A variation on a numerical model of the motor protein myosin V presentedin a paper by Craig and Linke (2009) is developed. An alternative potential is proposed. All aspects of the model development arederived in detail and tested. Two model tests are created and used toconfirm the correctness of the developed model. A simulation demonstratesthat the developed model is able to produce a myosin V step. Adidactical transposition is presented in the form of a compendium, in which a numerical model of myosin V by Craig and Linke (2009) isdescribed. The didactical transposition is developed using a methodologyof didactical engineering. The didactical study indicated thatthe content was well recieved by the target group of eight individualsin respect to the scientific complexity and that it evokes motivation forlearning. The study also indicated that unsuccessful areas of the didacticaltransposition existed. / En variation av en numerisk modell av motorproteinet myosin V presenteradav Craig och Linke (2009) utvecklas. En alternativ potential föreslås. Alla aspekter av modellutvecklingen härleds i detalj och testas.Två modelltester skapas och används för att bekräfta riktigheteni den utvecklade modellen. En simulering demonstrerar att myosin Vkan ta ett steg i den utvecklade modellen.En didaktisk transposition presenteras i form av ett kompendium,där en numerisk modell av myosin V av Craig och Linke (2009) beskrivs.Metodologiskt utvecklas den didaktiska transpositionen medhjälp av didaktisk ingenjörskonst. Den didaktiska studien indikeradeatt innehållet togs emot väl av målgruppen bestående av åtta personeri hänseende till vetenskaplig komplexitet och att det väckte motivationför lärande. Studien indikerade även att misslyckade områden av dendidaktiska transpositionen förekom.

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Master of Science / Department of Animal Sciences and Industry / Bradley J. Johnson / Zilpaterol hydrochloride (ZH) is a growth promotant that is approved for use in finishing cattle to improve growth performance and increase lean tissue accumulation. Little is known about the combined effects of ZH with anabolic steroid hormone implants. There is also little published data on the effect these growth promotants have on genes that play a role in skeletal muscle synthesis and degradation. Therefore, two separate studies were conducted to address these issues. The first study evaluated the effects of ZH and the steroid implant Revalor-S (RS) on animal performance and skeletal muscle gene expression in feedlot steers. Four treatments were used to analyze the effects of RS implanted 58 days before ZH, which was fed for 30 days with a 3 day withdrawal. It was determined that ZH and RS additively contribute to improved live and carcass performance; however these compounds had different effects on the abundance of the receptors for ZH as well as the abundance of myosin heavy chain (MHC) mRNA in skeletal muscle of feedlot steers. It was also determined that ZH can cause a transition in the abundance of MHC mRNA isoforms in skeletal muscle that are available for the translation of larger, faster, more glycolytic fiber types of MHC. The second study evaluated the effects of two types of anabolic steroid hormones on myosin heavy chain gene expression. Four treatments were used to measure the effects of trenbolone acetate (TBA) and estradiol (E[subscript]2) on performance and the amount of MHC mRNA in skeletal muscle of finishing steers. It was determined that anabolic steroid implants improve live animal performance, however there was no alteration in the abundance of MHC mRNA in skeletal muscle of feedlot steer for 28 days after implantation; however there was an increase in intermediate fiber type IIA of MHC mRNA in skeletal muscle with increasing days on feed. From these studies we concluded that ZH and anabolic steroids do have an effect on growth performance; however they may differ in the distinct mechanism of action utilized to enhance lean tissue deposition in feedlot steers.

