Thesis (doctoral)--Rijksuniversiteit te Groningen, 1980. / Summary and vita in Dutch. Highlights sheet in Dutch inserted. Vita. Includes bibliographical references (p. 125-140).

Thesis (Ph.D.)--York University, 2009. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 275-298). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR51670

Tissue regeneration in mammals does not occur via the process of dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state. McGann et al. (2001) showed that mouse C2C12 myotubes treated with newt extract derived from regenerating limbs can re-enter the cell cycle, fragment and proliferate ( a characteristic of muscle dedifferentiation). However, the validity of these studies has been called into question since others have been unable to repeat them. My research attempts to replicate the results of McGann et al, and to carry them further. I examined several strategies for tracking the extract-treated cells and I also repeated the studies in a primary muscle culture system. Furthermore, I examined the effect of the extract on myoblast differentiation. The most effective dedifferentiation assay that I developed involved the microinjection of myotubes with extract and with a GFP plasmid that allowed tracking of the injected cells. Cells were then examined for cell cycle re-entry using BrdU incorporation or Ki-67 immunostaining. In addition, immunocytochemistry and RT-PCR analysis were used to examine the expression or down-regulation of muscle-specific markers. Finally, a preliminary GeneChip analysis was conducted to examine which genes were up or down regulated following extract treatment. The results show that newt extract is able to block the differentiation of confluent myoblasts, resulting in fewer multinucleated, myosin heavy chain expressing myotubes. However, when myoblasts were differentiated into myotubes and subsequently treated with newt extract, the results suggest that cell cycle re-entry and down-regulation of differentiation markers can occur in C2C12 myotubes, but not in primary myotubes. Fragmentation though, was seen in both C2C12 and primary myotubes following treatment or injection with newt extract. Moreover, the fragmented cells appeared to be viable. Transcriptional profiling indicated that newt extract affects genes implicated in cell cycle, transcription, stress, chromatin modification, growth, cell adhesion, extracellular matrix, wound healing and microtubule binding. These findings confirm that mammalian myotubes can be induced to dedifferentiate following treatment with newt extract; however, a differential response was observed between C2C12 and primary muscle cells.

The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IκB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis.

L-type Ca2+ channels, found in vascular smooth muscle cells, function to control Ca2+ influx, which directly regulates the degree of tension in the vasculature. An influx of Ca2+ causes these cells to contract while inhibition of this channel causes muscle relaxation, a major goal in treating hypertension. Acute hypoxia inhibits, and chronic hypoxia enhances, Ca2+ channel currents. The mechanisms underlying these hypoxic responses were examined in HEK 293 cells by altering cellular levels of proposed mediators of 0 2 sensing which have previously been shown to be involved in the redox model of 02 sensing in various cell types. In these studies I investigated the roles of mitochondrial complexes and NADPH oxidase function, and changes in cellular ROS levels, on the acute and chronically hypoxic regulation of recombinant L-type Ca2+ channels. An increase in H202, a form of ROS, by exogenous application was found to enhance Ca2+ currents. However neither catalase nor H202 affected the acute hypoxic response. In contrast superoxide dismutase (SOD) abolished hypoxic inhibition of recombinant L-type Ca2+ channels, suggestive of a role of 02- production in 02 sensing. Altered production of this ROS during hypoxia may occur within the mitochondria since acute 02 sensing was abolished in mitochondria-depleted p0 cells. Alterations in NADPH oxidase activity via application of NADPH oxidase inhibitors such as DPl and P AO did not mediate the acute hypoxic response. Hypoxic regulation of mitochondrial complex I may also mediate the response to chronic hypoxia since current enhancement by this stimulus was abolished by rotenone. These findings support the involvement of altered mitochondrial function in the 0 2 sensing pathway which mediates the hypoxic responses of recombinant L-type Ca2+ channel a1c subunits. / Thesis / Master of Science (MSc)

