[Abstract]: Duchenne Muscular Dystrophy (DMD) is a fatal recessive genetic human disease affecting one in 3500 live male births. DMD is progressive, there is no cure, and patients typically die of respiratory or cardiac failure intheir second decade of life. Clinical disease symptoms are exacerbated at the onset of puberty and the physiological basis of this is unknown. The mdx mouse is the preferred experimental animal model of DMD, although aspectsof the model remain poorly understood. This dissertation characterises physiological and histological features of the dystrophic mdx mouse in response to manipulations of hormonal status including testosterone treatment, surgical castration, and oestrogen treatment. Sex-specificdifferences in the mdx were also examined. Furthermore, physiological and histological features of the dystrophic mdx mouse model throughout the mdx lifespan were evaluated. Cardiac muscle contractility, left atrial response to exogenous calcium, and the contractile properties of both fast-twitch (EDL)and slow-twitch (SOL) skeletal muscles were examined in male mdx miceranging from 14 to 330 days of age. Testosterone treatment produced a nonsignificant trend towards a dose-dependent decrease in both basal and maximal left atrial contractility in the mdx. Surgical castration produced nosignificant cardiac effects within mouse strains. The mdx castrates had a 45% lower maximum atrial force of contraction than control castrates (p<0.05). Conversely, oestrogen treatment significantly improved cardiaccontractility in the mdx. An increase in basal left atrial contractility was evident at doses of 0.08 mg/kg/day (p<0.05) and 0.16 mg/kg/day (p<0.01)and in maximum left atrial contractility at a dose of 0.16 mg/kg/day (p<0.01). Gender studies showed cardiac forces in mdx were not different between males and females at any age tested and that both sexes in mdx had adampened cardiac responsiveness to exogenous calcium. Skeletal muscle function studies showed that castration produced a 25% increase in mdx EDL specific force generation (p<0.01) and no increase in SOL forces.Oestrogen treatment produced a non-significant trend towards increased EDL forces and a 29% increase in SOL specific force at a dose of 0.16mg/kg/day (p<0.05). Gender studies revealed no differences between male and female mdx in terms of skeletal muscle force production. Further to the hormonal investigations, lifespan characterisation studies revealed that themdx mouse showed reduced basal and maximal left atrial contractility specifically at ages 14 and 90 days (p<0.05). Skeletal muscle studies showed that specific tetanic force production was significantly lower thancontrols at 19 (p<0.05), 21, 23, 27, and 330 days of age (p<0.01) for EDL muscles and at 19, 21, and 23 days of age in SOL muscles (p<0.01). These studies further improve our understanding of the mdx mouse as an experimental model of DMD and emphasises that the model is most appropriate a specific ages for specific muscles. These studies furtherillustrate that testosterone does not improve cardiac contractility in the mdx mouse but that oestrogen improves both cardiac and skeletal muscle function. Further research is warranted into the potential of oestrogen as atherapeutic agent in the treatment of both cardiac and skeletal musclemanifestations of DMD.

