Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 171 pages. Includes Vita. Includes bibliographical references.

Resumo: Sabendo-se do grande potencial dos bubalinos como produtores de carne e da importância da análise das fibras musculares para a caracterização da qualidade da carne, objetivou-se neste estudo avaliar as isoformas da miosina de cadeia pesada (MyHC) e os tipos de fibras dos músculos Longissimus dorsi (LD) e Semitendinosus (ST) de 28 bubalinos Mediterrâneo não-castrados e a possível modulação de acordo com os diferentes pesos de abate (450, 480, 510 e 540 kg). Amostras dos músculos foram coletadas e submetidas às técnicas de eletroforese (SDS-PAGE), m-ATPase e NADH-TR. Foram analisadas a porcentagem relativa das isoformas de MyHC, assim como, frequência, área e diâmetro das fibras musculares. Constatou-se que bubalinos Mediterrâneo não possuem a isoforma MyHC IIb, apresentando somente três isoformas de MyHC (IIa, IIx/d e I), cujas porcentagens não variaram significativamente (P > 0,05) entre os pesos de abate. A presença de fibras musculares híbridas (IIA/X) nos músculos LD e ST permitiu a classificação dos tipos de fibras pela atividade contrátil, em fast e slow. Para ambos os músculos, a frequência foi maior para as fibras fast em todas as categorias avaliadas, porém houve diferença (P < 0,05) na frequência dos tipos de fibras dos músculos LD e ST de acordo com os pesos de abate. Os animais abatidos aos 540 kg obtiveram maiores valores para área e diâmetro das fibras, tanto para o LD quanto para o ST. O músculo ST apresentou maiores valores de área e diâmetro que o músculo LD para todos os pesos de abate avaliados, revelando o crescimento mais tardio. / Abstract: Knowing of the great productive potential of the water buffalo as meat producer and of the importance of the research of muscle fiber types for the characterization of the meat quality, the aim of the study was to evaluate Myosin Heavy Chain Isoforms (MyHC) and the muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) muscles of 28 Mediterranean buffaloes not-castrated and its possible modulation according with the different slaughter weights (450, 480, 510 and 540 kg). Samples of the muscles were collected and submitted the electrophoresis (SDS-PAGE), m- ATPase and NADH-TR techniques. The relative percentage of isoforms MyHC were measured, as well as, frequency, area and diameter of muscle fiber types. The absence of MyHC IIb isoform in Mediterranean buffaloes was evidenced, which had only three MyHC isoforms (IIa, IIx/d and I), whose percentage had not varied significantly (P > 0.05) between the slaughter weights. The presence of hybrid fibers (IIA/X) in muscles LD and ST allowed to the classification of the fiber types for a contraction activity, in fast and slow. For both muscles, the frequency was higher for fiber fast in all the evaluated categories, however had differences (P < 0.05) in the frequency of muscle fiber types LD and ST in the slaughter weights evalueted. The animals of slaughter weight 540 kg showed higher values for area and diameter of the fibers, as much for the LD as well as the ST. Muscle ST presented greater values of area and diameter then muscle LD for all slaughtered weights evaluated, disclosing the more delayed growth. / Orientador: André Mendes Jorge / Coorientador: Maeli Dal Pai Silva / Banca: Cristiana Andrighetto / Banca: José Roberto Sartori / Mestre

Genome Wide Association Studies (GWASs) have identified variants associated with disease that promise to deliver insights into disease aetiology. However, because many GWAS variants lie in non-coding genomic regions, it is difficult to define the genes and pathways underlying a GWAS signal. The possibility of linking GWAS variants to molecular traits, combined with the development of high throughput assays, has motivated the mapping of molecular quantitative trait loci (QTLs), genetic associations with molecular traits such as gene expression (eQTLs) and DNA methylation (mQTLs). The Finland-United States Investigation of NIDDM (FUSION) tissue biopsy study is motivated by the desire to understand the molecular pathogenesis of Type 2 diabetes (T2D), a complex disease where the vast majority of the ~100 independent GWAS loci occur in non-coding regions. To elucidate the molecular mechanisms underlying