Dissertations / Theses on the topic 'Neuromuscular development'

The thesis presents contributions to the field of neuromuscular synaptic plasticity. Synaptic remodelling brings about changes in convergence and divergence in many different parts of the nervous system during development.  Neuromuscular junctions have proved to be accessible synapses in which to describe and explain the mechanisms. During development, muscle fibres initially receive convergent, polyneuronal innervation (π) by axons arising from different motor neurones. The characteristic mononeuronal innervation (µ) pattern of adult muscle is achieved by synapse elimination, a process of weakening of synaptic strength followed by withdrawal of synaptic boutons, until all but one of the motor neuron inputs to an endplate is lost. Similar hyperinnervation and elimination occur in adult muscle after nerve injury, collateral sprouting and regeneration. These processes are strongly influenced by activity, apparently in accordance with Hebbian rules of synaptic plasticity. But how decisive is activity in ultimately determining the pattern of neuromuscular connectivity? The amount of sprouting is increased and the rate of synapse elimination is decreased when muscle activity is blocked. Sprouts regress and synapse elimination resumes when muscles are stimulated, or once normal activity is restored. Selectively blocking or restoring activity in some motor neurones but not others supplying a π-junction gives a competitive advantage to the more active neuromuscular synapses. However, activity is not sufficient to effect synapse elimination because many muscle fibres retain π-junctions after activity resumes following a period of paralysis. Nor is activity strictly necessary, because – paradoxically – synapse elimination continues at some motor endplates even when muscles are completely paralysed. Competition for neurotrophic factors may play an important role in determining the outcome of synapse elimination, but factors intrinsic to the motor neurone, perhaps involving the selective trafficking of maintenance factors along specific axon collaterals, appear to be important also. In each motor neurone, synapses are eliminated or strengthened asynchronously.

During development of the nervous system an excess number of synapses are formed, most of which are subsequently pruned, resulting in functional neural networks. The precise mechanisms that determine which synapses are formed and which synapses are maintained are not thoroughly understood. The aim of this thesis is to investigate the intra-neuronal constraints and influences on synapse formation and elimination during development. In the first part of the thesis I investigated intra-neuronal influences on synapse elimination. Synapse elimination is known to occur at polyneuronally innervated neuromuscular junctions through competition, leading to mononeuronally innervated muscle fibres. However, whether synapse elimination ever occurs in the absence of competition, leading to muscle fibres becoming denervated, has not been resolved. The data presented in this thesis suggest that only large motor units undergo a reduction in motor unit size in the absence of competition. Using the Rasmussen and Willshaw (1993) version of the Dual Constraint Model I show that these data are consistent with the prediction that synapses will be eliminated from muscle fibres when a neuron's resources become stretched, for instance as a result of the normal growth of the animal. Larger motor units, which innervate more synapses, will thus be more vulnerable to the extra demand put upon them by the growth of each synapse. The model predicts that synapse elimination in the absence of competition should occur at least over the first 6 months of life and not only during the first two postnatal weeks, when most polyneuronal innervation is normally eliminated. In the second part of the thesis, I investigated intra-neuronal influences on synapse formation. Specifically I tested the hypothesis that each branch of a motor neuron forms synapses randomly and independently of other branches. If true there should be instances where two branches from the same neuron initially innervate the same endplate (sibling branch convergence). Sibling branch convergence was experimentally investigated in both regenerating and developing motor neurons. The evidence suggests that sibling branches can converge on the same muscle fibre and that they can competitively eliminate each other. However, it appears that convergence does not occur at the frequency that would be expected, suggesting that branches from the same motor neuron do not form synapses independently of each other. At present there are limitations in imaging immature networks due to the spatial resolution limit of light microscopy. The last part of this thesis explores thin serial sectioning and reconstruction as a possible technique for increasing resolution in the z-axis. This technique has potential to be developed but I show that at present it does not provide sufficient resolution to discriminate between developing motor axons in neonatal lumbrical muscles.

Vibration stimulation has been used as a tool to relieve muscle pain and spasm in physical therapy for many years. However recently, vibration, mainly Whole Body Vibration (WBV), has been increasingly studied and used as an exercise intervention in sports and rehabilitation. Although the physiological mechanisms which guide the body's response to this exercise modality are relatively poorly understood, evidence indicates that vibration can enhance muscle strength, power, and flexibility as well as increase bone mineral density in the general population. Evidence also suggests that the neuromuscular response to vibration stimulation depends on muscle length, stretch level (contraction) along with the vibration characteristics. One way to alter muscle length and contraction levels while receiving vibration is to superimpose the stimulation on graded isometric contraction. However, current WBV device designs cannot facilitate the delivery of vibration stimulation superimposed on graded isometric voluntary contraction. The aim of this PhD project was twofold, firstly to develop and evaluate a prototype WBV device which enables the delivery of vibration stimulation that can be superimposed on graded isometric contraction and secondly, to assess the neuromuscular responses to vibration superimposed on graded isometric contractions in lower limbs using this device. Due to the novelty of the device design and the method of the delivery, this study initially investigated the effects of different vibration frequencies and amplitudes combined with various effort levels on neuromuscular responses in lower limbs. The results of this study confirm that isometric contraction superimposed on vibration stimulation induce enhanced neuromuscular activity in the lower limbs. The results also confirm that although the neuromuscular responses to vibration depend on multiple factors the main determinants seem to be the vibration frequency, amplitude and muscle contraction /forc The results also confirm that although the neuromuscular responses to vibration depend on multiple factors the main determinants seem to be the vibration frequency, amplitude and muscle contraction /force level. Another limitation of most existing vibration devices is that they are not capable of delivering frequency of the vibration independent of amplitude and vice versa. Further, the evidence suggests that vibration amplitude can play an important role in neuromuscular response to vibration, especially when superimposed with graded contraction/force levels. To address the above limitation, the second aim of this PhD project was to develop and evaluate a prototype miniature upper limb vibration device capable of delivering precise and independent vibration frequency and amplitude stimulation. The miniature upper limb vibration (ULV) device with piezo actuators developed for this thesis, enables precise vibration stimulation to be delivered in a seated position with graded voluntary contraction superimposed. The neuromuscular responses to vibration superimposed on graded isometric contractions in upper limbs were also assessed by investigating the fatiguing effects of superimposed vibration stimulation using this newly developed device. This study is the first to investigate and compare the fatiguing effects of superimposed vibration stimulation pre and postvibration exercise in upper limbs. The results of this study confirm that isometric contraction superimposed on vibration stimulation lead to increased fatigue levels and neuromuscular activity in upper limbs. The results also indicate that post-vibration treatment the muscles display enhanced force generation capability associated with lower fatigue levels. In summary, two (WBV and ULV) novel vibration exercise devices were successfully developed and evaluated for this thesis. The results of the studies on these devices confirm that vibration stimulation superimposed on graded isometric contraction can induce higher neuromuscular activity compared to isometric contraction alone in both upper and lower limbs. However the effects of vibration frequency, amplitude and contraction/force levels seem to differ between the upper and lower limbs.

The dystrophin associated protein (DAP) complex has been implicated in such basic physiological processes as the maintenance of cellular homeostasis, the assembly of basement membranes (BM), and synaptogenesis. The dystroglycans, alpha and beta, constitute the functional core of the DAP complex since they establish the transmembrane link between dystrophin, whose mutations lead to Duchenne (DMD) or Becker (BMD) muscular dystrophy, and the extracellular matrix (ECM). The alpha and beta subunits of dystroglycan result from posttranslational processing of a single propeptide encoded by DAG1. No clinical cases or animal models have been identified with spontaneous mutations in that gene. Therefore, to gain insight to the function of dystroglycans I have used gene targeting to disrupt the coding sequence of Dag1 in murine embryonic stem (ES) cells. We find that dystroglycans are critical in the early stages of development and that a null mutation in Dag1 results in embryonic lethality at embryonic day (E) 6.5 because of a disruption in Reichert's BM. In culture however, the absence of dystroglycan in ES cells targeted for both Dag1 alleles does not obviously hinder their developmental potential. Consequently, I reasoned that it might be possible to circumvent the early lethality observed in 'classical' knockout mice, by injecting Dag1-null ES cells into wild-type blastocysts to generate chimeric mice only partially devoid of dystroglycan. Several chimeric mice developed to maturity and Dag1 -null ES cells were found to contribute extensively to the hindlimb musculature thus allowing the analysis of dystroglycan depleted muscles. These muscles are severely dystrophic, have low levels of dystrophin and a disrupted residual DAP complex, but have apparently normal BMs. Chimeric muscles also have disrupted neuromuscular junctions. In culture, myotubes derived from Dag1-null ES cells form clusters of acetylcholine receptors (AChRs) but these occupy a surface area three time

Traditional core stability training was developed as a method of treating and preventing back pain. It was however, seamlessly applied to healthy and athletic populations without scientific evidence supporting its efficacy. Traditional core stability focussed on isolating and training the anatomical region between the pelvis and diaphragm, using isometric or low load exercises to enhance spinal stability. Scientific research challenged this approach for healthy function and athletic performance, resulting in a more functional anatomical definition, which included pelvic and shoulder girdles. Hence, a revised definition of dynamic trunk stability; the efficient coordination, transfer and resistance by the trunk, of force and power generated by upper and lower appendicular skeletal extremities during all human movement. This led to an integrated exercise training approach to dynamic trunk stability. Although early evidence suggested loaded compound exercises preformed upright, in particular back squat, were effective in activating and developing trunk muscles, evidence was inconclusive. Accordingly, the aims of this PhD were to investigate neuromuscular trunk function in loaded, free barbell back squat to understand training implications for trunk stability in dynamic athletic activity. Five research studies were conducted; 4 are published and 1 is being prepared for re-submission. The literature review revealed evidence that back squat was an effective method of activating trunk stabilzers and showed that these muscles were load sensitive (study 1). A survey of practitioners reported an understanding and appreciation of the challenge against core stability training for athletic populations. Furthermore, perceptions were aligned with growing evidence for dynamic and functional trunk stability training (study 2). A test-retest neuromuscular study established interday reliability and sensitivity of electromyographical measurement of trunk muscle activity in squats (study 3). Trunk muscle activation in back squat was higher than hack squat at the same relative, but lower absolute loads (study 4). Trunk muscle activation was lower in squats and bodyweight jumps in the strong compared to weak group (study 5). Furthermore, activation of the trunk muscles increased in each 30o segment of squat descent and was highest in first 30o segment of ascent for all loads (study 5). In conclusion, this series of studies confirmed acute effect of squats on trunk stabilizers and demonstrated that external load increases activation in these muscles. Parallel squat depth is important in optimizing trunk muscle activation. Finally, high levels of squat strength result in lower trunk muscle activation in loaded squats and explosive jumps.