Endocytosis is essential for the pathogenic development of Ustilago maydis. It has been shown that the initiation of pathogenicity relies upon the ability of the cell to recognize pheromone (a1 or a2) released from its mating partner and subsequently to form conjugated hyphae. The actin and microtubule cytoskeleton plays an essential role in all aspects of cell growth. A component of the actin cytoskeleton, the filamentous actin is required for cell-cell fusion, whereas the molecular motors, kinesin and dynein, move along microtubules and provide the long distance transport of many proteins and they are important in cell growth and pathogenicity. In this thesis, we investigated the role of the cytoskeleton in endocytosis and a1 pheromone transport, using a fluorescently labelled derivative of the a1 pheromone. We confirmed that uptake of the a1 pheromone is also receptormediated. In addition, we have shown that pheromone transport towards the cellular vacuole requires the actin and microtubule cytoskeletons. Furthermore, we revealed that the microtubule-dependent motors kinesin-1 and kinesin-3 and dynein were shown to be essential in the delivery of the pheromone to vacuoles. Moreover, a mutation in the early endosomal protein Yup1 gene causes a stop in delivery of the synthetic pheromone to the vacuole. This suggests that it travels with early endosomes. Within the actin cytoskeleton, we analysed the dynamics of actin patches in the presence of the synthetic pheromone and found that the dynamics of the patches increased significantly. Additionally, in the presence of an over-expression of the tail domain of the molecular motor myosin-5, the dynamics of the patches were significantly reduced and their intensity diminished.

Critically ill patients admitted to the intensive care unit (ICU) commonly develop severe muscle wasting and weakness and consequently impaired muscle function. This not only delays respirator weaning and ICU discharge, but has deleterious effects on morbidity, mortality, financial costs, and quality of life of survivors. Acute Quadriplegic Myopathy (AQM) is one of the most common neuromuscular disorders underlying ICU muscle wasting and paralysis, and is a consequence of modern intensive care interventions, although the exact causes remain unclear. Muscle gene/protein expression, intracellular signalling, post-translational modifications, muscle membrane excitability, and contractile properties at the single muscle fibre level were explored in order to unravel the mechanisms underlying the muscle wasting and weakness associated with AQM and how this can be counteracted by specific intervention strategies. A unique experimental rat ICU model was used to address the mechanistic and therapeutic aspects of this condition, allowing time-resolved studies for a period of two weeks. Subsequently, the findings obtained from this model were translated into a clinical study. The obtained results showed that the mechanical silencing of skeletal muscle, i.e., absence of external strain (weight bearing) and internal strain (myosin-actin activation) due to the pharmacological paralysis or sedation associated with the ICU intervention, is likely to be the primary mechanism triggering the preferential myosin loss and muscle wasting, features specifically characteristic of AQM. Moreover, mechanical silencing induces a specific gene expression pattern as well as post-translational modifications in the motor domain of myosin that may be critical for both function and for triggering proteolysis. The higher nNOS expression found in the ICU patients and its cytoplasmic dislocation are indicated as a probable mechanism underlying these highly specific modifications. This work also demonstrated that passive mechanical loading is able to attenuate the oxidative stress associated with the mechanical silencing and induces positive effects on muscle function, i.e., alleviates the loss of force-generating capacity that underlie the ICU intervention, supporting the importance of early physical therapy in immobilized, sedated, and mechanically ventilated ICU patients.