these signals, we collected skeletal muscle biopsies, a T2D-relevant tissue, from 318 extensively phenotyped individuals who exhibit a range of glucose tolerance levels. From these biopsies, we generated genotype, gene expression, and DNA methylation information, enabling us to directly measure the effects of T2D on molecular traits, and to link non-coding T2D GWAS loci to candidate molecular targets. In this thesis, I present a catalogue of genetic effects on gene expression and DNA methylation. I use this catalogue firstly, to reveal basic biology of the genetic regulators of skeletal muscle molecular traits, and secondly, to identify molecular traits that are relevant to T2D, glycemic, and other complex traits. In regards to basic biology, I characterise the broader genomic context of QTLs by calculating the enrichment of QTLs in chromatin states across a diverse panel of cell/tissue types. I also identify key skeletal muscle transcription factors (TFs) and classify them as activators or repressors by aggregating the effects of QTLs predicted to perturb TF binding sites. In addition, I characterise the properties of methylation sites associated with gene expression and use inference models to dissect these methylation-expression relationships, classifying cases where the genetic effect is mediated by methylation, expression, or is independent. I also integrate molecular trait genetics with complex traits. First, I perform a conditional analysis, mapping GWAS variants for T2D and glycemic traits to molecular traits, prioritising disease relevant skeletal muscle molecular traits. Second, recognising QTLs may also be specific to a disease state or environmental context, I leverage the rich phenotyping of participants to map genotype by environment (GxE) effects on gene expression—eQTLs that exhibit effects specific to an environmental context. Altogether, these analyses form a thorough survey of the genetic regulators of skeletal muscle expression and DNA methylation, and provide an important resource for interpreting complex diseases.

Skeletal muscle is a highly metabolically active tissue, both in the adult and the fetus. Mitochondria are essential in providing energy in the form of ATP from the oxidative metabolism of carbohydrates, fats and amino acids. Mitochondrial function is influenced by the abundance and activity of the complexes comprising the electron transfer system (ETS) and the balance between mitochondrial fusion and fission. Any factors which affect the development of skeletal muscle, and mitochondria in particular, may have an impact not only on neonatal health but also on the metabolic health of the adult offspring. However, the normal developmental profile of skeletal muscle mitochondrial function as the fetus prepares for the increased metabolic challenges associated with extrauterine life, is not well characterised. The hormones, cortisol and triiodothyronine (T3) are known to be crucial in the maturation of several physiological processes during late gestation. Further, their role in regulating adult metabolism is well-documented. However, whether they play a role in regulating fetal mitochondrial function is unknown. Using fetal sheep, the aims of this project were twofold: 1) to determine any changes in skeletal muscle mitochondrial function which occur over the last third of gestation and in the first two days of neonatal life and 2) to determine any regulatory roles of cortisol and T3 in these developmental changes. Mixed fibre-type skeletal muscle was collected from fetuses at 3 time points over late gestation and from newborn lambs. In addition, skeletal muscle samples were taken from fetuses which had been thyroidectomised (TX) and fetuses infused with either T3 or cortisol. Respirometry, enzyme assays, qRT-PCR and western blotting were carried out on the skeletal muscle samples in order to assess mitochondrial parameters. Mitochondrial activity, as measured by carbohydrate- and fat- stimulated ADP-coupled oxygen uptake, increased with age in a thyroid hormone dependent manner, rising predominantly postnatally. Mitochondrial density, abundance of ETS complexes I-IV and ATP-synthase and expression of the adenine nucleotide transferase 1 and mitofusin 2 were all positively influenced by age, with the natural prepartum rise being prevented in the thyroidectomised fetuses. However, T3 infusion alone was insufficient to raise any of these factors prematurely. Cortisol infusion resulted in an increase in some aspects of mitochondrial oxidative capacity in a muscle-specific manner. Overall, the data presented shows that there are developmental changes in skeletal muscle mitochondria during the perinatal period. They also suggest that these changes are regulated by both cortisol and thyroid hormones in preparation for birth, although neither hormone alone was sufficient to induce all the functional changes.