Duchenne muscular dystrophy (DMD) is on the amendment to the X chromosome, has recessive character and affects 1: 3500 live births, especially males. Presents in infancy, delays engines. The genetic abnormality in the short arm of the X chromosome (Xp21.2 locus) is responsible for the lack of production of dystrophin protein linked in frame with the extracellular matrix, forming the oligométrico complex glycoproteins located on the inner surface of the sarcolemma and responsible for maintaining proper muscle functioning. The lack of dystrophin leads to muscle decreased permeability of the membrane, allowing an excessive amount of calcium to accumulate inside the cell. This condition leads to a super myofibrillar contraction, degradation of fibrils and metabolic disorders such as oxidative stress and altered energy metabolism, culminating in the death of the muscle fiber. The low-intensity exercise is known to decrease some parameters associated with macular degeneration in animal models of progressive muscular dystrophies. The objective of this study was to evaluate what the effects of an aerobic exercise protocol on the degeneration of muscle and neuronal tissue in an animal model of DMD. Male mdx mice were used and wild-type (wild) 28 days. The animals were submitted to a physical exercise protocol of moderate intensity for eight weeks. Twenty four hours after the last day of training, the brain structures of the striatum were removed, cerebellum, prefrontal cortex, hippocampus and cortex and gastrocnemius muscle, to evaluate the carbonylation of proteins, lipid peroxidation, glutathione activity, activity of complex respiratory chain and creatine kinase. It was found that physical exercise protocol of moderate intensity was able to reduce oxidative stress in muscle tissue and most of the structures analyzed the CNS, with significant increase in antioxidant activity in all analyzed structures. Furthermore, the protocol improved the activity of most of the mitochondria respiratory complexes both in muscle tissue and in the CNS, although it decreased the creatine kinase activity cerebellum only. In conclusion, it can be inferred that the use of a moderate intensity for 8 weeks exercise protocol was capable of reducing oxidative stress and improving energy metabolism in brain tissue and in the gastrocnemius muscle of animals with DMD. / Submitted by Rogele Pinheiro (rogele.pinheiro@unisul.br) on 2017-10-23T17:19:00Z No. of bitstreams: 1 111812_Andreza.pdf: 2125121 bytes, checksum: 72023555e215764aa8d0b95453babae7 (MD5) / Approved for entry into archive by Caroline Correa da Cruz (caroline.cruz@unisul.br) on 2017-10-23T20:16:28Z (GMT) No. of bitstreams: 1 111812_Andreza.pdf: 2125121 bytes, checksum: 72023555e215764aa8d0b95453babae7 (MD5) / Made available in DSpace on 2017-10-23T20:16:28Z (GMT). No. of bitstreams: 1 111812_Andreza.pdf: 2125121 bytes, checksum: 72023555e215764aa8d0b95453babae7 (MD5) Previous issue date: 2015 / Distrofia muscular de Duchenne (DMD) está ligada a alteração do cromossomo X, possui caráter recessivo e atinge 1:3500 nascidos vivos, principalmente indivíduos do sexo masculino. Apresenta-se na primeira infância, por atrasos motores. A anormalidade genética no braço curto do cromossomo X (lócus Xp21.2) é responsável pela falta de produção da proteína distrofina que, associada em trama com a matriz extracelular, forma o complexo oligométrico de glicoproteínas, localizada na superfície interna do sarcolema e responsável pela manutenção do bom funcionamento muscular. A ausência da distrofina provoca diminuição da permeabilidade da membrana muscular, permitindo que uma quantidade excessiva de cálcio se acumule no interior da célula. Essa condição leva a uma supercontratura miofibrilar, degradação das fibrilas e distúrbios metabólicos, como estresse oxidativo e alterações do metabolismo energético, que culminam com a morte da fibra muscular. O exercício físico de baixa intensidade é conhecido por diminuir alguns parâmetros associados à degeneração muscular em modelos animais de distrofias musculares progressivas. O objetivo deste estudo foi avaliar quais os efeitos de um protocolo de exercício físico aeróbio sobre a degeneração do tecido muscular e neuronal em um modelo animal de DMD. Foram utilizados camundongos machos mdx e wild-type (selvagens) com 28 dias. Os animais foram submetidos a um protocolo de exercício físico de baixa intensidade por oito semanas. Vinte quatro horas após o último dia de treinamento, foram retiradas as estruturas encefálicas do estriado, cerebelo, córtex pré-frontal, hipocampo e córtex e o músculo gastrocnêmio, para avaliação da carbonilação de proteínas, peroxidação lipídica, tióis livres, atividade dos complexos da cadeia respiratória e creatina quinase. Verificou-se que o protocolo de exercício físico de baixa intensidade foi capaz de modificar parâmetros de estresse oxidativo em tecido muscular e na maioria das estruturas analisadas do SNC, com aumento significativo da atividade antioxidante em todas as estruturas analisadas. Além disso, o protocolo modificou a atividade da maioria dos complexos respiratórios mitocôndrias, tanto em tecido muscular quanto em SNC, apesar de ter diminuído a atividade de creatina quinase somente de cerebelo. Em conclusão, pode-se inferir que a utilização de um protocolo de exercício de baixa intensidade durante 8 semanas foi capaz de modificar parâmetros de estresse oxidativo e melhorar o metabolismo energético em tecido encefálico e no músculo gastrocnêmio de animais com DMD.