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. / Diabetes mellitus, more commonly referred to as diabetes, is a cause for concern in the context of public health. Currently, 25.8 million or 8.3% of the American population is affected by some type of diabetes. While the development of new drugs, techniques, and surgeries have improved the survival rate of individuals with diabetes, the number of diabetes cases continues to rise. Type 2 diabetes (T2D) is a result of the inability of tissues to respond to insulin and a loss of insulin producing β-cell mass and function. T2D is always associated with an impairment in the storage and release of energy, causing increased fat content in skeletal muscle (SkM), liver, and fat cells, as well as pancreatic islets. As such, the search for new agents that simultaneously promotesthe ability of body tissues to respond to insulin 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 drug-like activities in incidence and distribution studies as well as in preclinical studies. However, to date, the studies regarding its effect on the onset and progression 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 added to the diet to determine whether it can prevent insulin resistance and high blood glucose in high fat (HF) diet-induced obese mice or chemically-induced obese diabetic mice. For the second animal study, kaempferol was given once daily via oral gavage to diet-induced obese and insulin-resistant mice or lean chemically-induced diabetic mice to evaluate its efficacy for treating diabetes and further determining its mechanism. The results demonstrated that dietary intake of kaempferol for 5 months (mo) improved insulin sensitivity and the ability of body tissues to respond to glucose, which were associated with increased expression of the insulin sensitive glucose transporter (Glut4) and a central regulator of metabolism (AMPKα) in muscle and adipose tissues in middle-aged mice fed a high-fat (HF) diet. In cell culture, kaempferol increased triglyceride breakdown and restored the ability of SkM cells to take up glucose and synthesize glycogen following long-term exposure to elevated fatty acids. These results were also associated with an improved AMPKα activity and Glut4 expression. In addition, kaempferol in the diet preserved functional pancreatic β-cell mass and prevented the development of high blood glucose and the inability of body tissues to respond to glucose in chemically-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 glucose production in the liver and an improved ability of the whole body to respond to insulin without altering body weight gain, food consumption, or fat storage. In addition, kaempferol treatment increased the activity of the final enzyme in glucose transport (Akt) and first enzyme (hexokinase) in glucose oxidation, but decreased the activity of the first and final regulatory enzymes in glucose production (pyruvate carboxylase and glucose-6 phosphatase respectively) without altering their protein expression. Consistently, kaempferol decreased pyruvate carboxylase activity and suppressed glucose production in HepG2 liver cells as well as primary liver isolated from obese mice. Kaempferol also directly blunted the activity of purified pyruvate carboxylase. In the last study, we found that kaempferol stimulates non-stimulated glucose uptake in primary human SkM. In C2C12 mouse muscle cells, kaempferol also increased insulin stimulated glycogen synthesis and prevented fatty acid impaired glycogen synthesis and glucose uptake stimulated by insulin. Kaempferol stimulated Akt phosphorylation (the active form of the enzyme) in a similar time-dependent manner as insulin in human SkM cells. Consistent with this, kaempferol increased Akt and AMPK phosphorylation in red SkM tissue from mice. The effect of kaempferol on glucose uptake was inhibited in the presence of chemical inhibitors of Glut4, phosphoinositide 3-kinase (an enzyme in the insulin signaling pathway), glucose transporter 1 (a basal glucose transporter), 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 responsiveness and glucose storage and breakdown, as well as by preserving functional β-cell mass.