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The primary aim of this dissertation was to describe the changes in microvascular hematocrit, as total[hemoglobin+myoglobin] (T[Hb+Mb] measured with near-infrared spectroscopy (NIRS), during post-occlusive reactive hyperemia (PORH). Mechanisms of reactive hyperemia within skeletal muscle were also explored. The investigation detailed in Chapter 2 of this dissertation found that the differing time courses of the kinetic responses of both oxy- and deoxy[Hb+Mb], are related to changes in T[Hb+Mb]. We also determined that adipose tissue thickness had no effect on a purely temporal analysis of NIRS data. In Chapter 3 we observed that brachial artery reactive hyperemia preceded changes in T[Hb+Mb] during reactive hyperemia. Assuming that myoglobin remained constant, we posited that changes in T[Hb+Mb] must reflect alterations in red blood cell concentration in the microvasculature, i.e., microvascular hematocrit. In Chapter 4 comparisons were made between brachial artery blood flow, cutaneous and skeletal muscle flux and T[Hb+Mb]. The conduit artery response was faster than the microvascular responses in all tissues. Within skeletal muscle, time to peak and the time constant for the on-kinetics were faster in T[Hb+Mb] compoared with intramuscular flux as measured with intramuscular laser-Doppler. We observed no differences in temporal responses between cutaneous and intramuscular measures and suggested that in a purely temporal analysis the cutaneous microvasculature could serve as an analog for the skeletal muscle microvasculature. Finally, in Chapter 5 we found that prostaglandin inhibition with ibuprofen altered the initial T[Hb+Mb] response during PORH without impacting cutaneous flux or brachial artery blood flow. Chapter 5 also discussed that the addition of a wrist cuff to our standard instrumentation prevented the accumulation of T[Hb+Mb] during the occlusion period.

abstract: Skeletal muscle (SM) mitochondria generate the majority of adenosine triphosphate (ATP) in SM, and help regulate whole-body energy expenditure. Obesity is associated with alterations in SM mitochondria, which are unique with respect to their arrangement within cells; some mitochondria are located directly beneath the sarcolemma (i.e., subsarcolemmal (SS) mitochondria), while other are nested between the myofibrils (i.e., intermyofibrillar (IMF) mitochondria). Functional and proteome differences specific to SS versus IMF mitochondria in obese individuals may contribute to reduced capacity for muscle ATP production seen in obesity. The overall goals of this work were to (1) isolate functional muscle SS and IMF mitochondria from lean and obese individuals, (2) assess enzyme activities associated with the electron transport chain and ATP production, (3) determine if elevated plasma amino acids enhance SS and IMF mitochondrial respiration and ATP production rates in SM of obese humans, and (4) determine differences in mitochondrial proteome regulating energy metabolism and key biological processes associated with SS and IMF mitochondria between lean and obese humans. Polarography was used to determine functional differences in isolated SS and IMF mitochondria between lean (37 ± 3 yrs; n = 10) and obese (35 ± 3 yrs; n = 11) subjects during either saline (control) or amino acid (AA) infusions. AA infusion increased ADP-stimulated respiration (i.e., coupled respiration), non-ADP stimulated respiration (i.e., uncoupled respiration), and ATP production rates in SS, but not IMF mitochondria in lean (n = 10; P < 0.05). Neither infusion increased any of the above parameters in muscle SS or IMF mitochondria of the obese subjects. Using label free quantitative mass