The combination of a severe systemic illness, corticosteroids, and neuromuscular blocking agents in patients on the mechanical ventilator often results in a condition known as Acute Quadriplegic Myopathy (AQM). While severe weakness of all spinal nerve innervated muscles is known to be a significant clinical characteristic of the disease, this symptom is typically not recognized until the disease has progressed to an advanced stage. End result effects have been classified, which include the loss of the thick filament, or myosin heavy chain, an in-excitable muscle membrane, and an up-regulation of protein degradation; however, there is little known about the acute stage of AQM. This project has focused on understanding the underlying mechanisms of AQM, specifically in regard to protein synthesis, both at the mRNA and nuclear transcription levels. To study the early stages of the disease two animal models have been developed: rat and pig. Further, we have examined AQM muscle tissue, to investigate the similarities of our animal models to patients, as well as to study the recovery process. Particular interest was directed on the myofibrillar proteins myosin (MyHC) and actin, as they are the primary proteins involved in muscle contraction, as well as the myosin associated proteins, myosin binding protein C and H. At the mRNA level, MyHC and actin are both down-regulated in response to AQM. The myosin binding proteins are affected differently, with H protein increasing during severe atrophy and C protein either being slightly down-regulated or unchanged. Nuclear transcription factors were also affected, with such factors as MuRF1 and MAFbx up-regulated. Thus far results have shown that protein synthesis is altered in AQM and largely contributes to both the development and recovery of the disease. The pathways of protein synthesis may prove to be an ideal target for the prevention of AQM and/or symptom alleviation.

Das Chemokine SDF-1α und sein Rezeptor CXCR4 sind in eine Vielzahl biologischer Prozesse, wie der Organogenese, der Hämatopoese und der Immunantwort involviert. Die Entdeckung des alternativen SDF-1α-Rezeptors CXCR7 bewirkte eine erneute Untersuchung der Funktion des SDF-1-Systems in diesen Vorgängen. CXCR7 ist in der Lage, je nach Gewebe- oder Zelltyp, als \"Scavenger\"-Rezeptor, Modulator des CXCR4 oder selbstständig aktiver Rezeptor zu agieren. In dieser Arbeit wurde untersucht, inwiefern beide Rezeptoren im Verlauf der Entwicklung der Muskulatur exprimiert werden, welche Aufgabe sie dabei übernehmen und ob sich Rückschlüsse auf die Muskelregeneration daraus ableiten lassen. Hierfür erfolgten in vitro-Untersuchungen an C2C12-Zellen und die anschließende Analyse der Expression von CXCR4, CXCR7 und SDF-1α in der sich entwickelnden Gliedmaßenmuskulatur von Wildtyp- und mdx-Mäusen. Die Untersuchung von C2C12-Zellen zeigte in allen Differenzierungsstadien eine detektierbare Menge von CXCR4 und eine zunehmende Expression des CXCR7. Die Behandlung der Zellen mit SDF-1α führte zu einer Phosphorylierung von Erk1/2 und PKCζ/λ und hemmte gleichzeitig deren Differenzierung. Nach einer Blockierung des CXCR4 mit seinem pharmakologischen Antagonist AMD3100 oder nach Hemmung der Expression durch spezifische siRNA blieb die Aktivierung des Signalweges aus und der hemmende Einfluss des SDF-1α auf die Differenzierung verschwand vollständig. Im Gegensatz dazu blieben nach der pharmakologischen Blockierung oder durch siRNA vermittelten Expressionshemmung des CXCR7 alle SDF-1α induzierten Effekte vollständig erhalten. Eine Untersuchung des Signalweges am dritten Tag der Differenzierung zeigte keine Aktivierung von Erk1/2. Ebenso blieb Erk1/2 nach einer Hemmung der Expression des CXCR4 unphosphoryliert. Im Gegensatz dazu fand nach einer Hemmung der Expression des CXCR7 mit spezifischer siRNA erneut eine Aktivierung des Signalweges statt. Weiterhin konnte in vivo festgestellt werden, dass die Expression des CXCR4 in der Muskulatur während der embryonalen Entwicklung am höchsten ist und bereits kurz nach der Geburt stark abnimmt, wenn die sekundäre Muskelentwicklung abgeschlossen ist. Die Expression des CXCR7 hingegen steigt perinatal an und bleibt bis zum Erwachsenenalter bestehen. Zusammengefasst zeigen diese