This dissertation titled Designing Project Business Intelligence focuses on data warehouse in a medium-sized company. The theoretical portion of the thesis describes the topic of data warehouse and dimensional modeling. In the first theoretical chapter, I compare and contrast two approaches to datawarehouse building - Bill Inmon's and Ralph Kimball's. Then, basic terminology used in the field, principles of dimensional modeling and various approaches of database use will be described. Furthermore, the thesis illustrates the role of the Mondrian system as a mediator between the relational database and the OLAP server. Possible uses of the MDX language in working with multidimensional databases are outlined. The theoretical portion of the thesis ends with a description of the applicational interface XMLA, which can be used for facilitation communication between applications and the OLAP server. The practice-oriented portion of the thesis includes a complete design of the Business Intelligence project. The project is divided into the following parts: Feasibility Study, Project Planing, Business Requirements, Design and Development, and Deployment and Maintenance. The entire project operates exclusively on Open Source Software. Consequently, the project shows that small and medium-sized companies can afford to run a fully operating Business Intelligence system.

No description available.

Duchenne muscular dystrophy (DMD) is a severe and fatal muscle wasting disease characterized by a high mutation rate in the gene that encodes the membrane-associated protein dystrophin that results in absence of expressed protein. Although the primary genetic defect for DMD is known, the mechanisms that initiate the onset of DMD are not currently understood. This study tested the hypothesis that pathophysiological processes involved in DMD could be identified by the global expression of mRNA in maturing dystrophin- and utrophin-deficient mouse (mdx:utrn-/-) muscles. Two potential dystrophic onset mechanisms targeted for analysis were (1) disrupted expression of calcium handling proteins; and, (2) increased expression of immune response markers. An mRNA expression profile was developed following isolation of total RNA from control and mdx:utrn-/- triceps surae (TS) muscles at ages 9-10 and 20-21 days using Affymetrix® Mu74Av2 GeneChips®. Compared to control, the mRNA expression profile in mdx:utrn-/- muscles revealed there was a 3-fold increase in the number of gene transcripts differentially expressed more than 2-fold (53 transcripts at ages 9-10 days; 153 at ages 20-21 days). However, there were no changes in the mRNA transcripts for calcium handling proteins. In distinct contrast, there was up-regulation of transcripts that corresponded to an immune response (40 transcripts), extracellular matrix activity (14), and proteolysis (8). Up-regulation of several transcripts corresponded to cytokines and their receptors (11), chemokines and their receptors (5), and lymphoid and myeloid markers (16) suggesting that dystrophic muscle is susceptible to invasion by macrophages, leukocytes, B- and T-cells. These results are consistent with several reports (Spencer et al., 1997; Chen et al., 2000; Porter et al., 2002; Porter et al., 2003a; Porter et al., 2003b; Porter et al., 2004) that indicate the immune system may play an important role in the early pathophysiology of DMD. Understanding the functional aspects of an immune response in DMD onset should lead to more effective therapeutics. / Master of Science

Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration and a chronic inflammatory response. Sphingolipid metabolites are associated with the generation or perpetuation of low-grade chronic inflammation critical in atherosclerosis, obesity and cancer. Dietary sphingolipids, however, can suppress intestinal inflammation. We hypothesized that dietary sphingomyelin (SM) from bovine milk can modulate the inflammatory signature and improve muscle function in mdx mice, a model of DMD. C57BL10 (WT) and mdx mice were fed AIN 76A diet ± 0.1% SM for 7 weeks starting at age 4 weeks (n=10/group: WT, WT + S, mdx, mdx + S). At ages 5, 7, and 9 weeks, ankle flexor torque was determined in vivo. Mice were euthanized at 11 wks. Serum creatine kinase and extensor digitorum longus (EDL) contractile properties in vitro were determined; Tibialis Anterior (TA) inflammatory markers were profiled by qRT-PCR; TA sections were stained with H&E and immunohistochemistry for p-Akt was performed. At age 9 weeks, in vivo ankle flexor torque at stimulation frequencies 50-150 Hz was greater in mdx+S vs. mdx (P=0.0160) and WT (P<0.0001). At 11 wks, only WT+S EDL stress in vitro was greater than all other groups at 50-150 Hz. The in vitro relative stress-frequency relationship of mdx+S EDL was left shifted from the other treatment groups. Inflammatory genetic markers were increased in mdx+S mice. These data suggest treatment of mdx mice with 0.1% SM improves ankle flexor torque in vivo, causes a left shift of the stress-frequency relationship in vitro, and modulates the inflammatory gene signature. / Master of Science