Thesis (PhD)--University of Stellenbosch, 2004. / 426 Leaves printed single pages, preliminary pages i-xxiv and i-xxvii and 399 numberd pages. Includes bibliography. List of figures, List of tables, List of abbreviations. / ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease. The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and an increased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring in thirteen genes, have been identified. As the vast majority of HCM-causing mutations occur in components of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere. Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed that HCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausing mutations in the gamma two subunit of adenosine monophosphate activated protein kinase highlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports a hypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion. Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second most prevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomeric thick filament, where it acts as a regulator of thick filament structure and cardiac contractility. Although cMyBPC has been studied extensively, the mechanisms through which it fulfill these functions have remained elusive, largely due to a lack of a comprehensive understanding of its interactions with other sarcomeric components and its quaternary structure. The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in a panel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC in which HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individuals was screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)- based single-strand conformation polymorphism method, as well as restriction enzyme digestion, DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands of domains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandand library-assays were performed. Three novel and two previously described putative HCM-causing mutations were identified in MYBPC3. Data generated in this and other studies, however, suggest that two of these “mutations” are likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausing mutations. Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This finding identified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specific C7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 and domain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no known sarcomeric ligands of the N-terminal region of cMyBPC. Identification of the ligands of specific domains of cMyBPC led to the development of detailed models of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fully phosphorylated. The integration of these models into an existing model of thick filament quaternary structure allows new insights into the functioning of cMyBPC as a regulator of both thick filament structure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HKM) is ‘n outsosomaal dominante primêre hartsiekte. Die primêre kenmerke van HKM is linker ventrikulêre hipertrofie, miokardiale wanorde, fibrose en ‘n verhoogde risiko van skielike dood. Tot dusver is 260 HKM-veroorsakende mutasies in 13 gene geïdentifiseer. Aangesien die oorgrote meerderheid van HKM-veroorsakende mutasies in komponente van die kardiale sarkomeer voorkom, is HKM as ‘n siekte van die kardiale sarkomeer beskryf. Funksionele analise van HKM-veroorsakende mutasies in sarkomeriese protein-koderende gene het aan die lig gebring dat hierdie mutasies ‘n wye spektrum van gevolge op kontraktiele funksie het. Die ontdekking van HKM-veroorsakende mutasies in die gamma-twee subeenheid van adenosien monofosfaat-geaktiveerde proteïen kinase het die feit dat mutasies nie-sarkomeriese proteïene ook HKM kan veroorsaak onderstreep en ondersteun ‘n hipotese dat HKM-veroorsakende mutasies energievermorsing en energie uitputting tot gevolg het. Mutasies in die kardiale miosien-bindingsproteïen C (kMiBPC) geen (MYBPC3) is die tweede mees algemene oorsaak van HKM. kMiBPC is ‘n modulêre protein wat ‘n integrale deel van die sarkomeriese dik filament vorm, waar dit die struktuur van die dik filament en kardiale kontraktiliteit reguleer. Nieteenstaande die feit dat kMiBPC intensief bestudeer is, word die meganismes hoe hierdie funksies vervul word swak verstaan, grotendeels weens die afwesigheid van ‘n in diepte begrip van sy interaksies met ander komponente van die sarkomeer asook sy kwaternêre struktuur. Die doelstellings van hierdie studie was, eerstens, om MYBPC3 vir HKM-veroorsakende mutasies in ‘n paneel van HKM-geaffekteerde individue te deursoek en tweedens, om die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is te identifiseer.