The anterior cruciate ligament (ACL) originates from posterior part of the medial side of the lateral condyle of femur to anterior intercondylar notch between a transverse meniscal ligament and medial side of medial meniscus of tibia. Once ACL is disrupted, pain, effusion and atrophy are commonly observable and cause functional disability. Because of the functional limitations, athletic participation is severely restricted. ACL injury is more prevalent in physically active females compared to their male counterparts in the sports of basketball and soccer in high school and collegiate levels. Several attributes of females are considered risk factors for the higher ACL injury incidences and include: anatomy, physiology and neuromuscular/biomechanics. Among them, neuromuscular/biomechanics is the only modifiable risk factor. Performing neuromuscular training may change muscular strength profiles, which may lead to reduction in ACL injury incidence in female athletes. However, this principle was not fully examined. Also, neuromuscular compliance may play a role in muscular strength development and ACL injury incidences. Thus, the purpose of this dissertation was to investigate the effects of neuromuscular training compliance on muscular strength development and ACL injury incidence. The influence of hip abductor, hamstrings and quadriceps strength was examined in this project. The results of these studies indicate neuromuscular training is an effective intervention to reduce ACL injury incidence in female athletes, and there is an inverse dose-response relationship between compliance of the neuromuscular training and number of ACL incidences in female athletes. The effect of compliance on muscular strength development was inconsistent. The results of these studies support that compliance of neuromuscular training is a key to reduce ACL injury incidences; however, more studies are need to conclude neuromuscular training compliance effects on muscular strength development in female athletes.

Therapeutic Botulinum neurotoxin preparations have found an increasing number of clinical uses in a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critil;al to ensure clinical efficacy and safety, and is currently achieved using a lethal dose (LDso ) assay in mice. Operational constraints and ethical concerns associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. One such tissue-based alternative, the 'Intercostal Neuromuscular Junction (Nl\JJ) Assay', uses nerve:muscle tissue preparations from the rat rib cage. The individual tissue preparations and the experimental protocol were subject to a detailed analysis. The tissue sections used were found to be highly reproducible as determined through the innervation pattern and the localisation of NMJs in situ and their distribution in skeletal muscle fibre types. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites ,vas critically assessed using molecular tracer molecules. Sonification during sample aplication resulted in an accelerated way of sample application.

The neuromuscular junction (NMJ) is the synaptic contact between motoneurons and muscle, where neurotransmission results in the contraction of the muscle fibres. The basal lamina instructs NMJ development at virtually every stage. Largely, the functions of the basal lamina are mediated through interactions with cell surface adhesion receptors; however, less is known about the identities and roles of these at the NMJ. Integrin-α3 is an extracellular matrix receptor that has previously been identified at the NMJ active zones, the sites of neurotransmitter release in the presynaptic terminus. As integrin-α3 binds to laminin-α4 and other active zone components, my hypothesis is that it may be important for relaying signals provided by the basal lamina during NMJ development. In this study, the integrin-α3 knockout mouse was used to explore the functions of this protein at the NMJ. Mutants displayed defects in active zone assembly and developmental motoneuron patterning. NMJs frequently resembled those found in aged animals, and in some cases, nerve terminals were detached from the synaptic cleft. Finally, electrophysiological analysis revealed defects in neurotransmission at mutant NMJs, including reduced efficiency of synaptic vesicle release, and impaired sensitivity of nerve terminals to external Ca2+. Previous studies have implicated integrin-α3 in muscle development; however, I find no expression of integrin-α3 in the muscle, and no defects in myogenesis in integrin-α3 mutants. These results indicate multiple roles for integrin-α3 at the NMJ, for active zone assembly, adhesion of nerve terminal, morphological integrity and neurotransmission. To my knowledge, this study identifies for the first time a cell surface receptor for the anchorage of pre- and postsynaptic elements at the NMJ. These results suggest that alterations in integrin-α3 expression or function may underlie some of the changes associated with ageing at the NMJ, and that mutations in its encoding gene may cause myasthenic syndromes.

Development and aging, two fundamental aspects of life, remain key biological processes that researchers try to understand. Drosophila melanogaster, thanks to its various merits, serves as an excellent model to study both of these processes. This thesis includes two parts. In the first part, I discuss our finding that the presynaptic neuron controls a retrograde signaling pathway by releasing essential components via exosomes. During synaptic development, postsynaptic cells send retrograde signals to adjust the activity and growth of presynaptic cells. It remains unclear what the mechanism is which triggers the release of retrograde signals; and how presynaptic cells are involved in this signaling event. The first part of this thesis demonstrates that a retrograde signal mediated by Synaptotagmin4 (Syt4) depends on the anterograde delivery of Syt4 protein from the presynaptic neuron to the muscle compartment likely through exosomes. This trans-synaptic transfer of Syt4 is required for the retrograde control of activity-dependent synaptic growth at the Drosophila larval neuromuscular junction. In the second part of this thesis, I talk about our discovery that the disruption of nuclear envelope (NE) budding, a novel RNA export pathway, is linked to the loss of mitochondrial integrity and premature aging in Drosophila. We demonstrate that several transcripts, which are essential for mitochondrial integrity and function, use NE-budding for nuclear export. Transgenic Drosophila expressing a LamC mutation modeling progeroid syndrome (PS), a premature aging disorder in humans, displays accelerated aging-related phenotypes including progressive mitochondrial degeneration as well as decreased levels of a specific mitochondrial transcript which is normally enriched at NE-budding site. The PS-modeled LamC mutants exhibit abnormal lamina organization that likely disrupts the egress of these RNAs via NE-budding. These results connect defective RNA export through NE-budding to progressive loss of mitochondrial integrity and premature aging in Drosophila.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2015.
"June 2015." Cataloged from PDF version of thesis.
Includes bibliographical references (pages 106-118).
Motor neurons located in the spinal cord and innervating muscle cells throughout the body are responsible for virtually all motor functions, from locomotion to respiration or speech. They arise from differentiation of progenitor cells within the neural tube under spatiotemporally well-defined morphogen concentration profiles, and extend axons into the peripheral nervous system following a precisely orchestrated sequence of events involving secreted chemo-attractants and repellents and dynamic expression of the corresponding ligand receptors. Finally, they form neuromuscular junctions, the synapses that transmit electrical signals to the muscle effectors. Failure for these motor neurons to develop or function properly, caused by developmental or neurodegenerative genetic disorders, or as a result of traumatic injuries, lead to highly incapacitating or even lethal malformation and conditions. Microfabricated platforms and optogenetic technologies have proven to be valuable tools to control the microenvironment, biochemical cues and the stimulation applied to neuronal tissues. Precise control of the geometry of microfluidic devices together with their ability to host 3D cell culture has enhanced the physiological relevance of such neuronal tissues relative to traditional 2D culture assays. And the ability to selectively excite neuronal cells with light has opened tremendous opportunities in the field of neuroscience. In this thesis, we combine these two technologies to stimulate and subject cells to chemical and physical microenvironments that emulate their in vivo counterpart. First, we present a microfluidic platform that generates orthogonal concentration gradients and emulates the confined appearance of motor neurons within the developing spinal cord. Then, we introduce a new device capable of forming a 3D compartmentalized neuron-muscle coculture and demonstrate remote stimulation of the myofibers by the motor neurons resulting in muscle contraction. By targeting the stem cells from which the motor neurons are derived with the light sensitive ion channel Channelrhodopsin, we form, in this microfluidic device, the first in vitro light-activatable neuromuscular junction. Keywords: microfluidics, optogenetics, morphogenesis, cell migration, neuromuscular junctions.
by Sébastien G. M. Uzel.
Ph. D.

Wheelchair-bound paraplegics are in an unnatural, almost all-day sitting position. This is physiologically disadvantageous as it may cause increased abdominal pressure, renal dysfunction, pressure sores, muscle atrophy and osteoporosis. Thus it would be beneficial, physiologically and psychologically, for a paraplegic to be able to stand for temporary periods of time. As a result of the muscle atrophy and functional degeneration that follows a spinal cord injury, it is essential for paraplegics to undergo a muscle restrengthening program, using Functional Neuromuscular Stimulation (FNS), before standing up under FNS control can be attempted. Six healthy spinal cord injured subjects with spinal lesions between CS and T9 (two tetraplegics and four paraplegics) exercised their quadriceps muscles at home using a portable two-channel FNS muscle stimulator. The muscles were exercised against an increasing load to maximise the training effect. Inclined standing exercise, under FNS control, was performed in the Inclistand. The subjects' general state of health and fitness were assessed, namely their responses during a maximal arm ergometry exercise test, arm muscle function, lung function, blood biochemistry and their dietary habits. Subjects have shown improvement in quadriceps muscle strength, fatigue resistance and muscle bulk to varying degrees - according to their individual circumstances. The tetraplegics responded in a different manner to that of the paraplegics. The muscle strength increased significantly by a mean (+SD) of 97,8 + 59,6% and 171,2 + 118,1% for the four paraplegics, left and right leg respectively. There was a mean improvement of 16% in fatigue resistance in the left leg (p=0,08), while the mean response of the right leg varied. Quadriceps muscle bulk increased by 4,43 + 3,4% (left) and 2,7 + 2,1% (right) (0,05<p<0,l). The amount of subcutaneous fat around the mid-thigh decreased significantly by 4,73 + 1,4% (left) and 3,43 + 1,1% (right leg). The group was in a state of general well-being, with the exception of one subject whose serum cholesterol concentration fell within the high risk category. This study therefore showed that the FNS was sucessful in improving the quadriceps muscle strength, bulk and fatigue response of the SCI people in our research group. The valuable experience gained from this FNS study will be used to improve the present program.

A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites. At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis. Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.

Development and aging, two fundamental aspects of life, remain key biological processes that researchers try to understand. Drosophila melanogaster, thanks to its various merits, serves as an excellent model to study both of these processes. This thesis includes two parts. In the first part, I discuss our finding that the presynaptic neuron controls a retrograde signaling pathway by releasing essential components via exosomes. During synaptic development, postsynaptic cells send retrograde signals to adjust the activity and growth of presynaptic cells. It remains unclear what the mechanism is which triggers the release of retrograde signals; and how presynaptic cells are involved in this signaling event. The first part of this thesis demonstrates that a retrograde signal mediated by Synaptotagmin4 (Syt4) depends on the anterograde delivery of Syt4 protein from the presynaptic neuron to the muscle compartment likely through exosomes. This trans-synaptic transfer of Syt4 is required for the retrograde control of activity-dependent synaptic growth at the Drosophila larval neuromuscular junction. In the second part of this thesis, I talk about our discovery that the disruption of nuclear envelope (NE) budding, a novel RNA export pathway, is linked to the loss of mitochondrial integrity and premature aging in Drosophila. We demonstrate that several transcripts, which are essential for mitochondrial integrity and function, use NE-budding for nuclear export. Transgenic Drosophila expressing a LamC mutation modeling progeroid syndrome (PS), a premature aging disorder in humans, displays accelerated aging-related phenotypes including progressive mitochondrial degeneration as well as decreased levels of a specific mitochondrial transcript which is normally enriched at NE-budding site. The PS-modeled LamC mutants exhibit abnormal lamina organization that likely disrupts the egress of these RNAs via NE-budding. These results connect defective RNA export through NE-budding to progressive loss of mitochondrial integrity and premature aging in Drosophila.

