERCA2 (Sarco / Endoplasmic Reticulum Ca2 + -ATPase) and IP3R1 , 5-Trisphosphate Receptor) in various vascular and skeletal tissues were studied at the molecular level as mRNA by Q-PCR or as protein by Western Blot.Our results show that IH induces a significant increase in blood pressure and hematocrit and a decrease in acetylcholine-induced aortic relaxation pre-contracted with phenylnephrine. This was consistent with our observation that HI increases the level of intracellular calcium in cultured aortic smooth muscle. On the other hand, IT induced a significant decrease in hematocrit and aortic vasoconstriction induced by phenylnephrine and endothelial-1, consistant with the observation that IT reduces the IH-N difference in the calcium response. On the molecular scale, IH induces a significant increase in the expression of RyR1, RyR2, RyR3, SERCA1, SERCA2, TRPV4 and IP3R1 at the mRNA level in the tissues of all groups with a greater amount of RyR1,RyR2,& RyR3 higher in IH tissue of smooth muscles (mainly in the thoracic and abdominal aorta) and SERCA1 (9-fold higher in IH tissues) and SERCA2 (10-fold higher in IH tissues) in the skeletal muscles (Gastrocnemius, plantaris and soléus). In addition, IH induces a significant increase in RYR1, RYR2 and TRPV4 at the protein level in the thoracic and abdominal aorta; And IT reduces the difference in expression between animals N and IH.Our results suggest that IT is a promising, non-pharmacological or complementary treatment for limiting cardiovascular complications induced by IH and muscle remodeling in patients with OSAS.

Hintergrund und Zielsetzung: DMD ist die häufigste Form der Muskeldystrophie im Kindesalter und bis heute unheilbar. Sie wird durch das Fehlen des Proteins Dystrophin verursacht, welches verschiedene Signaltransduktionswege beeinflusst. Das Anliegen der Arbeit ist die Untersuchung und Modulation von Signaltransduktionswegen, die als alternative Therapiestrategie den Verlust von Dystrophin kompensieren könnten. Experimentelle Strategie: Für die Charakterisierung von Dystrophin nachgeschalteten Prozessen wurden mRNA-Expressionsanalysen in Muskelgeweben von DMD-Patienten und einem DMD-Brüderpaar mit einem infrafamiliär unterschiedlichen Verlauf der DMD durchgeführt. Aus diesen Expressionsdaten wurde erstmalig ein Petri-Netz entwickelt, welches Dystrophin mit in diesem Zusammenhang bisher unbekannten Signaltransduktionswegen verknüpft. Das Petri-Netz wurde auf Netzwerkintegrität und –verhalten mittels Invarianten- (INA) und theoretischen Knockout- (Mauritius Maps) Analysen untersucht. Durch beide Methoden läßt sich der maßgebliche Teilsignalweg bestimmen. In diesem Signalweg wurden die Proteinaktivität und die Genexpression durch siRNA, Vektor-DNA und chemische Substanzen in humanen SkMCs moduliert. Anschließend wurden die Proliferation und die Vitalität der Zellen sowie auch die Expression auf mRNA- und Protein-Niveau untersucht. Ergebnisse: RAP2B und CSNK1A1 waren in dem DMD-Brüderpaar differentiell exprimiert und konnten erstmalig in einem neuen, komplexen Signalweg in Zusammenhang mit Dystrophin nachgeschalteten Prozessen dargestellt werden. Mittelpunkt dieses Signalweges ist die De- und Aktivierung des Transkriptionsfaktors NFATc. Seine Zielgene umfassen neben anderen den negativen Proliferationsfaktor p21, das Dystrophin homologe UTRN und den Differenzierungsfaktor MYF5. Folglich würde ein Anstieg von UTRN eine unerwünschte Reduktion der Proliferationsrate von Myoblasten implizieren. Letzteres konnte bereits nachgewiesen werden und stellte das Motiv für weitere Studien dar. Jedoch zeigten siRNA- und Vektor-DNA-Experimente, daß NFATc nicht der ausschlaggebende Faktor für diese Zielgene ist. Die Substanzen Deflazacort (DFZ) und Cyclosporin A (CsA) wurden dagegen beschrieben, die Aktivierung von NFATc zu beeinflussen. Die Ergebnisse zeigten, daß beide Substanzen die Proliferation von Myoblasten erhöhen können. Die gleichzeitige Applikation von DFZ und CsA führte zu einem Anstieg der UTRN-Expression. Schlußfolgerung: Die Modulation der Proliferation und UTRN-Expression ist unabhängig von einander möglich. Entsprechend der Grundidee der Arbeit zeichnet sich eine neue Therapiestrategie ab, welche Dystrophin nachgeschaltete Prozesse einbezieht. / Background and aim: DMD is the most common muscular dystrophy in childhood and incurable to date. It is caused by the absence of dystrophin, what influences several signal transduction pathways. The thesis is interested in the investigation and modulation of signal transduction pathways that may compensate the lack of dystrophin as an alternative therapy strategy. Experimental strategy: To study Dystrophin downstream pathways the mRNA expression of DMD patients and two DMD siblings with an intra-familially different course of DMD were analysed in muscle tissue. On the basis of these expression data a Petri net was first developed implicating signal transduction pathways and Dystrophin downstream cascades. Invariant (INA) and theoretical knockout (Mauritius Maps) analyses were applied for studying network integrity and behaviour. Both methods provide information about the most relevant part of the network. In this part modulation of protein activity and of gene expression using siRNA, vector-DNA, and chemical substances were performed on human SkMCs. Subsequently, the cells were studied by proliferation and vitality tests as well as expression analyses at mRNA and protein level. Results: RAP2B and CSNK1A1 were differently expressed in two DMD siblings, and first are part of a signal transduction pathway implicating Dystrophin downstream processes. The central point of this pathway is the de- and activation of the transcription factor NFATc. Its target genes are, among others, the negative proliferation factor p21, the Dystrophin homologue UTRN, and the differentiation factor MYF5. Consequently, an increase in UTRN implicates an undesirably reduced myoblast proliferation rate. Latter was found in DMD patients and was target for further studies. But, siRNA and vector DNA experiments showed that NFATc is not the decisive factor for the target genes. Deflazacort and cyclosporin A are known to influence the activation of NFATc. The results first showed that both substances do induce myoblast proliferation. The use of deflazacort in combination with cyclosporin A resulted in an increase of UTRN expression. Conclusion: The modulation of proliferation and UTRN-expression independently of each other is possible. According to the basic idea of this study, a new therapeutic strategy becomes apparent, which considers Dystrophin downstream processes.