Duchenne muscular dystrophy (DMD) is a fatal, x-linked disease that causes progressive muscle weakness and susceptibility to damage. DMD is caused by a lack of dystrophin, a large muscle protein that performs both structural and signaling functions. A promising treatment currently in clinical trials is microdystrophin gene therapy, which delivers a truncated version of dystrophin to muscle via a viral vector. Preclinical studies have established efficacy of microdystrophin to improve muscle quality and function. With clinical success of this treatment, patients affected by DMD could become more physically active. However, the effect of exercise on both dystrophic and gene therapy-treated muscles is unclear. Recently, we demonstrated that microdystrophin gene therapy with and without 21 weeks of voluntary wheel running (VWR) improved treadmill time to fatigue and in vivo plantarflexor torque output in young mdx mice, a mouse model of DMD. Although treated mice could run well, diaphragm force and power output were blunted by VWR. A subsequent study tested longevity of two different microdystrophin gene therapy constructs in combination with VWR. Versions of each construct are being tested in clinical trials. Construct 1 contained the nNOS-binding site found in full-length dystrophin, which localizes nNOS to the sarcolemma and reduces functional ischemia of exercising limb muscles, while construct 2 lacked the nNOS-binding site and was the same microdystrophin used in the previous study. Gene- therapy treated mice that were sedentary or performed 52 weeks of VWR demonstrated similar outcomes including increased plantarflexor torque and exceptional treadmill endurance capacity. However, ex vivo diaphragm and soleus force, as well as metabolic enzyme and mitochondrial respiration assays were differentially improved, revealing unique physiological adaptations to each microdystrophin construct. Together, the data demonstrated that response to exercise after gene therapy treatment was variable and dependent on age, microdystrophin construct, and muscle type. / Doctor of Philosophy / Duchenne muscular dystrophy (DMD) is a rare, fatal muscle disease that causes progressive muscle weakness and cardiorespiratory failure. Available treatments, such as corticosteroids, slow progression of the disease but do not address the underlying genetic cause. DMD is caused by a genetic mutation that results in the loss of the muscle protein dystrophin. Microdystrophin gene therapy aims to address the genetic cause of the disease by using a non-pathogenic virus to deliver microdystrophin, a small, functional version of dystrophin, to muscle. This gene therapy is in clinical trials, and, if it is successful, treated patients will likely want to engage in more physical activity than previously possible due to muscle weakness. However, the effects of physical activity on muscles treated with gene therapy are unclear. Therefore, we conducted two studies to test the effects of voluntary wheel running on microdystrophin gene therapy in the mdx mouse, a model of DMD. The first study demonstrated that voluntary wheel running was beneficial to whole-body muscle function in mice treated with microdystrophin gene therapy. However, adaptations to the gene therapy and voluntary wheel running were variable in individual muscles. In the second study, we tested two microdystrophin constructs, which each contain different structural components of full-length dystrophin. In addition, mice ran for 52 weeks, more than twice as long as the first study. The results of the second study found that adaptations in individual muscles depended on microdystrophin construct and activity level. Additionally, we confirmed that voluntary wheel running was beneficial to whole-body function of microdystrophin–treated muscles. Together, these studies demonstrated that adaptations of gene therapy-treated muscles were dependent on microdystrophin structure, activity level, and age.