The mouse nervous system undergoes a vast remodelling of synaptic connections postnatally, resulting in a reduced number of innervating axons to target cells within the first few weeks of life. This extensive loss of connections is known as synapse elimination and it plays a critical role in sculpting and refining neural connectivity throughout the nervous system, resulting in a finely tuned and well-synchronised network of innervation. This process has been well characterised at the mouse neuromuscular junction (NMJ), where synapse elimination takes place postnatally in all skeletal muscles. It has been well studied for the reasons that it is easily accessible for live imaging and post-mortem experimental analysis. Studies utilising this synapse to uncover regulators of synapse elimination have mainly focused on the importance of glial cell lysosomal activity, nerve conduction and target-derived growth factor supply. It is clear that non-axonal cell types play key roles in the success of developmental axon retraction at the NMJ, however the role of glial cells in the regulation of this process has not been fully explored, as lysosomal activity is thought of as a consequence of axon pruning rather than a molecular driver. Previous studies have shown that signals emanating from myelinating glial cells can modulate neurofilament composition and transport within the underlying axons. We know that these changes in neurofilament composition and transport are underway during developmental synapse elimination at the NMJ, so it seems logical to predict that myelinating glial cells may play a role in the regulation of axonal pruning. Myelinating glial cells are found along the entire length of lower motor neurons and form physical interactions with the underlying axons at regions known as paranodes. At the paranode, Neurofascin155 (Nfasc155: expressed by the myelinating glial cell) interacts with a Caspr/contactin complex (expressed by the axon). This site has been proposed as a likely site for axon-glial signalling due to the close apposition of the cell membranes. The main focus of this PhD project was to study the potential role of myelinating glial cells in the success of synapse elimination at the NMJ, using a mouse model of paranodal disruption (Nfasc155-/-). Chapters 3 and 4 show the results of this work. This work has revealed a novel role for glia in the modulation of synapse elimination at the mouse neuromuscular junction, mediated by Nfasc155 in the myelinating Schwann cell. Synapse elimination was profoundly delayed in Nfasc155-/- mice and was found to be associated with a non-canonical role for Nfasc155, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Loss of Nfasc155 was sufficient to disrupt axonal proteins contributing to cytoskeletal organisation and trafficking pathways in peripheral nerve of Nfasc155-/- mice and lower levels of neurofilament light (NF-L) protein in maturing motor axon terminals. Synapse elimination was delayed in mice lacking NF-L, suggesting that Nfasc155 influences neuronal remodelling, at least in part, by modifying cytoskeletal dynamics in motor neurons. This work provides the first clear evidence for myelinating Schwann cells acting as drivers of synapse elimination, with Nfasc155 playing a critical role in glial cell-mediated postnatal sculpting of neuronal connectivity in the peripheral nervous system. A small section of the results within this thesis are devoted to the study of axon-glial interactions in a mouse model of childhood motor neuron disease, otherwise known as spinal muscular atrophy (SMA). In SMA, there are reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein. The NMJ is a particularly vulnerable target in SMA, manifesting as a breakdown of neuromuscular connectivity and progressive motor impairment. Recent studies have begun to shed light on the role of non-neuronal cell types in the onset and progression of the disease, presenting SMA as a multi-system disease rather than a purely neuronal disorder. Recent evidence has highlighted that myelinating glial cells are significantly affected in a mouse model of SMA, manifesting as an impaired ability to produce key myelin proteins, resulting in deficient myelination. The final results chapter of this thesis (Chapter 5) is focussed on further exploring the effects that loss of SMN has in Schwann cells including their interactions with underlying axons. This work reveals a disruption to axon-glial interaction, shown by a delay in the development of paranodes, supporting the idea that non-neuronal cell types are also affected in SMA. The results within this thesis reveal a novel role for a glial cell protein, Nfasc155, in the modulation of synapse elimination at the NMJ. Mechanistic insight in to Nfasc155’s role in this process is also uncovered and likely involves axonal cytoskeletal transport systems and the filamentous protein NF-L, which have not previously been implicated in the process of synapse elimination. This work highlights an important role for axon-glial interactions during normal postnatal development of the mouse NMJ. This work also highlights a role for axon-glial interactions in disease states of the NMJ. Using a mouse model of SMA, axon-glial interaction was assessed with the finding of a delay in paranodal maturation due to loss of SMN.

Abstract Aim: The main objective of this study was to compare electro mechanical delay (EMD) and rate of torque development (RTD) of the knee extensors 6 weeks after rehabilitation of anterior cruciate ligament reconstruction (ACLR) with or without neuromuscular electrical stimulation (NMES). Further the feasibility of the study was examined. Method: 10 participants were randomized into two groups, one neuromuscular electrical stimulation group (NMESG) and one training group (TG). The NMESG used a NMES-device as a complement to the ordinary rehabilitation protocol. Regular meetings with a physiotherapist were scheduled during the rehabilitation. Measurements of RTD and EMD during knee extension were made in an isokinetic dynamometer with electromyography recordings (EMG) from the knee extensors 6 weeks after surgery. Results: All participants completed the study. The NMESG went to see the physiotherapist 6.7 ± 2.5 times and the TG 6.8 ± 1.8 times. The participants in the NMESG used the NMES-apparatus 28 ± 1.7 times. Total number of training days for the NMESG was 25 ± 4 and for the TG 35 ± 1. RTD did not significantly differ between the groups. For the TG, RDT was 901.1, 941.2 and 531.0 Nm/s, over the first 50, 100 and 200 ms, respectively. For NMESG: RTD was 824.3, 966.2 and 529.0 Nm/s, over the first 50, 100 and 200 ms, respectively. No significant difference between the groups or interaction between group and muscle was found in EMD. For both groups EMD was significantly larger for vastus medialis as compared to the vastus lateralis and rectus femoris. Conclusions: The study was feasible to perform, and despite fewer training days for the NMESG, no significant group differences were found in RTD or EMD. A larger study population is needed to evaluate the efficacy of the intervention.
Abstrakt Syfte: Huvudsyftet med denna studie var att jämföra elektromekaniska fördröjning (EMD) och vridmoment utveckling (RTD) för knäextensorerna 6 veckor efter rehabilitering av främre korsbandsrekonstruktion (ACLR) med eller utan neuromuskulär elektrisk stimulering (NMES). Vidare undersöktes genomförbarheten av studien. Metod: 10 deltagare randomiseras in i två grupper, en neuromuskulär elektrisk stimulerings grupp (NMESG) och en träningsgrupp (TG). NMESG använde en NMES - enhet som ett komplement till ordinarie rehabiliteringsprotokoll. Regelbundna möten med sjukgymnast var inplanerad under rehabiliteringen. Mätningar av RTD och EMD under knäets extension gjordes i en isokinetisk dynamometer med elektromyografi inspelningar (EMG) från knäextensorerna 6 veckor efter operationen. Resultat: Alla deltagare fullföljde studien. NMESG träffade sjukgymnasten 6,7 ± 2,5 gånger och TG 6,8 ± 1,8 gånger. Deltagarna i NMESG använde NMES - apparaten 28 ± 1,7 gånger. Totalt antal träningsdagar för NMESG var 25 ± 4 och för TG 35 ± 1. RTD skiljde sig inte signifikant mellan grupperna. För TG var RDT 901,1, 941,2 och 531,0 Nm/s, under de respektive första 50, 100 och 200 ms. För NMESG var RTD 824,3, 966,2 och 529,0 Nm/s, under de respektive första 50, 100 och 200 ms. Inga signifikanta skillnader mellan grupperna eller samspel mellan grupp och muskler hittades i EMD. För båda grupperna var EMD signifikant större för vastus medialis jämfört vastus lateralis och rectus femoris. Slutsats: Studien var möjligt att utföra, och trots färre träningsdagar för NMESG sågs inga signifikanta skillnader mellan grupperna i RTD eller EMD. Det behövs en större studiepopulation för att utvärdera effekten av interventionen.

Muscle can generate greater force with lower muscle activation during eccentric (ECC) than isometric (ISO) and concentric (CON) contractions (actions). Unaccustomed ECC exercise induces muscle damage represented by prolonged impairment of muscle function and delayed onset muscle soreness (DOMS), but also confers protection from muscle damage, known as the repeated bout effect (RBE). However, the neuromuscular characteristics of ECC contractions, and the mechanisms involved in the muscle damage and RBE induced by ECC are not fully understood. Therefore, the purpose of this PhD thesis was to examine the neuromuscular characteristics of ECC contractions of the knee extensors in relation to muscle damage and the RBE profiles. This thesis consisted of five studies as summarised below. Study 1: Short-interval intracortical inhibition (SICI) is often used to examine inhibitory responses in the primary motor cortex representation of the quadriceps, but appropriate pairedpulse transcranial magnetic stimulation (TMS) parameters to optimise SICI measurement were not clear. Using 9 men and 6 women (26.6 ± 4.4 y), responses to single and paired-pulse (3-ms interval) TMS were recorded from vastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM). Test stimulus intensity was 140% of active motor threshold (AMT), and conditioning stimulus intensities (CSIs) ranged from 55-90% (eight intensities) of AMT (5 test and 5 paired responses for each intensity). With CSI of 55% AMT, SICI was minimal (conditioned:test motor evoked potential [MEP]; 1.00, 0.96 and 0.95 for VL, RF and VM, respectively, -1.00 indicates inhibition). Inhibition was greater at 70-90% AMT for VL (0.67- 0.85), at 75-90% AMT for RF (0.70-0.78) and at 80-90% AMT for VM (0.59-0.68) when compared to 55% AMT. The CSIs that elicited maximal and 50% maximal inhibition were ~84% and ~75% AMT, respectively. This method was shown to provide a practical approach to investigate quadriceps inhibitory networks. Study 2: Responses to TMS, twitch forces (TF) and voluntary drive were compared between ECC, ISO and CON contractions of the knee extensors. Sixteen participants (20-33 y) performed submaximal and maximal voluntary contractions (MVCs) for ISO and isokinetic (30˚/s) CON and ECC of knee extensors. EMG was recorded from VL. Supramaximal femoral nerve stimulation during and after MVCs evoked superimposed (ST) and resting TF to calculate voluntary activation (VA). Maximal M-waves (MMAX) were recorded. During 30% MVCs, single- and paired-pulse TMS elicited MEPs and assessed SICI. MVC torque was greater (P Study 3: ECC-only and coupled concentric-eccentric contractions (CON-ECC) of the knee extensors were compared for changes in neurophysiological indices before, immediately after and 1-3 days post-exercise. Twenty participants (19-36 y) were randomly assigned to ECC (n=10) or CON-ECC (n=10) group. ECC group performed 6 sets of 8 ECC-only contractions at 80% of ECC one-repetition maximum (1-RMecc), while CON-ECC group performed 6 sets of 8 alternating CON and ECC (i.e., 8 CON and 8 ECC) contractions at 80% of CON 1-RM and 1-RMecc, respectively. Maximal voluntary isometric contraction (MVIC) force, rate of force development (RFD), TF elicited by femoral nerve stimulation, VA, MEPs, corticospinal silent period (CSP) and SICI assessed by single- and paired-pulse TMS, and muscle soreness were measured before, immediately after, and 1-3 days post-exercise. No significant (p>0.05) differences between ECC and CON-ECC were evident for the changes in any variables post3 exercise. MVIC force decreased immediately post-exercise compared to baseline (ECC: -20.7 ± 12.8%, CON-ECC: -23.6 ± 23.3%) and remained low at 3 days post-exercise (-13.6 ± 13.4%, -3.3 ± 21.2%), and changes in RFD were greater than those of MVIC force (immediately postexercise: ECC: -38.3 ± 33.9%, CON-ECC: -30.7 ± 38.3%). VA, TF and MEP/MMAX decreased and CSP increased post-exercise (p Study 4: Since the participants from Study 3 responded similarly for indirect indicators of muscle damage, the 20 participants were used to examine whether the magnitude of muscle damage indicated by changes in MVIC force 1-3 days after ECC could be predicted by changes in central and peripheral neuromuscular parameters immediately post-ECC. The criterion measures were the same as those in Study 2, and additional analyses of the rate of force development (RFDRT) and rate of relaxation (RRRT) of the TF were performed. Relationships between changes in the variables immediately post-ECC and changes in MVIC force at 1-3 days post-ECC were examined by Pearson product-moment (r) or Spearman correlations. MVIC force decreased (-22.2 ± 18.4%) immediately post-exercise, and remained below baseline at 1 (-16.3 ± 15.2%), 2 (-14.7 ± 13.2%) and 3 days post-ECC (-8.6 ± 15.7%). Immediately post-ECC, RFD (0-30-ms: -38.3 ± 31.4%), TF (-45.9 ± 22.4%), RFDRT (-32.5±40.7%), RRRT (-38.0±39.7%), VA (-21.4 ± 16.5%) and MEP/MMAX at rest (-42.5 ± 23.3%) also decreased, while CSP at 10%-MVIC increased by 26.0 ± 12.2% (p Study 5: Among the 20 participants described in Study 4, 10 participants (6 from the eccentriconly exercise group and 4 from the concentric-eccentric exercise group) returned two weeks after the first exercise bout to perform the second bout of the same exercise. Changes in MVIC force, RFD, muscle soreness, TF, VA, MMAX, MEPs, and SICI before, immediately after and 1–3 days post-exercise were compared between the first (ECC1) and the second bouts (ECC2). ECC2 induced less (P=0.01) muscle soreness (peak: 27.5 ± 26.6 mm) than ECC1 (50.7 ± 31.8 mm), and MVIC force decreased more immediately post-ECC1 (-21.6 ± 23.3%) than ECC2 (-11.0 ± 11.3%) and recovered faster to the baseline after ECC2 than ECC1. RFD decreased immediately after ECC1 (e.g. 0-50-ms: -50.5 ± 52.5%, P0.05). The non-significant differences in VA and MEPs between ECC1 and ECC2, and lack of changes in MMAX and SICI suggest that changes in neural factors after eccentric exercise do not explain the repeated bout effect. Collectively, these studies advance understanding of the characteristics of ECC muscle contractions and their relationship to muscle damage and the RBE, and can be used to inform future research directions.