Ergebnisse, dass CXCR4 aktiv das Signalgeschehen von SDF-1α in der Myogenese vermittelt und somit zur Differenzierungshemmung von Myoblasten beiträgt. CXCR7 hingegen wirkt als passiver SDF-1α-Scavenger und induziert somit durch eine Modulierung der extrazellulären SDF-1α-Konzentration die Differenzierung. In Übereinstimmung mit der Rolle des SDF-1α-Systems bei der Muskelentwicklung, konnte eine kontinuierliche SDF-1α- Expression im Bindegewebe um pränatale und im Endomysium von postnatalen und adulten Muskelfasern festgestellt werden. Diese SDF-1α-Expression stieg ebenso wie die CXCR4-Expression bei der Analyse der Muskulatur von dystrophin-defizienten Mäusen an und zeigte somit, dass dieses System auch für die Proliferation von Muskelvorläuferzellen in der regenerativen Muskulatur eine wichtige Rolle spielt. Bemerkenswerter Weise hatte diese Muskeldystrophie keinen Einfluss auf die Expression des CXCR7 und beeinflusst vermutlich dessen Funktion über einen anderen Mechanismus. Durch die offensichtlich enge Kontrolle von Muskelentwicklung und Regeneration durch CXCR4, CXCR7 und deren Liganden SDF-1α, bilden diese ein vielversprechendes therapeutisches Ziel für bestimmte Muskelerkrankungen. / The chemokine, SDF-1α, and its receptor, CXCR4, are assumed to control a multitude of biological processes such as organogenesis, haematopoesis, and immune responses. The previous demonstration that SDF-1α additionally binds to the chemokine receptor, CXCR7, currently urges a re-evaluation of the function of the SDF-1 system in these processes. In fact, depending on the tissue and cell type, CXCR7 either acts as a scavenger receptor, a modulator of CXCR4 or an independent active receptor. This thesis is dedicated to answer the following questions: Are both SDF-1α receptors expressed during muscle development? What is the actual function of these receptors during myogenesis? Is there a role of the SDF-1 system in muscle regeneration? To adress these issues both in vitro studies with the myoblast cell line, C2C12, as well as in vivo analyses on the expression of CXCR4, CXCR7 and SDF-1α in developing and regenerating limb muscles have been performed. At all stages of differentiation, C2C12 cells exhibited measurable amounts of CXCR4. In addition, in the course of differentiation C2C12 cells showed increasing expression levels of CXCR7. Treatment of the cells with SDF-1α resulted in the phosphorylation of Erk1/2 and PKCζ/λ and subsequently blocked their myogenic differentiation. Following inactivation of CXCR4 either by its antagonist, AMD3100, or by specific siRNA, SDF-1α failed to activate both pathways and in addition no longer inhibited the myogenic differentiation of C2C12 cells. By contrast, inactivation of CXCR7 remained without effects on SDF-1α-induced cell signalling and resulting inhibitory effects on myogenic differentiation. Interestingly, SDF-1α also failed to activate Erk1/2 signalling in differentiated C2C12 cells. Cell signalling in differentiated C2C12 cells was, however, restored following inhibition of CXCR7 expression, but not following inhibition of CXCR4 expression. The in vivo analysis further revealed that in limb muscles expression of the CXCR4 is highest during embryonic development with a decrease in expression levels shortly after birth when secondary muscle development is completed. Vice versa, expression levels of CXCR7 increased perinatally and remained high into adulthood. In summary, these findings unravel that CXCR4 actively mediates SDF-1α-signalling during myogenesis. The findings further provide evidence that CXCR7 acts as a scavenger receptor during myogenesis which controls myogenic differentiation by modulating extracellular SDF-1α concentration. In further agreement with a major role of SDF-1α in muscle development, SDF-1α is continously expressed by the endomysium of postnatal and adult muscle fibers. Moreover, expression of SDF-1α as well as CXCR4 is massively increased in muscles of dystrophin-deficient mice further implying that the SDF-1 system plays an equally important role during muscle development and regeneration. The pivotal role of SDF-1α in muscle development and regeneration points to the SDF-1 system as a promising therapeutical target for certain muscle diseases.