spectrometry, we determined differences in proteomes of SM SS and IMF mitochondria between lean (33 ± 3 yrs; n = 16) and obese (32 ± 3 yrs; n = 17) subjects. Differentially-expressed mitochondrial proteins in SS versus IMF mitochondria of obese subjects were associated with biological processes that regulate: electron transport chain (P<0.0001), citric acid cycle (P<0.0001), oxidative phosphorylation (P<0.001), branched-chain amino acid degradation, (P<0.0001), and fatty acid degradation (P<0.001). Overall, these findings show that obesity is associated with redistribution of key biological processes within the mitochondrial reticulum responsible for regulating energy metabolism in human skeletal muscle. / Dissertation/Thesis / Appendix A / Appendix B / Appendix C / Appendix D / Appendix E / Appendix F / Appendix G / Doctoral Dissertation Molecular and Cellular Biology 2017

Made available in DSpace on 2014-06-11T19:27:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-08Bitstream added on 2014-06-13T20:36:10Z : No. of bitstreams: 1 francisco_cl_me_botfmvz.pdf: 298670 bytes, checksum: 07cf29969b291dc9657e6127f73f1e6f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Sabendo-se do grande potencial dos bubalinos como produtores de carne e da importância da análise das fibras musculares para a caracterização da qualidade da carne, objetivou-se neste estudo avaliar as isoformas da miosina de cadeia pesada (MyHC) e os tipos de fibras dos músculos Longissimus dorsi (LD) e Semitendinosus (ST) de 28 bubalinos Mediterrâneo não-castrados e a possível modulação de acordo com os diferentes pesos de abate (450, 480, 510 e 540 kg). Amostras dos músculos foram coletadas e submetidas às técnicas de eletroforese (SDS-PAGE), m-ATPase e NADH-TR. Foram analisadas a porcentagem relativa das isoformas de MyHC, assim como, frequência, área e diâmetro das fibras musculares. Constatou-se que bubalinos Mediterrâneo não possuem a isoforma MyHC IIb, apresentando somente três isoformas de MyHC (IIa, IIx/d e I), cujas porcentagens não variaram significativamente (P > 0,05) entre os pesos de abate. A presença de fibras musculares híbridas (IIA/X) nos músculos LD e ST permitiu a classificação dos tipos de fibras pela atividade contrátil, em fast e slow. Para ambos os músculos, a frequência foi maior para as fibras fast em todas as categorias avaliadas, porém houve diferença (P < 0,05) na frequência dos tipos de fibras dos músculos LD e ST de acordo com os pesos de abate. Os animais abatidos aos 540 kg obtiveram maiores valores para área e diâmetro das fibras, tanto para o LD quanto para o ST. O músculo ST apresentou maiores valores de área e diâmetro que o músculo LD para todos os pesos de abate avaliados, revelando o crescimento mais tardio. / Knowing of the great productive potential of the water buffalo as meat producer and of the importance of the research of muscle fiber types for the characterization of the meat quality, the aim of the study was to evaluate Myosin Heavy Chain Isoforms (MyHC) and the muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) muscles of 28 Mediterranean buffaloes not-castrated and its possible modulation according with the different slaughter weights (450, 480, 510 and 540 kg). Samples of the muscles were collected and submitted the electrophoresis (SDS-PAGE), m- ATPase and NADH-TR techniques. The relative percentage of isoforms MyHC were measured, as well as, frequency, area and diameter of muscle fiber types. The absence of MyHC IIb isoform in Mediterranean buffaloes was evidenced, which had only three MyHC isoforms (IIa, IIx/d and I), whose percentage had not varied significantly (P > 0.05) between the slaughter weights. The presence of hybrid fibers (IIA/X) in muscles LD and ST allowed to the classification of the fiber types for a contraction activity, in fast and slow. For both muscles, the frequency was higher for fiber fast in all the evaluated categories, however had differences (P < 0.05) in the frequency of muscle fiber types LD and ST in the slaughter weights evalueted. The animals of slaughter weight 540 kg showed higher values for area and diameter of the fibers, as much for the LD as well as the ST. Muscle ST presented greater values of area and diameter then muscle LD for all slaughtered weights evaluated, disclosing the more delayed growth.