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Studies show that maternal obesity can affect fetal development, resulting in diseases in adult life, such as diabetes mellitus type 2, cardiovascular disease, obesity itslef. To reduce the effects of obesity and its comorbidities, bariatric surgeries stand out among the most effective interventions, with Roux-en-Y gastric bypass (RYGB) being the most frequently performed type of bariatric procedure. However, there are still few studies in the literature that evaluate the effects of obesity and bariatric surgeries on the morphology of skeletal muscle tissue in adult offspring. Thus, the objective of this study was to evaluate microscopic parameters of muscle fibers and neuromuscular junctions (NMJs) of the extensor digitorum longus muscle (EDL) in obese rats’ offspring submitted or not to RYGB surgery. Three-week Wistar rats were randomly divided into three groups: Control Group (CTL) which received a standard diet; 2) Cafeteria False Operated (CAF FO) and 3) Cafeteria RYGB (CAF RYGB), the latter two received a cafeteria diet before and after the surgical procedure until the weaning of the offspring. In the 18th of life, the surgical procedure and false operation were performed in the CAF RYGB and CAF FO groups, respectively. The mating of the animals occurred five weeks after the surgical procedure. The birth of the offspring was postnatal day 0, and weaning occurred at three weeks of age, and only the male offspring were separated for the experiment. The offspring of the first generation (F1) were named CTL-F1, OB-F1, CAF FO-F1 and CAF RYGB and received standard diet. At 17 weeks the animals were euthanized and the EDL muscle collected for analysis of fiber muscles and NMJs. When the CTL-F1 and OB-F1 groups were analyzed, the latter had an increase in body weight, retroperitoneal and periepididymal fats, and capillary/fiber ratio. Reduction in the number of nuclei, conjunctive and morphological changes in the parameters evaluated in the ultrastructure. The area and larger diameter of NMJs also showed reduction. The analysis between CAF RYGB-F1 and CAF FO-F1 groups showed reduction of body weight, ELD muscle weight, retroperitoneal and periepididimal fat, nasoanal length, fiber area and nuclei/fiber ratio in the CAF RYGB-F1 group. This group also presented increase in the number of fibers of type I and IIa and number of capillaries, as well as reduction in the area of the NMJs and morphological alterations in the ultrastructure. These results demonstrate that both obesity and bariatric surgery expose the offspring, through metabolic programming, to effects on the morphology of skeletal muscle tissue, being found greater aggravations in the muscular fiber of the offspring of mothers submitted to RYGB.
Estudos apontam que a obesidade materna pode afetar o desenvolvimento fetal, resultando em doenças na vida adulta, tais como diabetes mellitus tipo 2, doenças cardiovasculares e a própria obesidade. Para reduzir os efeitos da obesidade e as suas comorbidades, as cirurgias bariátricas destacam-se entre as intervenções mais eficazes, sendo a derivação gástrica em Y de Roux (DGYR) o tipo de procedimento bariátrico mais frequentemente realizado. No entanto, ainda são escassos na literatura estudos que avaliem os efeitos da obesidade e das cirurgias bariátricas na morfologia do tecido muscular esquelético da prole adulta. Diante disso, o objetivo deste estudo foi avaliar a morfologia e a morfometria das fibras musculares e as junções neuromusculares (JNMs) do músculo extensor longo dos dedos (ELD) da prole de ratas obesas submetidas ou não à cirurgia de DGYR. Para tanto, ratas Wistar de três semanas de vida foram separadas aleatoriamente em três grupos: 1) Controle (CTL), que recebeu dieta padrão; 2) Cafeteria Falso operado (CAF FO) e 3) Cafeteria DGYR (CAF DGYR); esses dois últimos receberam dieta de cafeteria antes e após o procedimento cirúrgico, até o desmame da prole. Na 18ª semana de vida, foi realizado o procedimento cirúrgico e a falsa operação nos grupos CAF DGYR e CAF FO, respectivamente. O cruzamento dos animais ocorreu cinco semanas após o procedimento cirúrgico. O nascimento dos animais foi considerado o dia zero pós-natal e o desmame se deu na terceira semana vida, quando somente os machos foram separados para o experimento. A prole da primeira geração (F1) foi nomeada em CTL-F1, OB-F1, CAF FO-F1 e CAF DGYR-F1 e todos os animais receberam dieta padrão. Na 17ª semana, os animais foram eutanasiados e o músculo ELD coletado para análise das fibras musculares e JNMs. Quando analisado os grupos CTL-F1 e OB-F1, esse último apresentou aumento do peso corpóreo, das gorduras retroperitoneal e periepididimal, e relação capilar/fibra. Além disso, houve a redução do número de núcleos, conjuntivo e alterações morfológica nos parâmetros avaliados na ultraestrutura. A área e diâmetro maior das JNMs também apresentaram redução. A análise entre os grupos CAF DGYR-F1 e CAF FO-F1 evidenciou redução do peso corporal, do peso do músculo ELD, da gordura retroperitoneal e periepididimal, don comprimento nasoanal, da área das fibras e relação núcleo/fibra no grupo CAF DGYR-F1. Esse grupo também apresentou aumento no número de fibras do tipo I e IIa e no número de capilares, assim como redução na área das JNMs e alterações morfológicas na ultraestrutura. Esses resultados demonstram que tanto a obesidade como a cirurgia bariátrica expõem a prole, por meio da programação metabólica, com efeitos na morfologia do tecido muscular esquelético, sendo encontrado maiores agravos na fibra muscular da prole de mães submetidas à DGYR.

Motor unit double discharges (i.e. doublets), which are excitatory potentials that occur at shorter-than-normal intervals (e.g. 5-10 ms) during normal muscle activation, are known to cause muscle force to exceed that predicted from a standard, linear summation of twitch forces. However, although a marked increase in the occurrence of motor unit doublets at the onset of a contraction has been observed after explosive-type exercise training, and has been correlated with changes in RFD (Van Cutsem et al., 1998), little is known about the influence of strength training on the physiological and biomechanical benefits derived from the phenomenon. The present research examined the effects of 4 weeks of ‘explosive’ isometric knee extensor strength training on voluntary and electrically-evoked contractile RFD (calculated as the time derivative of the moment-time curve) in 8 untrained male participants. Electrical stimulation (NMES) trains were delivered to the muscle at 20 Hz and 40 Hz and incorporated short (5 and 10 ms) inter-pulse intervals (IPIs) at the onset of stimulation (i.e. variable-frequency trains; VFT). The influence of the short inter-pulse interval was assessed by comparison to a constant frequency train (i.e. the VFT:CFT ratio). Following the training, substantial improvements in maximum isometric knee extensor strength (MVC) (24.3 ± 13.3%, p = 0.002) and RFD measured to time intervals of 50 (55.5 ± 50.3%, p = 0.011), 100 (34.0 ± 47.2%, p = 0.01) and 150 ms (31.9 ± 38.2%, p = 0.02) were observed. RFD normalised to MVC (RFDnorm), measured to time intervals of 50 and 100 ms from the onset of contraction, improved by 44.9 ± 38.8% (p = 0.04) and 13.8 ± 12.2% (p = 0.01), respectively. There was a significant reduction in the VFT:CFT ratio after training when a 10-ms IPI preceded a 20-Hz train when measured to 30 (-13.7 ± 11.3%, p = 0.03), 50 (-13.9 ± 8.4%, p = 0.007), 100 (-8.6 ± 10.2%, p = 0.04), and 200 ms (-8.1 ± 5.3%, p = 0.009) as well as in the interval 100-200 ms (-7.4 ± 6.6%, p = 0.02). However, no significant changes were observed for other stimulation frequency-IPI combinations. Moderate-to-very strong positive correlations were observed between changes in RFDnorm and changes in VFT:CFT when measured within some time periods, particularly in the early phase of the contraction (r = 0.02 – 0.91). In conclusion, the effect of a high-frequency double discharge at stimulation onset remained unchanged or, under some conditions, was reduced after 4 weeks of explosive-type knee extensor training. Additionally, training-dependent improvements in the ability to rapidly reach a specified torque level relative to peak MVC torque (i.e. RFDnorm) were greater for those participants whose VFT:CFT ratio either did not decline or declined the least. These data provide evidence that explosive training may reduce the effect of a high frequency discharge at the onset of a contraction, and that greater increases in RFD may occur in those who most retain this ability.