No description available.

Tendon transfer surgery is used to improve the hand function of patients with nerve injuries, spinal cord lesions, cerebral palsy (CP), stroke, or muscle injuries. The tendon of a muscle, usually with function opposite that of the lost muscle function, is transferred to the tendon of the deficient muscle. The aim is to balance the wrist and fingers to achieve better hand function. The position, function, and length at which the donor muscle is sutured is essential for the outcome for the procedure. In these studies the significance of the transferred muscle’s morphology, length and apillarization was investigated using both animal and human models. Immunohistochemical, biochemical, and laser diffraction techniques were used to examine muscle structure. In animal studies (rabbit), the effects of immobilization and of tendon transfers at different muscle lengths were analyzed. Immobilization of highly stretched muscles resulted in fibrosis and aberrant regeneration. A greater pull on the tendon while suturing a tendon transfer resulted in larger sarcomere lengths as measured in vivo. On examination of the number of sarcomeres per muscle fiber and the sarcomere lengths after 3 weeks of immobilization and healing time, we found a cut-off point up to which the sarcomerogenesis was optimal. Transfer at too long sarcomere lengths inhibited adaptation of the muscle to its new length, probably resulting in diminished function. In human studies we defined the sarcomere lengths of a normal human flexor carpi ulnaris muscle through the range of motion, and then again after a routinely performed tendon transfer to the finger extensor. A calculated model illustrated that after a transfer the largest force was predicted to occur with the wrist in extension. Morphological studies of spastic biceps brachii muscle showed, compared with control muscle, smaller fiber areas and higher variability in fiber size. Similar changes were also found in the more spastic wrist flexors comparing with wrist extensors in children with CP. In flexors, more type 2B fibers were found. These observations could all be due to the decreased use in the spastic limb, but might also represent a specific effect of the spasticity. In children and adults with spasticity very small fibers containing developmental myosin were present in all specimens, while none were found in controls. These fibers probably represent newly formed fibers originating from activated satellite cells. Impaired supraspinal control of active motion as well as of spinal reflexes, both typical of upper motor syndrome, could result in minor eccentric injuries of the muscle, causing activation of satellite cells. Spastic biceps muscles had fewer capillaries per cross-sectional area compared to age-matched controls, and also a smaller number of capillaries around each fiber. Nevertheless, the number of capillaries related to the specific fiber area was normal, and hence the spastic fibers are sufficiently supplied with capillaries. This study shows that the length of the muscle during tendon transfer is crucial for optimization of force output. Laser diffraction can be used for accurate measurement of sarcomere length during tendon transfer surgery. Wrist flexor muscles have more morphological alterations typical of spasticity compared to extensors.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

No description available.

