Thesis(M Med(Neurology))--University of Limpopo(Medunsa Campus),2009. / 1 BACKGROUND (geophagia/pica) Pica is a tendency to ingest nonnutritive substances eg. chalk, paper, wood, coal etc. The American Psychiatric Association in its diagnostic and statistical Manual defines Pica as persistent eating of non-nutritive substances that is inappropriate for developmental level, occurs outside culturally sanctioned practice and, if observed during the course of another mental disorder, is sufficiently severe to warrant independent attention. The term pica comes from the Latin word magpie- a bird known for its indiscriminating eating habits. The deliberate consumption of earth, soil or clay is known as geophagia which is the commonest form of pica. Hippocrates is credited with the oldest description of this ancient practice. An ancient Roman textbook, De Medicina, compiled by Celsus during the rein of Emperor Tiberius (13 – 37 AD), mentions earth eating as one of the reasons for bad skin colour. It is a practice that continues to exist in modern times, the full extent however, remains unrealized most probably due to underreporting. Geophagia has been practiced at some stage, virtually world wide eg. America (north & south), Asia, Australia, Britain, India, various parts of Africa. The epidemiology however, is still unknown due to the lack of studies in this regard.

BACKGROUND:Nemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases.RESULTS:We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.CONCLUSIONS:Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.

Skeletal muscle consists of an extremely regular organization of myofibers that are specialized in contraction. Development and maintenance of skeletal muscle function depends on the precise organization of sarcomeric contractile proteins that consist the myofibrils. Impaired or delayed myofibrillogenesis has been identified as the primary pathological mechanism of many skeletal muscle myopathies. Several members of the Kelch family of proteins have been implicated in skeletal muscle development and diseases, and mutations in these proteins have resulted in perturbations in the ubiquitin proteasome system (UPS), which is the primary means of proteasomal degradation in eukaryotes. In particular, KLHL41 of the BTB-BACK Kelch family is primarily expressed in skeletal muscle and has been identified as a regulator of the skeletal muscle differentiation process that results in the normal development and functioning of mature skeletal muscles. KLHL41 acts as a substrate-specific adaptor for Cullin 3 (Cul3) E3 ubiquitin ligase, implicating the role/s of KLHL41 in proteasomal ubiquitination processes in skeletal muscle. Recent studies have determined that the degradation of nebulin-related anchoring protein (NRAP), which was found to interact with KLHL41, is a critical process in skeletal myofibril maturation that is caused by KLHL41-mediated ubiquitination of the NRAP protein. Through this study, it was further confirmed that KLHL41 changes in localization as maturation occurs, which may provide insight into the mechanism of its functions in myofibril maturation. In addition, the study found that KLHL41 promotes the critical process of nebulin-related anchoring protein (NRAP) degradation. Lastly, mutations in the KLHL41, which are known to cause Nemaline Myopathy (NM) in patients, were modeled in murine C2C12 myoblasts to gain a greater understanding of how KLHL41 mutations may affect protein stability and Cul3 E3 ubiquitin ligase activity. Overall, the findings of this thesis support the critical role of KLHL41 in the formation of mature myofibrils, and provides insight into how deficiency of KLHL41 contributes to a disease state through regulation of the CUL3 protein complex. / 2022-06-30T00:00:00Z

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Characterized by the severe progressive wastage of skeletal muscle, Duchenne muscular dystrophy (DMD) is a crippling X-linked recessive disease that is caused by the absence of the protein dystrophin. This thesis aimed to critically evaluate the potential of different therapeutic options to combat this disease. Utrophin is a paralogue of dystrophin. The Fiona mouse is an mdx (dystrophin-deficient) transgenic mouse that overexpresses the full-length utrophin protein in skeletal muscle, and various studies have shown that it does not display a dystrophic phenotype. However, these studies have only been performed on sedentary mice. In this work it is demonstrated that utrophin’s protective effect is partially diminished after a sustained period of exercise-induced stress, highlighting for the first time a functional difference between dystrophin and utrophin. This thesis also presents results of two mdx mouse drug trials testing the ameliorative effects of the administration of the drugs GW501516 and C1100, which show that treatment with both drugs results in partial amelioration of the dystrophic phenotype. GW501516 administration results in a beneficial fast-to-slow fibre type switch and an in vivo increase in utrophin protein levels. We have also shown that C1100 treatment results in a significant increase in utrophin A promoter activity in vitro, and the mechanism of action of this drug on this promoter has been deciphered. The global dysregulation of microRNAs in skeletal muscle of mdx and dko (dystrophin- and utrophin-deficient) mice was evaluated by microarray analysis to identify microRNAs involved in the dystrophic pathological cascades. The results of detailed expression analyses of miR-31, miR-206 and miR-503 are presented, and two therapeutically-relevant predicted targets of miR-503 were validated. Overall, this thesis evaluates the potential of different and possibly complementary therapeutic options to combat DMD.