O objetivo do presente estudo foi comparar os efeitos da manipulação da ordem dos exercícios de força e aeróbico durante o treinamento concorrente na hidroginástica sobre as variáveis neuromusculares e cardiorrespiratórias de mulheres jovens e pósmenopáusicas. No estudo I, 26 mulheres jovens (25,12 ± 2,94 anos) foram aleatoriamente divididas em dois grupos de treinamento: força-aeróbico (FA) (n=13) e aeróbico-força (AF) (n=13). Para o estudo II, 21 mulheres pós-menopáusicas (57,14 ± 2,43 anos) foram divididas, também aleatoriamente, em dois grupos: força-aeróbico (FA) (n=10) e aeróbico-força (AF) (n=11). Em ambos os estudos os sujeitos realizaram o treinamento concorrente no meio aquático, duas vezes na semana durante 12 semanas, executando ambos os tipos de exercícios (aeróbico e força) na mesma sessão de treinamento. O treinamento de força foi realizado com séries em máxima velocidade e o treinamento aeróbico foi executado na frequência cardíaca do segundo limiar ventilatório. Todas as variáveis foram avaliadas antes e após o período de treinamento. Para análise dos dados foi utilizado o teste ANOVA para medidas repetidas com fator grupo (α=0,05). No estudo I, com as mulheres jovens, houve um aumento significativo da força muscular dinâmica máxima, avaliada através do teste de 1 repetição máxima (1RM), de todos os grupos musculares analisados (flexão e extensão de cotovelos e joelhos) após o período de treinamento. O grupo FA apresentou maiores ganhos da força muscular dinâmica máxima dos extensores de joelho em comparação ao grupo AF (43,58 ± 14,00% vs. 27,01 ± 18,05%, respectivamente). Após o treinamento houve um aumento do pico de torque isométrico (PT), avaliado no dinamômetro Biodex, de todos os grupos musculares avaliados (exceção PT extensores de cotovelo), sem diferença entre os grupos FA e AF. Além disso, após o treinamento, houve um aumento significativo da taxa de produção máxima e em diferentes janelamentos (50, 100, 250 ms), durante a contração isométrica voluntária máxima (CIVM) de extensão de joelho, sem diferença entre os grupos FA e AF. Houve um aumento significativo da amplitude máxima isométrica do sinal eletromiográfico (EMG), após o treinamento, dos músculos bíceps braquial e vasto lateral, sem diferença entre os grupos FA e AF. Além disso, observou-se uma diminuição significativa da amplitude submáxima isométrica do sinal EMG dos músculos bíceps braquial em 40% da CIVM, do vasto lateral em 40 e 80% da CIVM e do reto femoral em 80% da CIVM, sem diferença entre os grupos FA e AF após as 12 semanas de treinamento. Após o treinamento, houve um aumento significativo da espessura muscular do bíceps braquial, braquial, vasto medial e reto femoral, sem diferença entre os grupos FA e AF. O percentual de ganho da espessura muscular do vasto lateral e vasto intermédio diferiu significativamente entre os grupos, com maiores ganhos para o grupo que treinou na ordem FA em comparação a ordem AF (vasto lateral: 10,00 ± 7,64% vs. 5,28 ± 3,42%, vasto intermédio: 11,58 ± 5,36% vs. 4,40 ± 3,77%, respectivamente). Para os saltos, após o treinamento, houve um aumento significativo da altura do countermovement jump, sem diferença entre os grupos FA e AF. Por fim, ainda em relação ao estudo I, após o treinamento, houve um aumento significativo do consumo de oxigênio de pico (VO2pico) e referente ao primeiro limiar ventilatório (VO2LV1), sem diferença entre os grupos FA e AF. No estudo II, com as mulheres pós-menopáusicas, após o treinamento, houve um aumento significativo da força muscular dinâmica máxima dos flexores e extensores de cotovelo, sem diferença entre os grupos FA e AF. Para o teste de 1RM de extensão de joelhos, foi observado que o grupo FA apresentou maiores ganhos de força em comparação ao grupo AF (34,62 ± 13,51% vs. 14,16 ± 13,68%). Após o treinamento, houve um aumento significativo do PT dos flexores e extensores de joelho, sem diferença entre os grupos FA e AF. Além disso, houve um aumento significativo da taxa de produção máxima e também nos diferentes janelamentos (50, 100, 250 ms), durante a CIVM de extensão de joelho, sem diferença entre os grupos FA e AF. Houve um aumento significativo da amplitude máxima isométrica do sinal EMG dos músculos vasto lateral e reto femoral, sem diferença entre os grupos FA e AF após 12 semanas de treinamento. Também houve uma diminuição significativa da amplitude submáxima isométrica do sinal EMG do músculo reto femoral em 40% da CIVM, sem diferença entre os grupos FA e AF após o treinamento. Após o treinamento, houve um aumento significativo da espessura muscular de todos os músculos analisados, sem diferença entre os grupos FA e AF (exceto reto femoral). Por fim, no estudo II, após o treinamento, houve um aumento significativo do consumo de oxigênio referente ao segundo limiar ventilatório (VO2LV2), sem diferença entre os grupos FA e AF. Em suma, a ordem exercícios de força seguidos dos exercícios aeróbicos otimizou os ganhos de força muscular dinâmica máxima dos extensores de joelho tanto em mulheres jovens quanto em mulheres pós-menopáusicas, bem como a espessura muscular do quadríceps em mulheres jovens quando comparada com a ordem inversa (aeróbico-força).
The aim of the present study was to compare the effects of the intra-session exercise order (i.e., resistance-aerobic or aerobic-resistance) during water-based concurrent training on the neuromuscular and cardiorespiratory variables in young and postmenopausal women. Twenty-six young women (25.12 ± 2.94 years) were randomly assigned into two groups in study I: resistance-aerobic (RA) (n=13) and aerobicresistance (n=13). For study II, twenty-one postmenopausal women were also randomly assigned into two groups: resistance-aerobic (RA) (n=10) and aerobic-resistance (n=11). In both studies the subjects performed the water-based concurrent training two times a week during 12 weeks, performing both resistance and aerobic training in the same session. The resistance training was performed with sets at maximal effort and the aerobic training with exercises at heart rate corresponding to the second ventilatory threshold. All variables were evaluated before and after training. A repeated measure ANOVA with group factor was used to analyze the data of the present study (α=0.05). After training in study I, with the young women, there was a significant increase in the maximal dynamic strength in all muscle groups (elbow and knee flexors and extensors) evaluated using the one-repetition maximal test (1RM). The RA group presented grater relative gains of the knee extensors maximal dynamic strength compared to the AR group (43.58 ± 14.00% vs. 27.01 ± 18.05%, respectively). After training there was a significant increase of the maximal isometric peak torque (PT) of all muscle groups (except elbow extensor PT) evaluated using the Biodex dynamometer, with no difference between RA and AR groups. In addition, after training there was a significant increase of the maximal rate of force development (RFD) and of the RFD at different windows (50, 100, 250 ms) during the knee extension maximal isometric voluntary contraction (MIVC), with no difference between RA and AR groups. There was a significant increase of the maximal isometric electromyography (EMG) activity of biceps brachii and vastus lateralis after training, with no difference between RA and AR groups. Moreover, the submaximal isometric EMG activity of biceps braachi at 40% of MIVC, the submaximal isometric EMG activity of vastus lateralis at 40% and 80% of MIVC and the submaximal isometric EMG activity of rectus femoris at 80% of MIVC showed lower values after training, with no difference between RA and AR groups. After training, there was a significant increase of the muscle thickness of biceps brachii, brachialis, vastus medialis and rectus femoris, with no difference between RA and AR groups. The relative gains of the muscle thickness of the vastus lateralis and vastus intermedius were greater for the RA group compared to the AR group (vastus lateralis: 10.00 ± 7.64% vs. 5.28 ± 3.42%, vastus intermedius: 11.58 ± 5.36% vs. 4.40 ± 3.77%, respectively). The height of the countermovement jump improved after training, with no difference between RA and AR groups. In addition, the peak oxygen uptake (VO2peak) and corresponding to the first ventilatory threshold (VO2VT1) showed significant increases after training, with no difference between RA and AR groups. In study II, with the postmenopausal women, there was a significant increase in the maximal dynamic strength of the elbow flexors and extensors, with no difference between RA and AR groups. The knee extensors 1RM in the RA group showed greater increases than the AR group (34.62 ± 13.51% vs. 14.16 ± 13.68%). After training, there were significant increases of the knee flexors and extensors PT, with no difference between RA and AR groups. In addition, there was a significant increase in the knee extension maximal RFD and in the knee extension RFD at different windows (50, 100, 250 ms), with no difference between RA and AR groups. Moreover, there were increases of the maximal isometric EMG activity of vastus lateralis and rectus femoris, with no difference between RA and AR groups. Furthermore, the submaximal isometric EMG activity of rectus femoris at 40% of MIVC showed lower values after training, with no difference between RA and AR groups. Also, there were significant increases of the muscle thickness of all muscles evaluated, with no difference between RA and AR groups (except rectus femoris). Significant increase was also observed in the oxygen uptake corresponding to the second ventilatory threshold (VO2VT2) after training, with no difference between RA and AR groups. In summary, the intra-session exercise order with resistance exercises prior to aerobic exercises optimizes the knee extensors maximal dynamic strength gains in young and postmenopausal women, as well as the quadriceps femoris muscle thickness in young women when compared to the inverse order (i.e., aerobic-resistance).