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T19:53:30Z (GMT). No. of bitstreams: 1 Santos_AlineMarados_D.pdf: 7023687 bytes, checksum: ccd063ea57be631b6b555f86e24af597 (MD5) Previous issue date: 2011 / Resumo: A tirosino-quinase de adesão focal (FAK) tem papel crítico na mediação da migração, sobrevivência e proliferação celular. Estudos anteriores de nosso laboratório demonstraram que a FAK é ativada pelo estresse mecânico em miócitos cardíacos e que ela se coimunoprecipita com a miosina sarcomérica. No presente trabalho foi demonstrado que o domínio FERM da FAK medeia à interação com a miosina sarcomérica, sendo que esta interação leva a inibição da autofosforilação da FAK, enquanto que a ativação prévia da FAK reduz sua interação com a miosina in vitro. Ensaios de cross linking acoplado a espectrometria de massas e espalhamento de raios X a baixos ângulos demonstraram que a miosina interage em uma fenda localizada entre os subdomínios do domínio FERM. Experimentos de microscopia confocal demonstraram que estas proteínas estão colocalizadas em miócitos cardíacos de ratos neonatos e adultos. Ensaios de imunoprecipitação revelaram que aproximadamente 40% da FAK está basalmente associada à miosina sarcomérica enquanto que, após o estiramento celular esta associação reduziu paralelamente à ativação da FAK. A porcentagem de FAK associada à miosina não se alterou com a ativação da FAK após tratamento com fenilefrina, diferente da ativação pelo estresse mecânico. A interferência na interação FAK/miosina pelo silenciamento gênico da miosina culminou com a ativação da FAK e o tratamento dos miócitos cardíacos com o peptídeo FP-1, derivado do subdomínio F2 do domínio FERM, levou a uma diminuição na interação FAK/miosina e ao aumento na fosforilação/ativação da FAK. O tratamento prolongado com FP-1 resultou em hipertrofia dos miócitos cardíacos de ratos neonatos, efeito concomitante à ativação da via de sinalização Akt, TSC2 e S6Kinase. Tanto o silenciamento da FAK quanto o tratamento com rapamicina bloquearam a hipertrofia decorrente do tratamento com FP-1. Os dados deste trabalho indicam que a interação da FAK com a miosina sarcomérica é sensível ao estresse mecânico e que possui papel regulatório na manutenção da quiescência basal da FAK e no controle das vias de sinalização mediadas por esta quinase, como a via de crescimento celular AKT/mTOR/S6Kinase, em miócitos cardíacos em cultura / Abstract: The Focal Adhesion Kinase (FAK) plays a critical role in mediating the migration, survival and cell proliferation. Previous studies from our laboratory demonstrated that FAK is activated by mechanical stress in cardiac myocytes and it co-immunoprecipitate with sarcomeric myosin. Here, we demonstrated that the FAK FERM domain mediates the interaction with sarcomeric myosin, and that this interaction leads to inhibition of FAK autophosphorylation in vitro, whereas the previous activation of FAK reduces its affinity to myosin. A model based on small angle X-ray scattering analyses and crosslinking technology coupled with mass spectrometry indicated that a cleft in FERM domain is critical to the interaction of FAK to myosin. Confocal microscopy experiments showed that these proteins are colocalized in cardiomyocytes of neonatal and adult rats. Immunoprecipitation assays revealed that approximately 40% of FAK is basally associated with sarcomeric myosin while cardiomyocyte stretching reduced this association in parallel with FAK activation. The percentage of FAK associated with myosin was not change in response to FAK activation by treatment with phenylephrine, unlike in response to FAK by mechanical stress. The interference in the FAK/Myosin interaction by myosin silencing approach culminated with the activation of FAK. The treatment of cells with the FP-1 peptide, derived from the FAK FERM domain, lead to a decrease in the interaction with sarcomeric myosin and an increase in FAK activation. Prolonged treatment with FP-1 resulted in morphological hypertrophy of neonatal rat cardiomyocytes which was an effect concomitant with activation of the Akt, TSC2 and S6Kinase signaling pathway. Both FAK silencing and the rapamycin treatment blocked the morphological hypertrophy resulting of the treatment with FP-1. This study indicated that the interaction of FAK with sarcomeric myosin is sensitive to mechanical stress and it has regulatory role in maintaining of FAK quiescence and control of signaling pathways mediated by this kinase, such as cell growth via AKT/mTOR/S6Kinase in cardiac myocytes in culture / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

