Neuromuscular junction (NMJ) dysfunction has been identified as one of the earliest events in Amyotrophic Lateral Sclerosis (ALS) pathology. However, which tissue type induces NMJ disruption; be it the motoneurons (hMN), Schwann cells or skeletal muscle (hSKM) remains unresolved. While mechanisms by which ALS hMN contribute to NMJ dysfunction are well-described in literature, limited information exist on how the other tissue types in the tripartite synapse (hSKM and Schwann cells) induce and/or contribute to ALS NMJ disruption. A fair understanding of the role of each tissue type in NMJ dysfunction would help shape the trajectory of future ALS research and drug discovery. It is generally accepted that the observed ALS hSKM weakness and atrophy are a result of hMN axonal retraction. While recent findings postulate the active pathologic involvement of the hSKM in ALS onset via NMJ disruption, some scientists have questioned the validity of the transgenic model used in most studies and the translatability of their results. Contrarily, in vitro modeling of this phenomenon using patient hSKM samples has proved challenging due to the inability to reliably expand and maintain them before becoming senescent. Thus, this study sought to investigate the pathology of hSKM derived from patient ALS (ALS hSKM), its possible contribution to NMJ dysfunction, and whether hSKM-specific treatment confer any therapeutic benefit to the NMJ. To avoid the main challenges associated with human-based ALS hSKM studies i.e., tissue scarcity and established culturing challenges, patient iPSCs were utilized as the tissue source. This assured a single source for obtaining both cell types necessary for modeling the ALS NMJ. IPSC-derived wild type (WT) and ALS hSKM were differentiated using a serum-free, small molecule-directed protocol which centered on the concurrent modulation of the Wnt and bone morphogenetic protein pathways. To determine whether the ALS hSKM has intrinsic deficits independent of hMN, comparative assessment of WT and ALS hSKM were carried out. The myogenicity of resultant WT and ALS progenitors were confirmed via phase-contrast microscopy, immunocytochemistry, and flow cytometry, after which they were terminally differentiated in myotubes. WT and ALS hSKM were compared morphologically and functionally. The inner mitochondrial membrane potential (ΔΨM) and metabolic plasticity of both WT and ALS hSKM were also evaluated. After the hSKM-only characterization, both ALS and WT hSKM were co-cultured with either ALS or WT hMNs. This was to study how the previously outlined hSKM deficits affect NMJ formation, integrity and function in an effort to delineate the sole contribution to NMJ disruption. With knowledge of the pathologic contribution to NMJ dysfunction, hSKM-specific Creatine treatments were performed to investigate its therapeutic benefit to the ALS NMJ. This project advances the field's knowledge on the hSKM's role to ALS NMJ disruption and creates awareness about considering the hSKM in future research and drug discovery exploits. Additionally, this project resulted in the development of microphysiological platforms that recapitulate known phenotypic parameters of ALS while allowing the independent treatment and/or interrogation of each tissue type in the co-cultures.

No description available.

published_or_final_version / Sports Science / Master / Master of Science in Sports Science

The aims of this thesis were to 1) investigate the effect of SB431542 <i>in vitro</i> and <i>ex vivo</i> as a novel approach towards promoting the functional hypertrophy of skeletal muscle by inhibiting the myostatin-Smad pathway, 2) to investigate the expression and function of the Yes-associated protein (Yap) in skeletal muscle and C2C12 cells as a novel regulator of C2C12 differentiation and 3) to generate a GFP-RCASBP-hYAP1 S127A retrovirus to allow the study of the function of Yap in skeletal muscle differentiation <i>in vivo</i>. The results presented in this thesis show that SB431542 promotes the hypertrophy of C2C12 myotubes and mature <i>Xenopus</i> skeletal muscle fibres. However, SB431542 treatment also results in a reduction in specific force of mature <i>Xenopus</i> muscle fibres suggesting that SB431542 is not suitable as a treatment for skeletal muscle atrophy. These results also show that Yap is expressed in mouse skeletal muscle <i>in vivo</i> and that Yap is a novel regulator of C2C12 differentiation. Finally, these results descried the generation of a GFP-RCASBP-hYAP1 S127A retrovirus that can be used to assess the role of Yap <i>in vivo </i>during skeletal muscle formation in the chick embryo. Together, these results suggest that Yap is a novel regulator of C2C12 differentiation that should be studied as a potential therapeutic target in musculoskeletal diseases.