‘n Paneel van deoksiribonukleïensuur (DNS) monsters verkry van onverwante HKM-geaffekteerde individue is deursoek vir HKM-veroorsakende mutasies in MYBPC3, deur middel van die polimerase ketting-reaksie (PKR)-gebasseerde enkelstrand konformasie polimorfisme metode, sowel as restriksie ensiem vertering, DNS volgordebepaling en terugtranskripsie PKR tegnieke. Die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is, is geïdentifiseer deur middel van gis twee-hibried (G2H) kandidaat-ligand en biblioteek-siftings eksperimente. Drie onbeskryfde en twee voorheen beskryfde vermeende HKM-veroorsakende mutasies in MYPBC3 is geïdentifiseer. Data gegenereer in hierdie en ander studies dui daarop dat twee van hierdie “mutasies” eerder polimorfismes, of siekte-modifiserende faktore, as hoof-lokus HKMveroorsakende mutasies is. Onlangse bevindings het ‘n spesifieke interaksie tussend die C5 en C8 domeine van kMiBPC getoon. Hierdie bevindings het óf domein C6, óf C10, as kandidaat-ligande van domein C7 geïdentifiseer. G2H eksperimente het ‘n spesifieke interaksie tussen domains C7 en C10 getoon. Addisionele G2H eksperimente het ook C-zone titin as ‘n ligand van domein C7 sowel as domein C10 as ‘n ligand van domein C3 geïdentifiseer. Verdere G2H eksperimente het egter geen sarkomeriese ligande van die N-terminale gedeelte van kMiBPC geïdentifiseer nie. Die identifikasie van ligande van spesifieke domeins van kMiBPC het gelei tot die ontwikkelling van ‘n gedetaileerde model van kMiBPC kwaternêre struktuur wanneer kMiBPC beide ongefosforileerd en ten volle gefosforileerd is. Die intergrasie van hierdie modelle in bestaande modelle van dik filament kwaternêre struktuur werp nuwe lig op die funksionering van kMiBPC as ‘n reguleerder van beide dik filament struktuur en kardiale kontraktiliteit, sowel as die patofisiologie van kMiBPCgeassosieerde HKM.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality, and is a feature of common diseases, such as hypertension and diabetes. It is therefore vital to understand the underlying mechanisms influencing its development. However, investigating the mechanisms underlying LVH in such complex disorders can be challenging. For this reason, many researchers have focused their attention on the autosomal dominant cardiac muscle disorder, hypertrophic cardiomyopathy (HCM), since it is considered a model disease in which to study the causal molecular factors underlying isolated cardiac hypertrophy. HCM is a heterogeneous disease that manifests with various phenotypes and clinical symptoms, even in families with the same genetic defects, suggesting that additional factors contribute to the disease phenotype. Despite the identification of several HCM-causing genes, the genetic factors that modify the extent of hypertrophy in HCM patients remain relatively unknown. The gene encoding the sarcomeric protein, cardiac myosin binding protein C, cMyBPC (MyBPC3) is one of the most frequently implicated genes in HCM. Identification of proteins that interact with cMyBPC has led to improved insights into the function of this protein and its role in cardiac hypertrophy. However, very little is known about another member of the myosin binding protein family, myosin binding protein H (MyBPH). Given the sequence homology and similarity in structure between cMyBPC and MyBPH, we propose that MyBPH, like cMyBPC, may play a critical role in the structure and functionality of the cardiac sarcomere and could therefore be involved in HCM pathogenesis. The present study aimed to identify MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis and to verify these interactions using three-dimensional (3D) co-localisation and co-immunoprecipitation (Co-IP) analyses. We further hypothesized that both MyBPH and cMyBPC may be involved in autophagy. To test this hypothesis, both MyBPH and cMyBPC were analysed for co-localisation with a marker for autophagy, LC3b-II. The role of MyBPH and cMyBPC in cardiac cell contractility were analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to β-adrenergic stress after individual and concurrent siRNA-mediated knockdown of MyBPH and cMyBPC. In the present study we employed a family-based genetic association analysis approach to investigate the contribution of genes encoding the novel MyBPH-interacting proteins in modifying the hypertrophy phenotype. This study investigated the hypertrophy modifying effects of 38 SNPs and haplotypes in four candidate HCM modifier genes, in 388 individuals from 27 HCM