The main goal for my research was to challenge the long held belief that the mechanical properties of maturing dystrophic compared to control skeletal muscle membranes are weaker, leading to onset of Duchenne muscular dystrophy (DMD). We built on a previous report from our lab that suggested sarcolemmal membranes from dystrophic mice are not more susceptible to damage early in maturation (i.e., age 9-12 days) and determined if and when muscle mechanical properties change as the mice mature. Across four studies, I have helped define the role of dystrophin-deficient skeletal muscle membranes in the onset of DMD. A linear viscoelastic muscle model was used to determine passive stiffness and damping in control and dystrophic muscles from maturing mice aged 14-35 days. Results confirmed my hypothesis that there are no differences in passive mechanical properties between normal and dystrophic mice. Recognizing the limitations of the linear model, a nonlinear model was developed to determine the stiffness and damping of active and passive dystrophic muscles from maturing mice aged 21 and 35 days. The nonlinear model achieved a significantly better fit to experimental data than the linear model when muscles were stretched to 15% strain beyond resting length. Active and passive mechanical properties of dystrophic mice were not different than control at 14 and 28 days of age. The previously developed nonlinear model was used to determine a more complete time-course (14-100 days of age) of dystrophic muscle mechanical properties. There was no difference in passive stiffness between mdx and control muscles at each age. However, the mdx:utrn-/- muscles showed increased stiffness compared to control and mdx muscles at 21 and 28 days, suggesting a temporary change within the muscle that only occurs with a lack of both utrophin and dystrophin. Fast-twitch and slow-twitch muscle mechanical properties were compared in control and dystrophic mice aged 3, 5, and 9 weeks of age. Dystrophic and control slow-twitch muscles did not have different mechanical properties, suggesting that a lack of dystrophin does not affect slow-twitch muscles during maturation (3-5 weeks) or well after maturation (9 weeks). / Ph. D.

Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients. / Ph. D.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease affecting 1 in every 3500 boys. The disease is characterized by the absence of the dystrophin protein from the sarcolemma of muscle cells. Muscle cells lacking dystrophin go through cycles of degeneration and regeneration and are considered susceptible to contraction-induced injury 144. Eventually, the satellite cell proliferative capacity is exhausted and the muscle fibers are replaced by connective and adipose tissue that yields a progressive loss of force generating capability. DMD patients typically die by their early 20's, primarily due to respiratory or cardiac failure. The precise role of dystrophin is not presently known. However, its absence suggests that it may play a role in both cellular calcium regulation and oxidative stress 152. Recent studies suggest increased reactive oxygen species (ROS) may precede the initial wave of wasting that marks disease onset 49. Therefore, it is possible oxidative stress may contribute as a pathogenic mechanism of DMD. Strategies to reduce the deleterious effects of oxidative stress could be an effective therapeutic approach. Regular exercise is known to increase antioxidant capacity in humans and mice 146. Green tea extract (GTE) is a powerful antioxidant that is easily supplemented in the diet 83. The purpose of this study was to test the hypotheses that (1) voluntary endurance exercise alone, (2) a diet supplemented with 0.05% (wt/wt) GTE alone, or exercise and GTE combined will blunt the effects of ROS and improve muscle strength and endurance in young mdx mice. Male mdx mice at age 21-days were randomly divided into one of 4 treatment groups: Run Normal diet (RunNorm; n=8), Sedentary Normal diet (SedNorm; n=8), Run GTE diet (RunGTE; n=10), and Sedentary GTE diet (SedGTE; n=8). RunNorm and RunGTE mice were given free access to a running wheel for 3 weeks while SedNorm and SedGTE mice were restricted to normal cage movement. At the end of 3 weeks, mice in each treatment group were sacrificed and assessed for daily and weekly running distances, content of actin and myosin proteins and fiber type distribution (tibialis anterior), contractile/mechanical and fatigue properties (extensor digitorum longus), creatine kinase levels and antioxidant capacity (serum), lipid peroxidation (gastrocnemius), and citrate synthase and beta-hydroxyacyl-CoA dehydrogenase activities (quadriceps and soleus). The key findings of this study were: In normal diet running mice (RunNorm), average daily distance run was increased 300% (from 0.5 to 2.1 km/d, P<0.05) from week 1 to week 3. In GTE diet (RunGTE) compared to RunNorm mice, total distance over the 3 weeks was markedly improved 128% (61.2 vs. 26.8 km, P<0.0001). Running, independent of diet increased EDL muscle tetanic stress (18%), serum antioxidant capacity (22%), citrate synthase activity (35%), and beta-oxidation (37%; all P<0.05). GTE, independent of running decreased lipid peroxidation (gastrocnemius:-64%; heart: -29%) and serum creatine kinase (-36%), and increased citrate synthase activity (59%; all P<0.05). These findings in dystrophic mice suggest that voluntary endurance exercise with or without GTE supplementation blunted the deleterious effects of ROS. If similar positive effects are evident in human DMD patients, then these approaches may be beneficial therapies either alone or in combination. / Master of Science