Cardiovascular disease is the world’s number 1 killer. The cardiovascular system helps to pump blood throughout the human body and maintain a systemic balance. However, medial vascular calcification results when this system becomes off balance, such as in cases of high blood pressure leading to hypertension. Many factors are involved in this process, but the most important is the vascular smooth muscle cell phenotypic switch to osteoblast-like cells. When vascular smooth muscle cells are subject to mechanical stimuli, mechanotransduction occurs, causing an intracellular signaling cascade leading to a phenotypic switch associated with the Wnt signaling pathway and osteogenic markers. There is a lack of understanding of the defined linkages of pathways that lead to the development of the osteoblast-like cell type. Therefore, examining human aortic smooth muscle cells under hypertensive conditions could decrease the prevalence of cardiovascular disease worldwide.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-17T20:16:41Z No. of bitstreams: 1 Adriano Silvio dos Santos.pdf: 1418434 bytes, checksum: 4ac5ef78eb225e6693c107993f6ee978 (MD5) / Made available in DSpace on 2016-05-17T20:16:41Z (GMT). No. of bitstreams: 1 Adriano Silvio dos Santos.pdf: 1418434 bytes, checksum: 4ac5ef78eb225e6693c107993f6ee978 (MD5) Previous issue date: 2015-02-24 / Snakes venom of the Bothrops species induces a local inflammatory reaction, characterized by pain, edema, leukocyte migration and can be accompanied by tissue necrosis. The use of antivenom performs the function of neutralizing the greatest possible amount of circulating venom, thus minimizing its systemic effects, but its action does not extend to local manifestations, and thus require the use of another therapeutic option to control this reaction. The low level laser therapy (LLLT) is used as an alternative treatment in cases of muscle injury due to its biological effects, such as analgesics, anitinflamatory and healing. In a previous study of our lab it was found that LBP can enhance the viability of C2C12 muscle cells after the addition of B. jararacussu venom in the medium and that this effect of LBP is related to protection of the cell membrane. In the present study we analyzed the effect of LBP in the cell monolayer integrity, viability of muscle cells, exposed to injury by myotoxins BthTX - I - and BthTX - II isolated from Bothrops jararacussu venom. Cells received BthTX – I (75 μg / mL) and were immediately irradiated with LLLT Aluminum Indium Gallium Phosphate and Aluminium Gallium Arsenide, the wavelengths (λ) 685nm and 830 nm, power density 4 J/cm2, 100mW of power, total energy 1,3 J, application time of 13 and 35 seconds per point and the cells were incubated for 15, 30 and 60 minutes. The results demonstrated that BthTX – I affect cell viability in a dose dependent manner, but did not change cell integrity. The concentration of 75 μg/mL was chosen for the experiments with LBP. LLLT caused an significant increase in cell viability in all the analyzed period of time and in the λ 685 nm and 830 nm against Bothrops I toxin, however in the LBP λ 685 nm against Bothrops toxin II was effective only at 15 min, while the LBP at λ 830 was effective at 15 and 60 min. The LLLT was not able to change the LDH release at all times and wavelength used. Thus, LBP was able to protect C2C12 muscle cells against the miotoxic effect of isolated myotoxins isolated from B. jararacussu venom. Therefore, the results suggest that LLLT can be considered an effective therapeutic tool in patients bitten by snakes. / O veneno das serpentes do gênero Bothrops induz uma reação inflamatória local intensa, caracterizada por dor, formação de edema, migração leucocitária, podendo ser acompanhada por necrose tecidual. A utilização do soro antibotrópico desempenha a função de neutralizar a maior quantidade possível do veneno circulante, minimizando assim seus efeitos sistêmicos, porém sua ação não se estende às manifestações locais, sendo assim necessário o uso de outro recurso terapêutico para o controle dessa manifestação. A laserterapia de baixa potência (LBP) é uma alternativa de tratamento em situações de lesão muscular, devido a seus efeitos biológicos, tais como analgésicos, antinflamatórios e cicatrizantes. Em trabalhos anteriores realizados em nosso laboratório, verificou-se que o LBP foi capaz de aumentar a viabilidade de células musculares C2C12, após a adição do veneno de B. jararacussu e que esse efeito do LBP é relacionado a uma proteção da membrana celular. Assim, o objetivo deste trabalho foi analisar o efeito do LBP em células musculares C2C12 submetidas à lesão por miotoxinas (BthTX - I e BthTX - II) isoladas do veneno da serpente Bothrops jararacussu quanto a: viabilidade, descolamento celular e liberação da enzima LDH. As células receberam a BthTX – I e BthTX – II na dose 75 μg/mL e foram imediatamente irradiadas com LBP Índio Gálio Alumínio Fósforo e Arseneto de Gálio Alumínio, nos comprimentos de onda (λ) 685 nm vermelho e 830 nm infra-vermelho, de forma pontual, tempo de aplicação de 13 s e 35 s respectivamente e as células foram incubadas por 15, 30 e 60 minutos. Os resultados demonstraram que a BthTX - I e BthTX - II afetou a viabilidade celular de forma dose-dependente, sendo escolhida a dose 75 μg/mL para a realização dos experimentos com o LBP, porém não foi capaz de causar alterações na integridade. O LBP causou aumento significativo na viabilidade celular, em todos os tempos analisados no λ 685 nm e 830 nm frente à BthTX - I, entretanto o LBP no λ 685 nm e λ 830 frente a BthTX - II foi efetivo somente no tempo de 15 e 60. O LBP não foi capaz de diminuir a liberação de LDH em todos os tempos analisados e com os dois λ utilizados. Desta forma, verificou-se que o LBP foi capaz de proteger as células musculares C2C12 contra o efeito miotóxico das miotoxinas isoladas do veneno B. jararacussu e que esta proteção está relacionada ao efeito protetor a nível mitocondrial. Ainda, os resultados obtidos sugerem que o LBP pode ser considerado uma ferramenta terapêutica eficaz em pacientes picados por serpentes.

