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Šio darbo tikslas buvo nustatyti ar NAD(P)H fluorescencija, registruojama neinvaziniu fluorescenciniu metodu, gali būti naudojama kaip miogeninių ląstelių diferenciacijos žymuo, ištiriant fluorescencijos pokyčius miogeninių ląstelių diferenciacijos metu bei išaiškinant pagrindines šių pokyčių priežastis. Tyrimai parodė, kad 95 % mioblastų fluorescencijos, išmatuotos prie 460 nm emisijos, priklauso nuo NAD(P)H kiekio. Ląstelėms diferencijuojantis fluorescencija ženkliai didėja, tačiau susiejus fluorescenciją su didėjančiu baltymų kiekiu, ženklus fluorescencijos padidėjimas nustatytas tik po 10 diferenciacijos dienų. Panaudojus ląstelių metabolizmą ir NAD(P)H kiekį veikiančius veiksnius (skyriklį CCCP ir slopiklį KCN) buvo įvertinti santykiniai NADH ir NAD+ kiekiai bei NAD+/NADH santykis, kuris diferenciacijos metu mažėjo. 535 nm fluorescencijos pokyčiai diferenciacijos metu atskleidžia didėjantį FAD kiekį. Tiek NAD+/NADH santykis tiek 535 ir 460 nm fluorescencijos emisijų santykis yra patikimesni diferenciacijos rodikliai nei pati NAD(P)H fluorescencija. Šio darbo rezultatai patvirtina, kad tokie greiti, tikslūs ir neinvaziniai fluorescencijos matavimai gali būti naudingi nustatant mioblastų diferencacijos pradžią ir vyksmą bei taikytini įvairiems bandymams siekiant nustatyti vaistų ir kitų faktorių įtaką ląstelių metabolizmui. / The aim of this study is to check hypothesis that increase of fluorescence at 460 nm emission upon myoblast differentiation display alteration of NADH quantity that change NAD+/NADH ratio and could be a myoblast marker of entering into differentiation process. To evaluate NAD(P)H influence on total cell fluorescence emission intensity at 460 nm wavelenght were used metabolism effectors: carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to obtain minimal NADH fluorescence; and KCN to obtain maximal NADH fluorescence. Assuming, that CCCP caused maximal oxidation and that KCN caused maximum reduction, estimated that NAD+/NADH ratio decreased upon myoblast differentiation. CCCP effect showed that about 95 % of myoblast fluorescence at 460 nm emission is dependent on NAD(P)H. The fluorescence intensity at 460 nm was increasing with myoblast differentiation. Using fluorescence measuring estimated average NADH concentration significantly increased on 10 day of differentiation compared with control cells. Changes of fluorescence intensity at 535 nm reveal accumulation of FAD upon myoblast differentiation. Therefore, the best indicator of myoblast differentiation stage is ratio of fluorescence emission at 535 and 460 nm. Our results indicate that quick, quantitative and real-time fluorescence method may be useful for non-invasive detection and evaluation of myoblast differentiation stage.

RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.

The aim of this project was to create a novel biodegradable, synthetic scaffold that will provide the correct topographical cues for myoblast alignment and efficient differentiation into myotubes. Skeletal muscle repair after major surgery or serious burns is often overlooked leading to poor healing and consequent loss of power in movements of affected limbs. In order to overcome this problem a tissue engineered construct could be utilised as a grafting patch to encourage further regeneration and enhance possible power to the limb. Using a biodegradable polymer can provide structural support until the tissue is established, and will be excreted by the body's natural processes as it degrades. A synthetic polymer is desirable as it can reduce the risk of immunogenic responses thus reduce risk of graft rejection. For successful in vitro growth of skeletal muscle, the cells must be encouraged to arrange themselves into parallel arrays in order for efficient fusion and consequent contraction. By incorporating the correct topographical cues into the scaffold to promote contact guidance for cellular alignment this can be achieved. Electrospinning is a reliable technique which yields highly reproducible aligned fibres from the micro- to the nanoscale. This project focuses upon creating and characterising the electrospun scaffold, checking biocompatibility with myoblasts by monitoring the topography, residual solvent within the scaffold, the mechanical properties of the scaffold, and a brief investigation into the degradation profile of the electrospun fibres. The immunogenicity of the scaffold was investigated by monitoring cytokine release from macrophages. Myoblast morphology was monitored, as was the efficiency of the cells to differentiate and their potential to become contractile myofibres. Cellular adhesion to the scaffold was also looked into by measuring the expression of integrins during early and late adhesion and on substrates with different topographies. It was found that the electrospun scaffold did not contain a significant amount of residual solvent, and macrophages were not activated any more than on tissue culture plastic. Myoblasts responded to the topography of the aligned fibres by aligning along the length of the fibres, showing elongation and bi-axial cytoskeletal arrangement after just 30 minutes culture on the aligned fibres. This elongation prompted fusion and differentiation of the myoblasts to occur faster than cells which were not exposed to the aligned topography, and this global alignment was maintained in long term culture.

The NF-κB family of transcription factors can be activated through canonical (classical) or non-canonical (alternative) signalling pathways, which are regulated by the redundant ubiquitin ligases, cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2). While the canonical NF-κB pathway is needed for myoblast proliferation, it is inactivated during myoblast differentiation. However, the non-canonical NF-κB pathway is a major factor in promoting myoblast fusion, which is crucial to the processes of myogenesis and muscle repair. Ablation of cIAP1 levels through a chemical antagonist such as a SMAC- mimetic compound (SMC) activates non-canonical signalling to enhance myogenesis. The cytokine TNF-like weak inducer of apoptosis (TWEAK) has also been shown to activate primarily the alternative NF-κB pathway when signalling through its receptor Fn14. Here I show that alternative NF-κB signalling activity, stimulated by the addition of TWEAK or loss of cIAP1, can promote myogenesis. I also demonstrate that TWEAK is an endogenous myokine produced by myoblasts to promote their own differentiation, and suggest that targeting the alternative NF-κB pathway, with SMAC-mimetics or recombinant TWEAK for example, would be of therapeutic value in the repair and regeneration of muscle for various myopathies.