The disruption of the survival motor neuron (SMN1) gene leads to the children’s genetic disease spinal muscular atrophy (SMA). SMA is characterized by the degeneration of α-motor neurons and skeletal muscle atrophy. Although SMA is primarily considered a motor neuron disease, the involvement of muscle in its pathophysiology has not been ruled out. To gain a better understanding of the involvement of skeletal muscle pathophysiology in SMA, we have developed three aims: to identify cell-specific Smn-interacting proteins, to characterize postnatal skeletal muscle development in mouse models of SMA, and to assess the functional capacity of muscles from SMA model mice. We have used tandem affinity purification to discover Smn interacting partners in disease relevant cell types. We have identified novel cell-specific Smn interacting proteins of which we have validated myosin regulatory light chain as a muscle-specific Smn associated protein in vivo. We have taken advantage of two different mouse models of SMA, the severe Smn-/-;SMN2 mouse and the less severe Smn2B/- mouse, to study the postnatal development of skeletal muscle. Primary myoblasts from Smn2B/- mice demonstrate delayed myotube fusion and aberrant expression of the myogenic program. In addition, the expression of myogenic proteins was delayed in muscles from severe Smn-/-;SMN2 and less severe Smn2B/- SMA model mice. Muscle denervation and degeneration, however, are not the cause of the aberrant myogenic program. At the functional level, we demonstrate a significant decrease in force production in pre-symptomatic Smn-/-;SMN2 and Smn2B/- mice indicating that muscle weakness is an early event in these mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant levels of developmentally regulated proteins important for muscle function, which may impact muscle force production in skeletal muscle of SMA model mice. The present study demonstrates early and profound intrinsic muscle weakness and aberrant expression of muscle proteins in mouse models of SMA, thus demonstrating how muscle defects can contribute to the disease phenotype independently of and in addition to that caused by motor neuron pathology.

A trauma frequency analysis was undertaken on a skeletal sample (n = 75) from the skeletal collections of the medieval Augustinian Priory of St. Mary Merton and the post-medieval lower cemetery of St. Bride’s Church. Fourty-four individuals exhibited trauma on one or more cranial elements. Cranial bones were arranged in different groupings for analysis: inclusive samples of 100% complete, at least 75% complete, at least 25% complete, entire sample; and independent samples of 100% complete, 75 - <100% complete, 25 - <75% complete, and <25% complete. Crania were categorized as 100% complete and incomplete. Four frequencies were calculated (frequency of lesions, of individuals with lesions, of individuals with multiple lesions, and the number of lesions per injured individuals) for each category and cranial element. The results illustrate a general trend towards a decrease in frequency as more fragmentary material is included, illustrating that including the more fragmentary material may bias the results towards underestimating trauma frequencies. However, Fisher’s exact tests do not show statistically significant differences between frequencies in the independent samples analysis, except for individuals with lesions on the right nasal bone. Further research into the effect of fragmentation and poor preservation in skeletal research, cranial trauma research in particular, is required. / Graduate

Osteological studies have identified scarring on the bone surface of the human pelvic bone as evidence of childbirth, termed parturition scarring. It remains unknown whether a single or multiple births cause parturition scarring. Such scarring has also been found on male pelvic bones. This study examines parturition scarring within the broader morphometric and musculoskeletal context of the pelves of both sexes. This project investigates the influence of body size (stature and body mass) and pelvic size (individual pelvic measurements and pelvic canal size) and shape (pelvic canal shape) on the presence of parturition scarring on the pelvic bones of females and males. Two skeletal collections of known-age and sex were chosen for this project on the basis of access to parity (childbirth) records: the Maxwell Museum Documented Skeletal Collection and the Christ Church, Spitalfields collection. The dimensions of articulated and disarticulated pelves, femoral measurements and scores for six types of parturition scarring were recorded for all individuals (n=292). Skeletal proxies for body mass and stature were calculated for all individuals. Univariate, bivariate and multivariate statistical analyses were used to identify significant differences in parturition scarring between sexes, correlation between body size variables, parity status, pelvic canal size and pelvic canal shape (as represented by principal components analysis) and parturition scarring. Parity status and pelvic canal shape do not associate with parturition scarring. Pubic tubercle variables associated variously with femoral head diameter and pelvic canal size in females or males only. Dorsal pitting correlates weakly with four pelvic dimensions in females. The results of this study suggest that the term ‘parturition scarring’ should be revised to reflect its non-connection with parity status and that future investigations should examine musculoskeletal interactions based on body and pelvic size variation that affect the presence of such scarring in males. / Graduate / 0327 / 0287 / decrausa@uvic.ca

Three languages with polymorphic type disciplines are discussed, namely the λ-calculus with Milner's polymorphic type discipline; a language with imperative features (polymorphic references); and a skeletal module language with structures, signatures and functors. In each of the two first cases we show that the type inference system is consistent with an operational dynamic semantics. On the module level, polymorphic types correspond to signatures. There is a notion of principal signature. So-called signature checking is the module level equivalent of type checking. In particular, there exists an algorithm which either fails or produces a principal signature.

Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

Sarcolipin (SLN) is a small sarcoplasmic reticulum (SR) integral membrane protein that regulates the SR Ca2+-ATPase (SERCA). Previous studies indicate that the functional interaction between SLN and SERCA is thermogenic in nature. Recently, SLN knock-out (SLNKO) mice have been shown to develop excessive obesity and glucose intolerance when placed on a high-fat diet (HFD; 42% kcal derived from fat) relative to wild-type (WT) littermates, implicating SLN in diet-induced obesity. The purpose of this thesis was two-fold: 1) to determine whether an excessively obese phenotype persists when SLNKO mice are given access to voluntary exercise, and 2) to determine if SLN ablation results in a deficit in skeletal muscle oxidative capacity, given the integral role cellular Ca2+ plays in mitochondrial metabolism. Mice were fed either standard chow or a HFD for 8 weeks, and remained sedentary or given access to voluntary running wheels during this period. Glucose tolerance was assessed pre- and post-diet, along with weight gain and adiposity. Skeletal muscle succinate dehydrogenase (SDH), citrate synthase (CS), cytochrome c oxidase (COX), and 3-hydroxyacyl CoA dehydrogenase (ß-HAD) activities were measured in the soleus (SOL) and extensor digitorum longus (EDL) of both chow- and high-fat fed sedentary mice. Both average daily running distance and total exercise volume were not different between WT and SLNKO mice given voluntary running wheels. As before, sedentary SLNKO mice gained more mass following the HFD relative to WT counterparts (P < 0.05); however, no difference in mass gain existed between genotype for voluntary exercising mice on a HFD. Despite this, SLNKO animals were more obese and glucose intolerant following high-fat feeding, regardless of activity status (P < 0.05). Under chow-fed conditions COX activity was higher in the EDL of SLNKO mice (P < 0.05), while no differences in SDH, CS, or ß-HAD existed between genotype in either muscle group. Following the HFD, no changes in mitochondrial enzyme activities within the SOL existed. COX activity in the EDL remained elevated in SLNKO mice post-HFD (P < 0.001), while ß-HAD increased in both WT and SLNKO animals relative to chow-fed controls (P < 0.05). These findings suggest that increasing energy expenditure through voluntary activity cannot compensate for increased basal SERCA Ca2+-pumping efficiency during caloric excess. Additionally, ablation of SLN does not result in a metabolic deficit within skeletal muscle, nor does it limit the adaptive enzymatic response of SLNKO mice to high-fat feeding. Thus, the findings of this study provide further support of the view that SLN’s thermogenic role is the primary mechanism of diet-induced obesity in SLNKO mice.

Changes in intracellular Ca2+ ([Ca2+]f) and high-energy phosphates are known to induce adaptive changes in skeletal muscle during endurance exercising training, including mitochondrial biogenesis. Levels of [Ca2+]f are regulated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) which are further regulated by sarcolipin (SLN), through a reduction in the apparent affinity of SERCAs for Ca2+. Furthermore, SLN reduces the efficiency of Ca2+ transport by SERCAs supporting a thermogenic role for SLN in skeletal muscle. Thus, it is possible SLN ablation could reduce Ca2+ and metabolic signaling during exercise training and attenuate increases in mitochondrial content. To investigate the potential role of SLN in the exercise-induced adaptive response of skeletal muscle, mice devoid of SLN (SLNKO) underwent endurance training for 8 weeks and were compared to WT controls. Maximal oxygen uptake (V̇O2 max) was measured with an exercise stress test while mitochondrial content was assessed through measurement of protein expression and maximal enzyme activities of several mitochondrial enzymes in soleus and extensor digitorum longus (EDL) muscles, which express high and low levels of SLN, respectively. All data were analyzed using a two-way analysis of variance (ANOVA) and student t-tests were conducted on enzyme data. V̇O2 max was found to not be significantly altered with exercise training in either genotype. Exercise training significantly increased the contents of adenine nucleotide translocase (ANT), cytochrome-c (cyt-c) and cytochrome-c oxidase subunit IV (COXIV) in soleus independent of genotype. Likewise, exercise training significantly increased cyt-c and COXIV expression (P<0.04), while increases in ANT expression were not significant (P=0.13) in the EDL. Two-way ANOVAs of mitochondrial enzymes in soleus revealed an interaction existed for succinate dehydrogenase (SDH) where its activity was increased only in the SLNKO mice (P<0.02). In comparison, exercise training significantly elevated activities of cytochrome c oxidase (COX) and citrate synthase (CS) activities (P<0.02) but not β-hydroxyacyl-CoA dehydrogenase (β-HAD; P=0.08), independent of genotype. Upon closer examination using student t-tests, it was determined that exercise training induced greater increases in COX and CS activity in SLNKO compared to WT controls (P<0.02), similar to and consistent with SDH data. In EDL, only SDH activity increased following exercise training, an effect that was independent of genotype. In conclusion, these data suggest that SLN ablation does not attenuate exercise-induced mitochondrial adaptations and may increase mitochondrial enzyme adaptations to exercise training in slow-twitch muscle. Further examination of the effects of SLN on Ca2+ and metabolic signaling may provide mechanisms explaining the results of this thesis.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : University, 2003. / Härtill 5 uppsatser.