Movement is a prerequisite for normal fetal development and growth. Intrauterine movement restrictions cause a broad spectrum of disorders in which the unifying feature is a reduction or lack of fetal movement, giving rise to the term fetal akinesia deformations sequence (FADS [OMIM 208150]). FADS corresponds to a clinically and genetically heterogeneous constellation of properties and is characterized by multiple joint contractures, facial abnormalities, and lung hypoplasia as a result of the decreased in utero movement of the fetuses. Affected babies are often prematurely and stillborn, and those born alive typically die within minutes or hours after birth. The genetic causes for this fatal disorder are ill-defined as a genetic diagnosis is rarely executed, but mutations in three genes, namely RAPSN, DOK7, and MUSK, as well as in the subunits of the muscular nicotinic acetylcholine receptor (AChR) have been described. These mutations are thought to affect neuromuscular junctions, although this has not been proven experimentally.The nucleoporin NUP88 is a constituent of the nuclear pore complex (NPC), the gate for all trafficking between the nucleus and the cytoplasm. NUP88 resides on both the cytoplasmic and the nuclear side of NPCs, and it is found in two distinct subcomplexes. It associates with NUP214 and NUP62 on the cytoplasmic face, while on the nuclear side NUP88 binds NUP98 and the intermediate filament protein lamin A. The NUP88-NUP214-NUP62 complex plays an essential role in the nuclear export of a subset of proteins and pre-ribosomes, which is mediated by the nuclear export receptor CRM1. NUP88 in particular somewhat participates in the nuclear export of NF-κB proteins in a CRM1-dependent manner. Moreover, NUP88 is frequently overexpressed in a variety of human cancers, and its role in cancer appears linked to the deregulation of the anaphase-promoting complex. Here, we report the first Mendelian disorders caused by mutations in NUP88 and with that the first lethal developmental human disease due to mutations in a nuclear pore component. We demonstrate that biallelic mutations in NUP88 are likely to cause fetal akinesia of the Pena-Shokeir subtype. We confirm in zebrafish that loss of NUP88 impairs movement and the mutations identified in the affected individuals resemble a loss-of-function phenotype. We show that loss of NUP88 affects expression and localization of rapsyn, the protein encoded by RAPSN, in human and mouse cell lines, and patient samples. Consistent with altered rapsyn, AChR clustering and neuromuscular junction formation in zebrafish are abnormal. We therefore propose that defective NUP88 function cause FADS by affecting neuromuscular junction formation.Keywords: Nuclear pore complex, NUP88, Fetal Akinesia Deformation Sequence, rapsyn, acetylcholine receptor clustering, synaptic transmission, fetal development, inherited developmental disorder.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Les ossifications hétérotopiques (OH) sont des formations osseuses bénignes anormalement situées dans les tissus mous du corps humain. Les OH peuvent être d’origine génétique, acquise et idiopathique. Elles sont appelées paraostéoarthropathies neurogènes (POAN) lorsqu’elles se développent après une lésion du système nerveux central. Cette thèse a pour objectif de préciser les facteurs inflammatoires locaux et systémiques, impliqués dans la survenue de POAN. Nous avons tout d’abord étudié l’effet de l’injection de composants de paroi membranaire bactérienne dans un modèle de souris développant des ossifications hétérotopiques neurogènes (OHN) après section médullaire et injection intramusculaire d’un myotoxique chimique. L’injection locale intramusculaire comme systémique d’un composant de paroi d’Escherichia Coli ou de Staphylococcus Aureus a permis d’augmenter de façon significative le volume des OHN. A l’issu de ces expériences, il semble que le niveau d’inflammation intramusculaire influence de façon importante le volume des OHN, selon un effet-dose. La provenance des agents de paroi bactérienne fait supposer que l’inflammation pourrait être spécifiquement induite par des pathogènes infectieux. Il semblerait qu’il existe un niveau inflammatoire seuil à partir duquel, la section médullaire ne soit plus obligatoire à la survenue d’OH. Toujours dans le même modèle murin, nous avons montré que le blocage de la jonction neuro-musculaire par une injection de toxine botulique augmente le volume des OHN. La jonction neuro-musculaire semble réguler le développement de OHN dans un muscle pathologique chez la sourie avec section médullaire. Il est possible que cette régulation s’effectue via un mécanisme de neuro-inflammation locale. Suite à ces découvertes en recherche fondamentale, nous avons mené une étude cas-témoins recherchant des facteurs pourvoyeurs d’inflammation à la phase très précoce suivant l’accident neurologique, qui pourraient être corrélés à l’apparition de POAN. Cette étude a été la première à montrer que les patients avec POAN présentent de façon plus importante des infections à Pseudomonas Aeruginosa. La présence de traumatismes associés et de chirurgies était corrélée à la survenue de POAN, tout comme la longueur de la ventilation mécanique, du coma, du séjour en réanimation et la présence d’une trachéotomie. En revanche, aucun critère neurologique n’était associé à une augmentation du risque de POAN. A gravité neurologique égale, il semble donc que les patients développant des POAN présentent un niveau d’inflammation plus élevé (infections, polytraumatisme, chirurgies multiples, réanimation) que les patients sans POAN. Comme pour d’autres pathologies articulaires avec une composante inflammatoire, nous avons infiltrer des dérivés cortisonés localement, dans le but de traiter les douleurs induites par certaines POAN. Un mois après l’injection, 80% des patients présentaient un soulagement des douleurs. Ce traitement local parait être une alternative intéressante à la prise en charge antalgique des POAN douloureux.Les POAN sont toujours, à l’heure actuelle, diagnostiqués et prises en charge tardivement lors de l’apparition de complications. L’enjeu actuel est d’identifier les patients à risque de développer des POAN, le plus tôt possible après leur accident neurologique de façon à leur assurer un suivi et une prise en charge adaptée et précoce. En se basant sur les études cliniques et les données issues du modèle murin développant des POAN, nous allons relever des critères cliniques et biologiques reconnus comme étant corrélés à l’apparition de POAN dans une base de données clinico-biologique. Ce tout premier travail prospectif sur les POAN permettra de repérer les patients à risque de développer des POAN et peut être à terme d’identifier des cibles thérapeutiques qui permettrons de prévenir ou de guérir les POAN
Heterotopic ossifications (H0) are abnormal ectopic bone formations that develop in soft tissues. HO can be related to genetic factors or acquired pathologies. HO occurring after central nervous system lesion are called neurogenic HO (NHO). The objective of this project is to identify local and systemic inflammatory factors that may be associated with occurrence of NHO. We study first, the effect of bacterial membrane components on the development of NHO in a mouse model of spinal cord injury triggered by injection of a myotoxic compound into muscle. Local and systemic administration of membrane components from Escherichia coli or Staphylococcus aureus significantly increased the volume of NHO. Changes in the level of inflammation, which was dose responsive, correlated with changes in NHO volume suggesting that inflammation influences NHO formation. As bacterial membrane components were also linked to increased volumes of NHO, it is possible that inflammation triggered by infectious pathogens could also be involved in NHO development. Furthermore, we identified that after reaching a certain threshold of inflammation, triggered by administration of bacterial membrane components, spinal cord injury was not required for NHO formation. Further experiments with this model involved determining the effect of blocking neuromuscular signaling on NHO formation. Botulinum toxin injection increased the size of NHO. Therefore, neuromuscular signaling also modulates NHO development in damaged muscles of spinal cord-injured mice. By extension, local neuroinflammation was implicated in regulating neuromuscular signals received by affected muscles. Based on these preclinical results, we carried out a case-control study to look for factors inducing inflammation that could be linked with NHO occurrence, and which occur at early stages after neurological trauma. This study identifies for the first time that patients with Pseudomonas Aeruginosa-positive infections were more likely to develop NHO. NHO patients more frequently experience surgery and polytrauma, compared to patients without NHO. Furthermore, extended stays in intensive care, long periods of mechanical ventilation, enduring coma, or patients with a tracheotomy were more frequent in patient with NHO. In contrast, no neurological factors were associated with a higher risk of developing NHO. Patients with comparable neurological trauma severity were more susceptible to develop NHO when they were experiencing a high level of inflammation (infection, polytrauma, surgeries, intensive care). Like for other inflammatory joint pathologies, we performed a further study which involved the infiltration of NHO with corticosteroid locally, in order to treat pain induced by NHO formation. One month after treatment, 80% of patients reported an improvement of pain. Therefore, we demonstrate that corticosteroid infiltration at the site of NHO is an effective treatment for pain associated with NHO. Detecting patients that are at risk to develop NHO as early as possible after an accident is imperative, to adapt rehabilitative strategies or treatment needs specific for patients that develop NHO. However, NHO diagnosis occurs during late phase of disease, when complications are occurring. To address this shortfall in the detection of NHO formation, we are undertaking the first prospective study of NHO, where clinical and biological information will be recorded to make a database. The specific data to be collected has been defined by our previous research in the mouse model and earlier clinical studies, and will identify specific biological and clinical factors that can be monitored to identify patients at risk to develop NHO. The outcomes of this project have specific implications in the understanding the drivers of NHO formation and its detection. Global outcomes of this project include improving patient management and possibly the prevention of NHO formation in patients

El desenvolupament del sistema nerviós perifèric implica una inicial exuberant producció de neurones i, una posterior reducció dependent de l'activitat del nombre de sinapsis de les unions neuromusculars (NMJ). Aquest procés s’anomena eliminació sinàptica. Al final de la primera setmana postnatal, cada fibra muscular està innervada per una sola motoneurona. Els receptors muscarínics d’acetilcolina (mAChR), els receptors d’adenosina (AR) i el receptor cinasa de tropomiosina B (TrkB) podrien permetre la competició entre terminals nerviosos durant el procés d’eliminació sinàptica mitjançant la modulació de l’alliberament d’acetilcolina. En aquesta tesi s’ha investigat, mitjançant microscòpia confocal i un anàlisi morfològic quantitatiu, el paper dels receptors mAChRs (M1, M2 i M4), dels AR (A1 i A2A) i del receptor TrkB en el procés d’eliminació en el desenvolupament de la NMJ. Els resultats mostren que els receptors mAChRs, AR i el receptor TrkB promouen una desconnexió axonal al principi de la segona setmana postnatal independentment de la maduració dels receptors d’acetilcolina postsinàptics. En resum, els receptors mAChRs, AR i el receptor TrkB endarrereixen el procés d’eliminació sinàptica a P7 però l’acceleren a P9. Pel que fa la cooperació d’aquests receptors, M4 produeix un efecte oclusiu sobre M1 i un efecte additiu sobre a P7. La cooperació entre els receptors M1, A1 i A2A promou la pèrdua axonal a P9, mentre que, l’efecte de M2 és independent dels altres receptors. El M1 i TrkB treballen junts per incrementar la pèrdua axonal a P9 independentment dels receptors M2 i TrkB. En conclusió, l’eliminació sinàptica postnatal és regulada per un mecanisme que depèn de varis receptors, involucrant la cooperació dels diferents subtipus de receptors muscarínics, d’adenosina i el receptor TrkB, els quals garanteixen la monoinnervació de les sinapsis neuromusculars al final del procés.
El desarrollo del sistema nervioso periférico implica una inicial exuberante producción de neuronas y, una posterior reducción dependiente de actividad del número de sinapsis en las uniones neuromusculares (NMJ). Este proceso se denomina eliminación sináptica. Al final de la segunda semana postnatal, cada fibra muscular esta inervadas por una solo motoneurona. Los receptores muscarínicos de acetilcolina (mAChR), los receptores de adenosina (AR) y el receptor quinasa de tropomiosina B (TrkB) podrían permitir la competición entre los terminales nerviosos durante el proceso de eliminación sináptica mediante la modulación en la liberación de acetilcolina. En esta tesis se ha investigado, mediante microscopía confocal y un análisis morfológico cuantitativo, el papel de los receptores mAChRs (M1, M2 y M4), de los receptores de adenosina (A1 y A2A) y del receptor TrkB en el del proceso de eliminación en el desarrollo de la NMJ. Los resultados muestran que los receptores mAChRs, AR y el receptor TrkB promueven una desconexión axonal al inicio de la segunda semana postnatal independientemente de la maduración de los receptores de acetilcolina postsinápticos. En resumen, los receptores mAChRs, AR y el receptor TrkB retrasan el proceso de eliminación sináptica en P7 pero lo aceleran en P9. En la cooperación de estos receptores, se ha demostrado que M4 produce un efecto oclusivo sobre M1 y aditivo sobre A1 en P7. La cooperación entre M1, A1 y A2A promueve la pérdida axonal en P9, mientras que M2 es independiente de los otros receptores. M1 y TrkB cooperan para incrementar la pérdida axonal en P9 independientemente de M2 y TrkB. En conclusión, la eliminación sináptica postnatal está regulada por un mecanismo que depende de varios receptores, involucrando la cooperación de diferentes subtipos de receptores muscarínicos, de adenosina y el receptor TrkB, los cuales garantizan la monoinnervación de las sinapsis neuromusculares al final del proceso. saludable.
The development of the peripheral nervous system involves an initially exuberant production of neurons and a subsequent activity-dependent reduction in the number of synapses at the neuromuscular junctions (NMJ). This process is called synaptic elimination. At the end of the first postnatal week, each muscle fiber is innervated by a single motoneuron. Muscarinic acetylcholine receptors (mAChR), adenosine receptors (AR) and the tropomyosin-related kinase B (TrkB) receptor may allow the direct competition between nerve endings during synapse elimination through the modulation of acetylcholine release. Here, it has been investigated by confocal microscopy and quantitative morphological analysis the involvement of the individual and synergic or oclusive effect of M1-, M2- and M4-subtypes of mAChRs, A1 and A2A of ARs and TrkB in the control of the axonal elimination in developing NMJ. The results show that mAChRs, ARs and TrkB promote axonal disconnection at the beginning of the second postnatal week without affecting the postsynaptic maturation of the nicotinic receptor cluster. In summary, mAChRs, ARs and TrkB delay axonal loss at P7 but accelerate it at P9. In terms of receptor cooperation, M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway at P7. The cooperation between M1, A1 and A2A receptors promotes axonal loss at P9, whereas the effect of M2 is independent of the other receptors. M1 and TrkB receptors work together to increase axonal loss rate at P9 but the effect of M2 is largely independent of the TrkB receptor. In conclusion, postnatal synapse elimination is a regulated multireceptor mechanism involving the cooperation of several muscarinic, adenosine and TrkB receptor subtypes that guarantees the monoinnervation of the neuromuscular synapses in the end of the process.