Made available in DSpace on 2014-06-11T19:22:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-05Bitstream added on 2014-06-13T20:09:53Z : No. of bitstreams: 1 bonfim_mr_me_prud.pdf: 1651509 bytes, checksum: 6ef623e67889d7c91d851d5f95098ebe (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Investigar o efeito do uso e da descontinuidade da atorvastatina associadas a um programa de atividade física nos parâmetros bioquímicos de ratos. Métodos: 56 ratos machos Wistar, divididos em 4 grupos: sedentário, exercitado, sedentário com estatina e exercitado com estatina, foram submetidos ao uso da Atorvastatina (10 mg/Kg/dia) ou placebo sete dias na semana por 4 semanas, e a um programa de atividade física em esteira de 60 minutos diários, 5 dias na semana por 4 semanas. Após este período, 8 animais de cada grupo foram sacrificados; os demais animais foram submetidos à descontinuidade da estatina ou placebo, permanecendo a atividade física por 2 semanas. Nos três momentos distintos das intervenções, foram realizadas coletas de sangue para verificação dos parâmetros bioquímicos lipídicos e de biomarcadores de lesão hepática e muscular. Resultados: O uso da atorvastatina por 4 semanas reduziu efetivamente os níveis de colesterol total, fração não HDL-c e triacilgliceróis sem alteração dos biomarcadores de lesão muscular e hepática; a descontinuidade de seu uso acarretou em aumento dos níveis lipídicos. A adição da atividade física reduziu significantemente os níveis de triacilgliceróies e elevou os de HDL-c, permitindo que não houvesse aumento dos níveis lipídicos após a descontinuidade do uso da estatina. Conclusão: Conclui-se que o uso da estatina em conjunto com a prática de atividade física promove efeitos benéficos no perfil lipídico, que são sustentados até mesmo após a descontinuidade do fármaco. / Investigate the effect of atorvastatin use and its discontinuity associated to a physical activity program in the biochemical profiles of rats. Methods: 56 male wistar rats, divided into 4 groups: sedentary, exercised, sedentary with statin, and exercised with statin, were submitted to Atorvastatin use or placebo seven days per week for four weeks, and a physical activity program in treadmill consisted of 60 minutes daily of activity, 5 days per week for four weeks. After this period, eight animals of each group were sacrificed and the others were submitted to the discontinuance of statin or placebo, maintaining the physical activity for 2 weeks. In the three different intervention moments, the blood was collected to verify the lipid and muscle and liver lesion biomarkers parameters. Results: The use of Atorvastatin for 4 weeks effectively reduced total cholesterol levels, non HDL-c fraction and triglyceride levels with no alteration on muscle and liver biomarkers; the discontinuance of its use led to the rise of lipids levels. Adding the physical activity in the statin treatment significantly reduced triglyceride levels and raised HDL-c levels, and allowed the maintenance of the lipids levels after the discontinuance of the statin. Conclusion: The results of this study suggest that statin use together with physical activity practice promote beneficial effects in the lipid profile, that are sustained even after the discontinuance of the drug.