families, in which three unique South African HCM-causing founder mutations segregate. Yeast two-hybrid analysis identified three putative MyBPH-interacting proteins namely, cardiac β-myosin heavy chain (MYH7), cardiac α-actin (ACTC1) and the SUMO-conjugating enzyme UBC9 (UBC9). These interactions were verified using both 3D co-localisation and Co-IP analyses. Furthermore, MyBPH and cMyBPC were implicated in autophagy, since both these proteins were being recruited to the membrane of autophagosomes. In addition, a cardiac contractility assay demonstrated that the concurrent siRNA-mediated knockdown of MyBPH and cMyBPC resulted in a significant reduction in cardiomyocyte contractility, compared to individual protein and control knockdowns under conditions of β-adrenergic stress. These results indicated that MyBPH could compensate for cMyBPC, and vice versa, further confirming that both these proteins are required for efficient sarcomere contraction. Results from genetic association analyses found a number of SNPs and haplotypes that had a significant effect on HCM hypertrophy. Single SNP and haplotype analyses identified SNPs and haplotypes within genes encoding MyBPH, MYH7, ACTC1 and UBC9, which contribute to the extent of hypertrophy in HCM. In addition, we found that several variants and haplotypes had markedly different statistical significant effects in the presence of each of the three HCM founder mutations. The results of this study ascribe novel functions to MyBPH. Cardiac MyBPC and MyBPH play a critical role in sarcomere contraction and have been implicated in autophagy. This has further implications for understanding the patho-etiology of HCM-causing mutations in the gene encoding MyBPH and its interacting proteins. This is to our knowledge the first genetic association analysis to investigate the modifying effect of interactors of MyBPH, as indication of the risk for developing LVH in the context of HCM. Our findings suggest that the hypertrophic phenotype of HCM is modulated by the compound effect of a number of variants and haplotypes in MyBPH, and genes encoding protein interactors of MyBPH. These results provide a basis for future studies to investigate the risk profile of hypertrophy development in the context of HCM, which could consequently lead to improved risk stratification and patient management. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is 'n primêre risikofaktor vir kardiovaskulêre morbiditeit en mortaliteit asook 'n kenmerk van algemene siektes soos hipertensie en diabetes. Daarom is dit van kardinale belang om te verstaan wat die onderliggende meganismes is wat die ontwikkeling van LVH beïnvloed. Die ondersoek na die onderliggende meganismes wat lei tot LVH in sulke komplekse siektes is ‟n uitdaging. Om hierdie rede fokus baie navorsers hul aandag op die autosomaal dominante hartspier siekte, hipertrofiese kardiomiopatie (HKM), wat beskou word as 'n model siekte om die molekulêre oorsake onderliggend tot geïsoleerde kardiovaskulêre hipertrofie te ondersoek. HKM is 'n heterogene siekte wat manifesteer met verskeie fenotipes en kliniese simptome, selfs in families met dieselfde genetiese defekte, wat impliseer dat addisionele faktore bydra tot die modifisering van die siekte fenotipe. Ten spyte van die identifisering van verskeie HKM-versoorsakende gene, bly die genetiese faktore wat die mate van hipertrofie in HKM pasiente modifiseer relatief onbekend. Die geen wat kodeer vir die sarkomeriese proteïen, kardiale miosien-bindingsproteïen C (kMyBPC) is die algemeenste betrokke in HKM. Die identifisering van proteïene wat bind met kMyBPC het gelei tot verbeterde insigte tot die funksie van hierdie proteïen en die rol wat hierdie proteïen in hipertrofie speel. Ten spyte hiervan, is daar baie min inligting beskikbaar oor 'n ander lid van die miosien-bindingsproteïen families, miosien-bindingsproteïen H (MyBPH). Gegewe die ooreenstemming tussen die DNA basispaar-volgorde en struktuur tussen hierdie twee proteïene, stel ons voor dat MyBPH, net soos kMyBPC, 'n kritiese rol in die struktuur en funksie van die kardiale sarkomeer speel en kan daarom betrokke wees in die patogenese van HKM. Die huidige studie het beoog om proteïene wat met MyBPH bind te identifiseer deur die gebruik van gis-twee-hibried (G2H) kardiale biblioteek sifting en om hierdie interaksies te verifieer met behulp van drie-dimensionele (3D) ko-lokalisering en ko-immunopresipitasie eksperimente. Ons het verder gehipotiseer dat beide MyBPH and kMyBPC betrokke kan wees in outofagie. Om hierdie hipotese te toets is beide MyBPH en