In parasitic platyhelminths, including Schistosoma mansoni the coordination of neuromuscular system is critical for continued propagation, development and successful completion of the lifecycle. Neuromuscular signaling in these parasites is mediated by a variety of neurotransmitters, both small molecules ("classical") transmitters and neuropeptides. Biogenic amines (BA) constitute the largest subset of classical neurotransmitters and play several key roles in the control of schistosome muscle function and movement. There are several putative BA receptors identified in the S. mansoni genome, the majority of which are Class A G protein coupled receptors (GPCRs). Here we report the functional role of these putative BA receptors in parasite development and motility by developmental expression analysis and RNAi screening. We performed an expression analysis of several putative BA receptors at the RNA level in different developmental stages of the parasite, using reverse-transcription coupled to quantitative PCR (RT-qPCR). One of these proteins is a previously described serotonin receptor of S. mansoni (named Sm5HTR) and the others are novel "orphan" BA-like receptors. The analysis showed that the BA receptors tested are expressed in all developmental stages, however the majority are preferentially expressed in cercaria and schistosomula, suggesting these receptors play particularly important roles in parasite larvae. Next we performed RNA interference (RNAi) targeting the same BA receptors by transfecting S.mansoni larvae with small interfering RNA (siRNA) and analyzed for effects on motor activity by comparing with the control groups. Given that BAs are known modulators of schistosome movement, we hypothesized that the RNAi would produce a motor phenotype in the larvae and this was confirmed by the data. The results identified strongly hypoactive phenotypes for three out of four receptors tested, including Sm5HTR (Smp_126730), Smp_150180 and Smp_120620), all showing significant reduction in movement compared to control larvae transfected with irrelevant (scrambled) siRNAs. The RNAi phenotype correlated with a significant and specific knockdown in transcript levels as determined by RT-qPCR. To elucidate the mode of action of Sm5HTR we also performed confocal immunolocalization analysis using a specific peptide antibody. The expression pattern suggests Sm5HTR is highly abundant in the central and peripheral nervous system of the parasite, including the peripheral innervation of the body wall muscles responsible for movement. Together the results suggest that Sm5HTR and other BA receptors play an important role in the control of schistosome motility, particularly the larvae, and could be potential targets for new drug discovery.
En plathelminthes parasites, y compris Schistosoma mansoni la coordination de système neuromusculaire est essentiel pour continuer à se propager, le développement et la réussite du cycle de vie. Signalisation neuromusculaire chez ces parasites est médiée par une variété de neurotransmetteurs, les petites molécules (classique) des émetteurs et des neuropeptides. Les amines biogènes (BA) constituent le plus grand sous-ensemble de neurotransmetteurs classiques et jouent plusieurs rôles clés dans le contrôle de la fonction musculaire schistosome et le mouvement. Il RNA ya plusieurs récepteurs BA putatifs identifiés dans le génome de S. mansoni, dont la majorité sont des récepteurs couplés aux protéines de classe AG (GPCR). Nous rapportons ici le rôle fonctionnel de ces récepteurs putatifs de BA dans le développement du parasite et de la motilité par analyse de l'expression du développement et de dépistage RNAi. Nous avons effectué une analyse de l'expression de plusieurs récepteurs de BA putatifs au niveau de l' dans les différents stades de développement du parasite, en utilisant la transcription inverse couplée à une PCR quantitative (RT- qPCR). Une de ces protéines est un récepteur de la sérotonine décrit précédemment de S. mansoni (nommé Sm5HTR) et les autres sont de nouveaux récepteurs "orphelins" BA -like . L'analyse a montré que les récepteurs de la BA testés sont exprimés dans tous les stades de développement mais la majorité sont préférentiellement exprimé dans les cercaires et schistosomule, suggérant que ces récepteurs jouent un rôle particulièrement important dans les larves de parasite. Suivant nous avons effectué l'interférence RNA (RNAi) de cibler les mêmes récepteurs de BA par transfection larves S.mansoni avec petits RNA interférents (siRNA) et analysé les effets sur l'activité motrice par comparaison avec les groupes témoins. Étant donné que le BAs sont des modulateurs du mouvement schistosome connu, nous avons supposé que l' RNAi serait de produire un phénotype de moteur dans les larves et cela a été confirmé par les données. Les résultats identifiés phénotypes fortement hypoactif pour trois des quatre récepteurs testés, y compris Sm5HTR (Smp_126730), Smp_150180 et Smp_120620), tous montrant une réduction significative de mouvement par rapport à lutter contre les larves transfectées avec non pertinentes (brouillés) siRNA. Le phénotype RNAi corrélée avec un effet de choc important et spécifique dans les niveaux de transcription tel que déterminé par RT- qPCR. Pour élucider le mode d'action de Sm5HTR nous avons également effectué une analyse de immunolocalisation confocale en utilisant un anticorps anti- peptide spécifique. Le profil d'expression suggère Sm5HTR est très abondant dans le système nerveux central et périphérique du parasite, y compris l'innervation périphérique des muscles de la paroi du corps chargés de mouvement. Ensemble, les résultats suggèrent que Sm5HTR et d'autres récepteurs de la BA jouent un rôle important dans le contrôle de la motilité schistosome, en particulier les larves, et pourraient être des cibles potentielles pour la découverte de nouveaux médicaments.

Background: Dyspraxia, also included under the term Developmental Coordination Disorder (DCD), is a condition characterised by an impairment in motor skills function which impacts negatively on other aspects of daily living such as athletic capability, handwriting, self-esteem and social interaction. However, no effective therapy currently exists to address all of these issues within this group. The aim of the present study therefore was to investigate whether a complementary therapy, called Fascia Bowen therapy, would improve neuromuscular function and psychological wellbeing in males aged 8-11 (at Primary School) diagnosed with this condition. Methods: A group of 10 participants meeting the criteria of 15th centile or below in motor skills functioning, received a Fascia Bowen therapy treatment session from a qualified Fascia Bowen practitioner each week for 6 weeks. All participants’ motor skills function were assessed by an occupational therapist before and after the end of the intervention using the Motor Skills Assessment Battery for Children test (MABC-2). Additionally, parents, teachers and participants completed questionnaires measuring self-esteem, social skills, social interaction, behaviour and scholastic function before and after the intervention. Results: The participants showed significant improvement in neuromuscular function over time using the MABC-2. However, no significant changes were shown in the other measures of functioning. Although parents did provide some anecdotal reports about positive changes in real life, these were not reflected in the measures. The results suggest that while improvements were shown as significant in the motor domain, which was the focus of the therapy, the results did not translate to other domains of life over time. 13 Conclusions: Further research is necessary to test the efficacy of the treatment’s effects using a larger sample, a control group and a longer intervention timescale. A six week intervention period may not be sufficient to show significant changes in self-esteem, social skills, social interaction, behaviour and scholastic functions which have deep-rooted constructs developed over many years. These may therefore take a long time to change.

Although nestin has served as a marker of neural stem/progenitor cells for close to twenty years, its function is still poorly understood. During development, this intermediate filament protein is expressed in many different progenitors including those of the central nervous system, heart, skeletal muscle and kidney. The adult expression of nestin is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction and renal podocytes. I have used two approaches of gain of function and loss of function to elucidate the role of nestin in vivo. Although I was able to generate transgenic lines in which the transgene was ubiquitously expressed at the RNA level, over-expression of nestin at the protein level was not achieved possibly due to post transcriptional regulation of this gene. My data from loss of function approach indicates that nestin-deficient mice have impaired coordination. Balance and muscle strength are not affected and there are no apparent anatomical defects. I found that nestin deficiency is compatible with normal development of the central nervous system but results in abnormal clustering of acetylcholine receptors in the neuromuscular junctions, similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5) a candidate downstream effector of nestin. In renal podocytes, where both nestin and Cdk5 are normally expressed, we found reduced branching and abnormally contoured podocyte processes. To further connect the phenotype of nestin deficiency to Cdk5, I demonstrated that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knockouts, indicating that the observed nestin deficiency phenotypes are the consequence of aberrant Cdk5 activity.

RNA helicases are enzymes that bind or remodel RNA and RNA-protein complexes. They are involved in numerous cellular functions including RNA metabolism, transcription, translation, and mRNA decay. Defects in helicase function or disregulated expression, can cause diseases. DEAD-box (DDX) RNA helicases are highly conserved and are known to be involved in muscle development and disease, by interacting with muscle specific transcription factors and genes in humans. The Gupta Lab is currently studying zebrafish (an established and reliable model to study muscle diseases) with a mutation in ddx27. These fish have impaired motility behavior, skeletal muscle hypotrophy, and extensive central nucleation. They also exhibit disorganization of skeletal muscle, abnormalities in the brain, eyes, and heart. These phenotypes mimic the abnormalities seen in human myotonic dystrophy. It is known that ddx27 is necessary for regulation of rRNA maturation. Recent studies have pointed to it’s non-ribosomal roles of nucleolar genes. IGHMBP2, another RNA helicase, is known to result in spinal muscular atrophy (SMARD1) or Charcot-Marie Tooth disease when mutated. We used zebrafish and patient myoblast cells to determine the role of ddx27 in myogenesis and diseases. As a basis for future studies, the 43 known human DDX genes were outlined for their functions. Immunofluorescence studies in ddx27 mutant zebrafish showed drastic skeletal muscle and nucleolar assembly defects with large numbers of cells with transcriptionally active euchromatin, suggesting altered gene regulation. In addition, IF with Pax7 (a marker for satellite cells) and MF20 (a marker for myosin heavy chain antibodies) showed a significant increase in the number of Pax7 positive cells that suggest perturbed satellite cell regulation. Nucleolar defects were also seen in cells isolated from myotonic dystrophy patients. While the cause of these defects is not known, the results lead us to believe that ddx27 may be involved in cell cycle regulation or apoptosis events. Finally, while this study also attempted to develop a zebrafish model of IGHMBP2 deficiency in order to study and develop therapies for SMARD1, a consistent phenotype was not observed and further work is required to characterize this model. More than one million Americans suffer from neuromuscular disorders, however many of these conditions have no known treatments. By studying the molecular pathways involved we can attempt to develop therapies for these diseases.