by Cheung Man Kit. / Errata slip inserted / Bibliography: leaves 83-84 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1985

by Shuk-man Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Bibliography: leaves 108-121. / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Neural Regulation of Muscle Development --- p.3 / Chapter 1.3 --- "Changes in Motoneuron Number, Motoneuron Properties and Innervation during Development" / Chapter 1.3.1 --- Decrease in Motoneuron Number --- p.6 / Chapter 1.3.2 --- Reduction of Synaptic Connections --- p.7 / Chapter 1.3.3 --- Changes in Motoneuron Properties during Development --- p.9 / Chapter 1.4 --- Neural Influence on Muscle Properties in Adult Mammals / Chapter 1.4.1 --- Effect of Denervation on Muscle Properties --- p.10 / Chapter 1.4.2 --- Plasticity of Muscle Properties --- p.12 / Chapter 1.5 --- Partial Denervation --- p.15 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- General Plan --- p.22 / Chapter 2.2 --- Normal Unoperated Group / Chapter 2.2.1 --- Innervation of the SOL and EDL --- p.23 / Chapter 2.2.2 --- Tension Recordings --- p.24 / Chapter 2.2.3 --- Contributions of L4 and L5 to Normal Tension of the SOL and EDL --- p.26 / Chapter 2.3 --- Young Denervated and Adult Denervated Groups / Chapter 2.3.1 --- Unilateral Partial Denervation --- p.27 / Chapter 2.3.2 --- Tension Recordings --- p.28 / Chapter 2.3.3 --- Measurement of Contraction and Relaxation Speeds --- p.28 / Chapter 2.3.4 --- Recovery in Tension of Partially Denervated Muscles --- p.29 / Chapter 2.3.5 --- Motor Unit Number and Size --- p.29 / Chapter 2.3.6 --- Resting Membrane Potential --- p.31 / Chapter 2.3.7 --- Histometric Analysis and Histochemical Study --- p.32 / Chapter 2.3.8 --- Immunocytochemical Study on Different Types of Myosin --- p.34 / Chapter 2.3.9 --- Histological Study on Innervation of Muscle Fibres --- p.35 / Chapter 2.4 --- Retrograde HRP Labelling for HRP Group --- p.37 / Chapter 2.5 --- Statistic Analysis --- p.39 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Contribution of Spinal Nerves to Tension in Normal Unoperated Group --- p.44 / Chapter 3.2 --- Results from Young Denervated and Adult Denervated Groups / Chapter 3.2.1 --- Recovery in Tension --- p.45 / Chapter 3.2.2 --- Changes in Other Contractile Properties --- p.48 / Chapter 3.2.3 --- Motor Unit Number and Size --- p.49 / Chapter 3.2.4 --- Resting Membrane Potential of Adult Denervated Group --- p.51 / Chapter 3.2.5 --- Changes in Cross-sectional Area of Whole Muscle and Number of Muscle Fibres --- p.52 / Chapter 3.2.6 --- Histochemical Properties and Types of Myosin --- p.53 / Chapter 3.2.7 --- Histological Study on Innervation of Partially Denervated Muscles --- p.56 / Chapter 3.3 --- Results of HRP Group --- p.58 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Contribution of Spinal Nerves to Tension Development --- p.91 / Chapter 4.2 --- Recovery of Partially Denervated Muscles --- p.93 / Chapter 4.3 --- Time Courses of Functional Recovery --- p.98 / Chapter 4.4 --- Effect of Partial Denervation on Properties of Muscles --- p.100 / Chapter CHAPTER 5 --- CONCLUSION --- p.106 / REFERENCES --- p.108

No description available.

Abstract not available

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Stuart A. Binder-Macleod, Dept. of Physical Therapy. Includes bibliographical references.