kMyBPC geanaliseer vir ko-lokalisering met 'n merker vir outofagie, LC3b-II. Verder het ons beplan om die rol van MyBPH en kMyBPC in kardiale spiersel-sametrekking te ondersoek deur die oppervlak van gedifferensieerde H9c2 rot kardiomiosiete in reaksie op β-adrenergiese stres te meet, na individuele en gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC. In hierdie studie het ons 'n familie-gebaseerde genetiese assosiasie analise benadering gevolg om vas te stel of MyBPH en gene wat kodeer vir die geverifieerde bindingsgenote van MyBPH bydra tot die modifisering van die hipertrofiese fenotipe. Die doel van hierdie studie was om die hipertrofiese effek van 38 enkel nukleotied polimorfismes (SNPs) en haplotipes in vier kandidaat HKM modifiserende gene in 388 individue van 27 HCM families te toets, waarin drie unieke Suid-Afrikaanse HKM-stigters mutasies segregeer. G2H analise het drie verneemde MyBPH bindingsgenote geidentifiseer, naamlik miosien (MYH7), alfa kardiale aktien (ACTC1) en die SUMO-konjugerende ensiem UBC9 (UBC9). Hierdie interaksies is geverifieer deur middel van 3D ko-lokalisering en ko-immunopresipitasie analises. Verder is bewys dat MyBPH en kMyBPC betrokke is in outofagie, siende dat beide proteïene gewerf is tot die membraan van die outofagosoom. 'n Kardiale sametrekkings eksperiment het gevind dat die gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC 'n merkwaardige vermindering in die kardiomiosiet sametrekking veroorsaak het in reaksie op β-adrenergiese stres kondisies, in vergelyking met die individuele proteïen en kontrole uitkloppings eksperimente. Hierdie resultate bevestig dat MyBPH vir kMyBPC kan instaan en ook andersom, wat verder bevestig dat beide proteïene benodig word vir effektiewe sarkomeer sametrekking. Resultate van die genetiese assosiasie studie het gevind dat 'n aantal SNPs en haplotipes 'n beduidende effek of HKM hipertrofie het. Enkel SNP en haplotipe analises in gene wat kodeer vir MyBPH, MYH7, ACTC1 en UBC9 het SNPs en haplotipes geidentifiseer wat bydra tot die omvang van hipertrofie in HKM. Verder het ons gevind dat sekere SNPs en haplotipes kenmerkend verskillende statisties beduidende effekte in die teenwoordigheid van elk van die drie HKM-stigter mutasies gehad het. Die resultate van hierdie studie skryf twee nuwe funksies aan MyBPH toe. Kardiale MyBPC en MyBPH speel 'n kritiese rol in sarkomeer sametrekking en is betrokke in outofagie. Hierdie resultate het verdere implikasies vir die verstaan van die pato-etiologie van die HKM-veroorsakende mutasies in die MyBPH, MYH7, ACTC1 en UBC9 gene. So vêr dit ons kennis strek is dit die eerste genetiese assosiasie studie wat die modifiserende effek van bindingsgenote van MyBPH ondersoek as risiko aanduiding vir die ontwikkeling van LVH in die konteks van HKM. Ons bevindinge bewys dat die hipertrofiese fenotipe van HKM gemoduleer word deur die komplekse effekte van SNPs en haplotipes in die MyBPH geen en gene wat MyBPH proteïen-bindingsgenote enkodeer. Hierdie resultate verskaf dus 'n basis vir toekomstige studies om die risiko profiel van hipertrofie ontwikkeling met betrekking tot HKM te ondersoek, wat gevolglik kan bydra tot die verbeterde risiko stratifikasie en pasiënte bestuur.

<p>The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study.</p><p>Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.</p>

Hyperkalemic periodic paralysis is characterized by myotonic discharges followed by paralysis. Caused by a mutation in the gene encoding for NaV1.4 channel, patients do not experience symptoms during infancy, but the onset starts between 1-10 years of age. The symptoms severity then increases with age until adolescence. A large increase in gene expression marked by an increase in oxidative capacity of muscles has also been reported in HyperKPP. It is possible that the onset of symptoms is related solely to NaV1.4 channel content/activity reaching a critical level. It is also possible that the onset of some symptoms are due to defective NaV1.4, while other symptoms and the increase in severity with age are related to changes in membrane components as a result of changes in gene expression. To test these possibilities, the progression of paralysis and changes in fiber types were followed with age in HyperKPP mice in relation to changes in NaV1.4 content and activity. Changes in fiber types (index of changes in gene expression), started after the onset of paralysis was observed, which coincided with NaV1.4 channels reaching maximum expression. Therefore, the onset of symptoms was related to defective NaV1.4 channels.