Before major athletic events, a taper is often prescribed to facilitate recovery and enhance performance. However, it is unknown which taper model is most effective for peaking maximal strength and positively augmenting skeletal muscle. Thus, the purpose of this study was to compare performance outcomes and skeletal muscle adaptations following a step vs. an exponential taper in strength athletes. Sixteen powerlifters (24.0 ± 4.0 years, 174.4 ± 8.2 cm, 89.8 ± 21.4 kg) participated in a 6-week training program aimed at peaking maximal strength on back squat [initial 1-repetition-maximum (1RM): 174.7 ± 33.4 kg], bench press (118.5 ± 29.9 kg), and deadlift (189.9 ± 41.2 kg). Powerlifters were matched based on relative maximal strength, and randomly assigned to either (a) 1-week overreach and 1-week step taper or (b) 1-week overreach and 3-week exponential taper. Athletes were tested pre- and post-training on measures of body composition, jumping performance, isometric squat, and 1RM. Whole muscle size was assessed at the proximal, middle, and distal vastus lateralis using ultrasonography and microbiopsies at the middle vastus lateralis site. Muscle samples ( = 15) were analyzed for fiber size, fiber type [myosin-heavy chain (MHC)-I, -IIA, -IIX, hybrid-I/IIA] using whole muscle immunohistochemistry and single fiber dot blots, gene expression, and microRNA abundance. There were significant main time effects for 1RM squat ( < 0.001), bench press ( < 0.001), and deadlift, ( = 0.024), powerlifting total ( < 0.001), Wilks Score ( < 0.001), squat jump peak-power scaled to body mass ( = 0.001), body mass ( = 0.005), fat mass ( = 0.002), and fat mass index ( = 0.002). There were significant main time effects for medial whole muscle cross-sectional area (mCSA) ( = 0.006) and averaged sites ( < 0.001). There was also a significant interaction for MHC-IIA fiber cross-sectional area (fCSA) ( = 0.014) with comparisons revealing increases following the step-taper only ( = 0.002). There were significant main time effects for single-fiber MHC-I% ( = 0.015) and MHC-IIA% ( = 0.033), as well as for MyoD ( = 0.002), MyoG ( = 0.037), and miR-499a ( = 0.033). Overall, increases in whole mCSA, fCSA, MHC-IIA fCSA, and MHC transitions appeared to favor the step taper group. An overreach followed by a step taper appears to produce a myocellular environment that enhances skeletal muscle adaptations, whereas an exponential taper may favor neuromuscular performance.

No description available.

Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. The genus Orthohepevirus A of the family Hepeviridae includes all mammalian strains of HEV and consists of 8 recognized genotypes. Genotypes 1 and 2 HEVs only infect humans and genotypes 3 and 4 infect humans and several other animal species including pigs and rabbits. An ever-expanding host range of genetically-diversified strains of HEV now include bat, fish, rat, ferret, moose, wild boar, mongoose, deer, and camel. Additionally, the ruminant species goats, sheep, and cattle have been implicated as potential reservoirs as well. My dissertation research investigates a novel animal model for HEV, examines the immune dynamics during acute infection, and evaluates the possibility of additional animal reservoirs of HEV. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans and evaluated the pathogenesis of HEV infection in this novel animal model. The dynamics of acute HEV infection in gnotobiotic pigs were systematically determined with a genotype 3 human strain of HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and immune dynamics during the acute stage of virus infection. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. The objective of the second project for my PhD dissertation was to determine if cattle in the United States are infected with a bovine strain of HEV. We demonstrated serological evidence of an HEV-related agent in cattle populations with a high level of IgG anti-HEV prevalence. We demonstrated that calves from a seropositive cattle herd seroconverted to IgG binding HEV during a prospective study. We also showed that the IgG anti-HEV present in cattle has an ability to neutralize genotype 3 human HEV in vitro. However, our exhaustive attempts to detect HEVrelated sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research. / Ph. D. / Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. According to the World Health Organization, there are approximately 20 million HEV infections annually, which result in 3.3 million cases of acute hepatitis E and >44,000 HEV-related deaths. Hepatitis E is a self-limiting acute disease in general, but carries the ability to cause high mortality in pregnant women and chronic hepatitis in immunocompromised individuals. The underlying mechanisms of HEV host tropism and progression of disease to chronicity are unknown. My dissertation work investigates a novel animal model for HEV, evaluates the possibility of additional animal reservoirs of HEV, and examines the immune dynamics during acute infection. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans. The dynamics of acute HEV infection were determined in both the knock-out and wild-type piglets with a genotype 3 strain of human HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and virus infection. In the second project, we determined if cattle in the United States are infected with a bovine strain of HEV. We showed serological evidence of an HEV-related agent in cattle as well as calves born in a seropositive herd. Despite the detection of specific antibodies recognizing HEV in cattle, definitive evidence of virus infection could not be demonstrated. Our exhaustive attempts to detect HEV-related sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation research delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research.