Made available in DSpace on 2014-06-11T19:22:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-14Bitstream added on 2014-06-13T20:49:21Z : No. of bitstreams: 1 kodama_fy_me_prud.pdf: 3657773 bytes, checksum: e2020e59e5ae04245371bee87b21d60a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Pro-Reitoria de Auxilio A Pesquisa / A imobilização é geralmente considerada como o método de escolha para o tratamento de lesões musculoesqueléticas, embora possa incorrer em alterações estruturais indesejáveis. Na literatura poucos estudos avaliam a recuperação da resistência do músculo após a imobilização, principalmente do músculo envelhecido, desta forma o objetivo do presente estudo é descrever os efeitos da imobilização, da remobilização livre e por meio de exercício físico sobre as propriedades mecânicas e histológicas do músculo esquelético de ratos de duas faixas de idade. Para tanto, foram utilizados 69 ratos machos Wistar (Rattus novergicus) divididos em dois grupos de acordo com a idade, sendo um grupo adulto (cinco meses) e um idoso (15 meses). Estes grupos foram subdivididos de acordo com os procedimentos experimentais em controle, imobilizado, remobilizado livre e remobilizado por meio de exercício físico. A imobilização foi realizada com membros posteriores dos animais em posição de encurtamento por um período de sete dias. O protocolo de exercícios foi composto por 5 sessões de natação, 1 vez por dia e 25 minutos por sessão. Para obtenção dos valores das propriedades mecânicas, o músculo gastrocnêmio foi submetido a ensaios mecânicos de tração na máquina universal de ensaio EMIC® (modelo DL 2000) e calculadas as respectivas propriedades mecânicas: alongamento no limite máximo, carga no limite máximo e rigidez. Foram realizadas também medidas de morfometria por meio de técnica histológica. Os resultados do ensaio mecânico demonstram diferenças significantes dos valores de carga no limite máximo entre os subgrupos controle e os demais, tanto no grupo adulto como no idoso. Não houve diferença significante com relação à rigidez... / Immobilization is generally regarded as the method of choice for the treatment of musculoskeletal injuries, but may incur undesirable structural changes. In literature, few studies evaluate the recovery of muscle strength after immobilization, mainly aged muscle, so the purpose of this study is to describe the effects of immobilization, the free remobilization and physical exercise on the mechanical and histological properties in the skeletal muscle of two age groups of rats. To this end, we used 69 Wistar rats (Rattus norvegicus) divided into two groups according to age, being an adult group (five months) and old group (15 months). These groups were subdivided according to the experimental procedures in control, immobilized, free remobilized and remobilized through exercise. Immobilization was performed with the hindquarters of the animals in a shortened position for a period of seven days. The exercise protocol consisted of five sessions of swimming, 1 time per day and 25 minutes per session. To obtain the values of mechanical properties, the gastrocnemius muscle was subjected to mechanical testing machine EMIC universal test ® (model DL 2000) and assessed their mechanical properties: stretching at the maximum limit, load at the maximum limit and stiffness. Morphometry measurements were also taken using histological technique. The results of mechanical tests show significant differences in the values of the maximum load between the control and the other subgroups, both in the adult and the elderly ... (Complete abstract click electronic access below)

abstract: Skeletal muscle injury may occur from repetitive short bursts of biomechanical strain that impair muscle function. Alternatively, variations of biomechanical strain such as those held for long-duration are used by clinicians to repair muscle and restore its function. Fibroblasts embedded within the unifying connective tissue of skeletal muscle experience these multiple and diverse mechanical stimuli and respond by secreting cytokines. Cytokines direct all stages of muscle regeneration including myoblasts differentiation, fusion to form myotubes, and myotube functionality. To examine how fibroblasts respond to variations in mechanical strain that may affect juxtapose muscle, a myofascial junction was bioengineered that examined the interaction between the two cell types. Fibroblasts were experimentally shown to increase myoblast differentiation, and fibroblast biomechanical strain mediated the extent to which differentiation occurred. Intereleukin-6 is a strain-regulated cytokine secreted by fibroblasts was determined to be necessary for fibroblast-mediated myoblast differentiation. Myotubes differentiated in the presence of strained fibroblasts express greater number of acetylcholine receptors, greater acetylcholine receptor sizes, and modified to be more or less sensitive to acetylcholine-induced contraction. This study provides direct evidence that strained and non-strained fibroblasts can serve as a vehicle to modify myoblast differentiation and myotube functionality. Further understanding the mechanisms regulating these processes may lead to clinical interventions that include strain-activated cellular therapies and bioengineered cell engraftment for mediating the regeneration and function of muscle in vivo. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2014