Skeletal muscles are composed of thousands of muscle fibres (muscle cells), densely packed together in parallel and surrounded by connective tissue sheaths. These fibres are multinuclear in nature, which allows for the control and regulation of the highly organised, protein rich cellular interior. The primary function of skeletal muscle is to produce force, which allows for movement to occur or posture to be maintained, and the regulation of this function is in turn reliant on the expression of specific genes and proteins. Skeletal muscle exhibits a high degree of plasticity, and can adapt in response to stimuli such as increased/decreased use, metabolic perturbations or changes in the systemic environment which often occur as a result of exercise, ageing, disuse or disease. Examining responses and adaptations in skeletal muscle in vivo are challenging due to experimental restrictions, and studies are limited by ethical issues surrounding experimentation on human beings and indeed on animals following the principals of refinement, reduction and replacement. Thus in vitro studies are often conducted in order to further understand mechanisms involved in adaptation. However, the environment to which skeletal muscle cells are exposed to in vitro is far removed from that in the body, and the resulting cellular architecture is often abnormal in morphology. Tissue engineered skeletal muscle has shown much promise in rectifying these issues, as cells can be grown on/within a matrix which is biologically relevant and engineered to grow in a uniaxial manner in parallel to one another. However, this field is in its relative infancy, and to date little data exists with regards the behaviour and characteristics of human muscle derived cells (MDCs) in tissue engineered constructs. In this thesis, human skeletal MDCs were obtained, characterised and subsequently cultured in a suitable model for tissue engineering purposes. MDCs were seeded on to a fibrin based hydrogel, which self-assembled over time to form a cylindrically shaped construct held in place between two anchor points. In ii this model, the cells were shown to align uniaxially and in parallel to one another in a fascicular like structure. The model was improved in terms of biomimicity and maturation by both increasing the seeding density of the MDCs, and by increasing the ratio of myogenic to non-myogenic cells. These models appear to promote the development of a slow muscle, as evidenced by the favourably high levels of MYH7 transcription in comparison to other isoforms, and showed suggestions of sarcomeric organisation as indicated by the classically striated pattern of protein organisation when myosin heavy chain immunostaining was conducted. The work conducted in the final chapter of this thesis focussed on developing a system capable of assessing and quantifying the force produced by these tissue engineered human skeletal muscle constructs when electrically stimulated. Further work in this area should aim to determine these functional characteristics and thereafter use the model for physiological, cellular and molecular studies in exercise science.

Muscle wasting is a significant complication of many diseases including diabetes mellitus, renal and liver failure, HIV/AIDS, and cancer. Sustained loss of skeletal muscle can severely impair a patient's quality of life and often results in poor tolerance and responsiveness to disease treatments. The increased protein breakdown observed during muscle atrophy has been attributed to accelerated activity of the ubiquitin-proteasome pathway, but the precise mechanisms by which this activation stimulates muscle protein loss are poorly understood. Previous work showed that the deubiquitinating enzyme USP19 is upregulated in rat skeletal muscle in various forms of muscle wasting, including streptozotocin induced diabetes, cancer, and dexamethasone treatment. 1 To further explore the role of USP19 in muscle wasting, siRNA-mediated depletion of the enzyme was carried out in L6 myotubes. Knockdown of USP19 resulted in more rapid differentiation of myoblasts into myotubes, with a greater extent of myoblast fusion. It also produced tubes that were visibly larger than those formed by myoblasts transfected with a control siRNA. At the molecular level, silencing of USP19 increased the amount of myosin heavy chain (MHC) and tropomyosin proteins. It also increased levels of MHC transcript, suggesting that USP19 acts at the level of gene transcription or mRNA stability rather than protein degradation. USP19 may mediate its effects on muscle-specific gene expression through the myogenic transcription factor myogenin, since depletion of USP19 increased protein and mRNA levels myogenin but did not affect protein levels of the related transcription factor Myf5. Moreover, the increased tropomyosin and MHC observed upon USP19 knockdown could be abolished when myogenin was simultaneously depleted using siRNA. Collectively, these results suggest that USP19 functions to inhibit the synthesis of key muscle proteins and may therefore be a promising target for the treatment of muscle atrophy.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.