As a front-line treatment for cardiovascular disease, statins are among some of the most widely prescribed drugs worldwide. Statins are effective at lowering cholesterol, but approximately 7-29% of patients report some form of adverse muscle effect during the course of treatment. The severity of these side effects ranges from low-level to life-threatening myopathy. The mechanism of statin myopathy remains ill-defined, but muscle-specific E3 ubiquitin ligases have been implicated. In addition, statins have been shown to activate caspase-1 (and increase IL-1β) in immune cells, which is a key effector of the NLRP3 inflammasome. The relevance of this inflammatory response in statin myopathy remains unknown. Using C2C12 myotubes, an in vitro model of statin-induced myopathy was developed to test the impact of NLRP3 inflammasome activation on markers of statin myopathy. Gene expression of the muscle-specific E3 ubiquitin ligases atrogin-1 and MuRF-1 (atrogenes) were used as markers of statin-induced myopathy. Lipopolysaccharide priming of the NLRP3 inflammasome was found to lower the effective dose of fluvastatin required to augment atrogene expression. This effect correlated with reduced phosphorylation of Akt and FOXO3a, a transcription factor regulating atrogene expression. Statin-induced atrogene expression was also found to be dependent on an isoprenoid that is required for protein prenylation rather than cholesterol biosynthesis pathways. Fluvastatin increased caspase-1 activity in a prenylation-dependent manner and selective inhibitors of NLRP3 and caspase-1 were able to prevent increased atrogene expression with fluvastatin treatment. Therefore, the NLRP3 inflammasome contributes to markers of statin-induced myopathy through a prenylation-dependant pathway in muscle cells. This work presents a novel mechanism involved in statin myopathy, and has shown that the inflammasome may represent a new drug target to mitigate muscle symptoms in patients taking statins. / Thesis / Master of Science (MSc) / Statins are a class of widely prescribed cholesterol-lowering drugs that reduce the risk of heart attack and stroke. However, many patients often complain of statin-induced muscle side effects (myopathy) that impact their quality of life. Symptoms of this statin-induced myopathy can manifest as muscle pain and weakness. The underlying biology causing this condition is still not well understood. Independent of its cholesterol-lowering effect, statins can activate an immune receptor called the NLRP3 inflammasome, indicating that inflammation may contribute to myopathy. Therefore, the primary goal of this study was to determine if this immune response contributes to statin-induced myopathy. It was found that inhibition of the NLRP3 inflammasome lowers markers of statin myopathy. Results from this study will provide further insight into mechanisms regulating this myopathy, and may lead to new treatments that can help alleviate statin side effects in muscle.

Chicken breast samples, (n=90; n=30 normal, n=30 moderate woody breast (WB), n=30 severe WB) were collected from a commercial processing plant on 5 separate occasions. After 5 days of storage at 2-4 ºC, 84% of severe WB fillets dissipated to moderate WB, which was greater (P<0.05) than all other storage times. In comparison, 40-52% of the moderate WB fillets dissipated to slight WB or normal breasts after 3 to 5 days of storage. Shear force was greater (P<0.05) for normal breast meat than moderate and severe WB meat on day 0. After 2, 3, 4, and 5 days of storage the upper position (cranial part) of severe WB had greater shear force than normal fillets (P<0.05). Therefore, the dissipation that occurred in woody breast meat over refrigerated storage was apparent through palpation but did not result in improved texture in the cranial portion of the breast, based on shear force results.

Congenital myopathies are a rare and heterogeneous group of diseases. They are primarily characterised by skeletal muscle weakness and disease-specific pathological features. They harshly limit ordinary life and in severe cases, these myopathies are associated with early death of the affected individuals. The congenital myopathies investigated in this thesis are nemaline myopathy and myofibrillar myopathy. These diseases are usually caused by missense mutations in genes encoding myofibrillar proteins, but the exact mechanisms by which the point mutations in these proteins cause the overall weakness remain mysterious. Hence, in this thesis two different nemaline myopathy-causing actin mutations and one myofibrillar myopathy-causing myosin-mutation found in both human patients and mouse models were used to investigate the cascades of molecular and cellular events leading to weakness. I performed a broad range of functional and structural experiments including skinned muscle fibre mechanics, small-angle X-ray scattering as well as immunoblotting and histochemical techniques. Interestingly, according to my results, point mutations in myosin and actin differently modify myosin binding to actin, cross-bridge formation and muscle fibre force production revealing divergent mechanisms, that is, gain versus loss of function (papers I, II and IV). In addition, one point mutation in actin appears to have muscle-specific effects.  The presence of that mutant protein in respiratory muscles, i.e. diaphragm, has indeed more damaging consequences on myofibrillar structure than in limb muscles complexifying the pathophysiological mechanisms (paper II). As numerous atrophic muscle fibres can be seen in congenital myopathies, I also considered this phenomenon as a contributing factor to weakness and characterised the underlying causes in presence of one actin mutation. My results highlighted a direct muscle-specific up-regulation of the ubiquitin-proteasome system (paper III). All together, my research work demonstrates that mutation- and muscle-specific mechanisms trigger the muscle weakness in congenital myopathies. This gives important insights into the pathophysiology of congenital myopathies and will undoubtedly help in designing future therapies.

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