Impaired mass transfer characteristics of blood borne vasoactive species such as ATP in regions such as an arterial bifurcation have been hypothesized as a prospective mechanism in the aetiology of atherosclerotic lesions. Arterial endothelial (EC) and smooth muscle cells (SMC) respond differentially to altered local hemodynamics and produce coordinated macro-scale responses via intercellular communication. Using a computationally designed arterial segment comprising large populations of mathematically modelled coupled ECs & SMCs, we investigate their response to spatial gradients of blood borne agonist concentrations and the effect of micro-scale driven perturbation on the macro-scale. Altering homocellular (between same cell type) and heterocellular (between different cell types) intercellular coupling we simulated four cases of normal and pathological arterial segments experiencing an identical gradient in the concentration of the agonist. Results show that the heterocellular calcium (Ca2+) coupling between ECs and SMCs is important in eliciting a rapid response when the vessel segment is stimulated by the agonist gradient. In the absence of heterocellular coupling, homocellular Ca2+ coupling between smooth muscle cells is necessary for propagation of Ca2+ waves from downstream to upstream cells axially. Desynchronized intracellular Ca2+ oscillations in coupled smooth muscle cells are mandatory for this propagation. Upon decoupling the heterocellular membrane potential, the arterial segment looses the inhibitory effect of endothelial cells on the Ca2+ dynamics of underlying smooth muscle cells. The full system comprising hundreds of thousands of coupled nonlinear ordinary differential equations simulated on the massively parallel Blue Gene architecture. The use of massively parallel computational architectures shows the capability of this approach to address macro-scale phenomena driven by elementary micro-scale components of the system.

Numerous macroscale models of arteries have been developed, comprised of populations of discrete coupled Endothelial Cells (EC) and Smooth Muscle Cells (SMC) cells, an example of which is the model of Shaikh et al. (2012), which simulates the complex biochemical processes responsible for the observed propagating waves of Ca2+ observed in experiments. In a 'homogenised' model however, the length scale of each cell is assumed infinitely small while the population of cells are assumed infinitely large, so that the microscopic spatial dynamics of individual cells are unaccounted for. We wish to show in our study, our hypothesis that the homogenised modelling approach for a particular system can be used to replicate observations of the discrete modelling approach for the same system. We may do this by deriving a homogenised model based on Goldbeter et al. (1990), the simplest possible physiological system, and comparing its results with those of the discrete Shaikh et al. (2012), which have already been validated with experimental findings. We will then analyse the mathematical dynamics of our homogenised model to gain a better understanding of how its system parameters influence the behaviour of its solutions. All our homogenised models are essentially formulated as partial differential equations (PDE), specifically they are of type reaction diffusion PDEs. Therefore before we begin developing the homogenised Goldbeter et al. (1990), we will first analyse the Brusselator PDE with the goal that it will help us to understand reaction diffusion systems better. The Brusselator is a suitable preliminary study as it shares two common properties with reaction diffusion equations: oscillatory solutions and a diffusion term.