碩士
國立臺灣大學
分子醫學研究所
99
In response to environmental and physiological stimulations or fluctuations, the synapse has to perform plasticity and also maintains homeostasis. Accumulating evidences support the role of the ubiquitin-proteasome system (UPS) in regulating synapse formation and remodeling, thus maintaining long-term neural circuit plasticity and homeostasis. Here we characterize the function of BTB-Kelch protein, the substrate receptor in the Cullin3 (Cul3)-organized ubiquitin E3 ligase, in the formation and neurotransmission of Drosophila neuromuscular junctions (NMJs). Mutant NMJs lacking henji activity show a dramatic increase in bouton number, including the appearance of numerous satellite boutons. Ultrastructurally, the electron-dense membrane area delineating the presynaptic active zone and the postsynaptic density (PSD) is expanded and the periactive zone is concurrently decreased in henji mutants. The PSD houses glutamate receptors (GluRs) IIA and IIB that show distinct transmissions at Drosophila NMJs. In henji mutants, the GluRIIA abundance is upregulated and the GluRIIB is downregulated. Our electrophysiological results also support a composition shift toward a higher GluRIIA/IIB ratio at henji mutant NMJs. By rescue experiments, we show that Henji acts in the postsynapse to regulate proper NMJ growth and GluRIIA/IIB composition. We further show that Henji controls NMJ growth and GluRIIA/IIB ratio by downregulation of dPak at PSDs. The postsynaptic dPAK marks the PSD area and regulates the GluRIIA abundance. Losing one copy of dpak suppressed the bouton phenotype and GluRIIA abundance in the henji mutant. Also, the intensity and area of dPAK punctates at PSDs were increased in henji mutants. We found that dPAK interacts with Henji, which promotes ubiquitination and degradation of dPak. Therefore, Henji acts at PSDs to restrict both the presynaptic bouton growth and the postsynaptic GluRIIA clustering via controlling dPAK protein level. Several questions remain to be addressed: the subcellular localization of Henji; how dPAK is regulated by Henji during its activation; and how the GluRIIA/IIB balance is controlled by Henji-regulated dPak levels. Some preliminary results and future works will be presented in the thesis. In addition, we also addressed the role of integrin betanu in restricting NMJ growth. In the loss of betanu activity, drastic increase in bouton number was observed. Further, we dissected the downstream signaling of betanu/FAK56 and reported a bifurcating cascade of NF1-regulated cAMP/PKA and Vap-mediated Ras/MAPK pathways.

Drosophila melanogaster is an excellent model system for studying the molecular mechanisms of neuronal development and function, since many of the basic components and processes within the nervous system are highly conserved between Drosophila and vertebrates. Thus, characterization of genetic and environmental factors affecting the nervous system of Drosophila may provide valuable information about the the nervous system of vertebrates, possibly leading to treatments for neuronal injury or neurodegenerative diseases in humans. In an effort to identify novel genes involved in synaptic transmission, a transposable element mutagenesis procedure was initiated. Two potential genes were identified and partially characterized, but neither were cloned. In addition, previously identified factors affecting neuronal development and function, including genetic components, like calmodulin and kinesin, and environmental factors, like gravitational forces were examined. Calmodulin is a ubiquitous, Ca$\sp{2+}$-binding protein expressed at high levels in the developing nervous system of Drosophila. Behavioral, physiological, and morphological studies of a heteroallelic combination at the Calmodulin (Cam) locus, denoted Cam$\sp{3cl}$/Cam$\sp{null},$ revealed several defects. Cam$\sp{3cl}$/Cam$\sp{null}$ adults exhibit abnormal behavior. Third instar larvae of this genotype exhibit increased sensitivity to the K$\sp{+}$ channel blocking drug quinidine at low Ca$\sp{2+}$ concentrations and a striking abnormality in the structure of the synaptic junction in longitudinal muscle 13. There are several possible targets for this heteroallelic combination, including GAP-43-1ike proteins, Ca$\sp{2+}$/Calmodulin-dependent kinases and phosphatases, and ion channels. Kinesin is an anterograde motor protein distributed throughout the axonal processes of Drosophila. Mutations in the kinesin heavy chain gene interacted with mutations in the Shaker K$\sp{+}$ channel gene and with the application of the K$\sp{+}$ channel blocking drug quinidine to produce both synergistic and antagonistic phenotypes. These results suggest that kinesin is responsible for the transport of several factors, possibly including Na$\sp{+}$ channels, K$\sp{+}$ channels, and their regulators. Gravitational forces affect the development and function of the nervous system in several organisms, including Drosophila. However, preliminary studies revealed no gross abnormalities in neuronal development or function in wildtype Drosophila exposed to 8 x g during various stages of embryogenesis.

碩士
中山醫學大學
物理治療學系碩士班
104
Background: The connection between core stability and lower extremity injury and lower back pain has been reported.Currently, most assessment methods are used to test muscle endurance and core stability.However,because the activities of athletes tend to exhibit dynamic patterns, muscle strength, muscle power, and neuromuscular control are highly vital components of core stability; therefore, an assessment system that considers multiple components should be established for athletes. Purpose: The aim of this study was to develop acore stability assessment technique for athletes and prove its reliability and applicability in discriminating the core stability conditions among different subjects. Method: This study recruited 20 student athletes who had undergone formal training for more than 5 years.An electromyographic and force platform were used to record data whilethe athletes executed thesecore stability exercise, and their center of pressure (COP) were assessed. Result: Analysis results showed that when the athletes performed the Bird Dog exercise with the right hand and left leg raised, both the COP path length and COP path area demonstrated a positive moderate correlation. Conclusion: Only theBird Dog exercise with the right hand raisedinvolves a correlation between the COP and electromyographic signals, but the difference in core stability among the athletes can be determined because of the significant difference existon core stability movementswas found.

Hormonally regulated cell death is thought to underlie many sex differences in the nervous system, but little is known about the molecular mechanisms involved. This question was investigated in a sexually dimorphic neuromuscular system in mice. Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) innervate striated perineal muscles including the bulbocavernosus (BC) and levator ani (LA). In rats, SNB cell number and BC/LA muscle size are similar in males and females prenatally, but the postnatal persistence of this neuromuscular system is androgen dependent. Androgens reduce cell death of SNB motoneurons during a perinatal critical period and, as a result, males have more SNB motoneurons than do females in adulthood. The Bcl-2 family of proteins, which includes anti-apoptotic (e.g., Bcl-2) and pro-apoptotic (e.g., Bax, Bak) family members, regulates cell death in neurons and other tissues. I found that Bax is essential for the normal sex difference in SNB motoneuron number in adult mice. Experiments to characterize the time course of developmental cell death in the SNB of mice proved inconclusive based on counts of retrogradely-labeled SNB motoneurons and pyknotic cells in the SNB region, but suggest that developmental cell death may overlap with a period of late secondary SNB migration. Further, SNB motoneuron location differs between perinatal male and female mice. It's controversial whether the sex difference in perineal muscle size is due to sexually dimorphic cell death or to androgen dependent growth, especially of the LA. We find that Bax deletion slightly increased LA fiber number in adult females, but did not eliminate the gross sex difference in perineal muscle size. Modest effects of Bax deletion on the perineal muscles in adulthood may result from functional redundancy between Bax and Bak. In confirmation, I found that Bax/Bak DKO females have a more than 10-fold increase in LA fiber number over that in wild type females. In addition, wild type females have a higher density of TUNEL-positive cells than do males in both the BC and the LA during perinatal life. Thus, cell death makes an important contribution to the sexually dimorphic development of the BC and the LA.

abstract: The human shoulder plays an integral role in upper limb motor function. As the basis of arm motion, its performance is vital to the accomplishment of daily tasks. Impaired motor control, as a result of stroke or other disease, can cause errors in shoulder position to accumulate and propagate to the entire arm. This is why it is a highlight of concern for clinicians and why it is an important point of study. One of the primary causes of impaired shoulder motor control is abnormal mechanical joint impedance, which can be modeled as a 2nd order system consisting of mass, spring and damper. Quantifying shoulder stiffness and damping between healthy and impaired subjects could help improve our collective understanding of how many different neuromuscular diseases impact arm performance. This improved understanding could even lead to better rehabilitation protocols for conditions such as stroke through better identification and targeting of damping dependent spasticity and stiffness dependent hypertonicity. Despite its importance, there is a fundamental knowledge gap in the understanding of shoulder impedance, mainly due to a lack of appropriate characterization tools. Therefore, in order to better quantify shoulder stiffness and damping, a novel low-inertia shoulder exoskeleton is introduced in this work. The device was developed using a newly pioneered parallel actuated robot architecture specifically designed to interface with complex biological joints like the shoulder, hip, wrist and ankle. In addition to presenting the kinematics and dynamics of the shoulder exoskeleton, a series of validation experiments are performed on a human shoulder mock-up to quantify its ability to estimate known impedance properties. Finally, some preliminary data from human experiments is provided to demonstrate the device’s ability to collect the measurements needed to estimate shoulder stiffness and damping while worn by a subject.
Dissertation/Thesis
Doctoral Dissertation Mechanical Engineering 2020

The nervous system is made up of specialized cells which receive and respond to environmental stimuli. Intercellular communication in the nervous system is achieved predominantly through chemical synaptic transmission. Within the chemical synapse, the actin cytoskeleton plays a major role in regulating synaptic activities, although the extent and clarity in our understanding of these processes is still limited. Using the genetically pliable model, Drosophila melanogaster, this thesis begins to unravel contributions of actin binding proteins to synaptic development and physiology at the larval neuromuscular junction (NMJ). Two actin binding proteins, Moesin and Nonmuscle Myosin II (NMMII) were selected for study based on previous studies implicating them in synaptic development. Combining genetics, fluorescent imaging and electrophysiological recordings this thesis unveils previously unidentified functions for Moesin and NMMII in morphology and physiology of the Drosophila NMJ. Moesin was found to help restrain synaptic growth but did not affect synaptic physiology. By correlating morphological and electrophysiological measurements in Moesin mutants, it was determined that physiology and morphology can be independently regulated at the NMJ. NMMII was used to investigate a role for actin binding proteins in physiology at the Drosophila NMJ. By using the fluorescent imaging technique, FRAP, this becomes the first research to implicate NMMII in unstimulated synaptic vesicle mobility. FRAP indicated that vesicle mobility was highly dependent on the expression level of NMMII. Electrophysiological analysis of NMMII indicated distinct mechanisms for spontaneous and evoked vesicle release. NMMII expression exhibited a positive correlation with basal synaptic transmission and was important in mobilizing vesicles for synaptic potentiation. In addition, NMMII was found to be involved in a high frequency dependent low frequency depression. This work begins to identify how vesicles traverse within boutons and suggests differential mechanisms of synaptic release, both of which are partially dependent of NMMII expression. Studying Moesin and NMMII have revealed a complex interplay between the actin cytoskeleton and synaptic function and together this research furthers our understanding of how the actin cytoskeleton regulates synaptic activity.

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
O desenvolvimento do músculo, ou miogénese, é um processo bastante conservado entre os vertebrados. Todos os músculos-esqueléticos do tronco e dos membros são provenientes dos sómitos, estruturas epiteliais que se formam em ambos os lados do tubo neural. Os sómitos são posteriormente padronizados em diferentes compartimentos que darão origem a diferentes linhagens celulares. A porção mais ventral do sómito perde a sua estrutura epitelial e forma o esclerótomo, fonte de células precursoras do esqueleto axial. A porção mais dorsal, o dermamiótomo, permanece epitelial e é constituído pelos precursores miogénicos (MPCs) e os percursores da derme, entre outros. Os músculos-esqueléticos iniciam o seu desenvolvimento quando os progenitores no dermamiótomo, que expressam os factores de transcrição Pax3 e/ou Pax7 são induzidos a activar o programa de diferenciação miogénica, controlado pelos factores regulatórios da miogénese (MRF), nomeadamente Myf5, MyoD, Mrf4 e Miogenina. O dermomiótomo encontra-se dividido em três compartimentos distintos: (1) dermomiótomo dorsomediano (2) dermomiótomo central e (3) dermomiótomo ventrolateral. O desenvolvimento dos músculos epaxiais inicia-se no ratinho por volta de E8.5 com a formação do miótomo através da adição das células do dermamiótomo quando estas delaminam do dermomiótomo e povoam a zona ventral ao dermamiótomo para constituir o miótomo. As células precursoras musculares no dermamiótomo, ou células musculares estaminais, que passam a expressar os MRFs, entram no miótomo como mioblastos, mas no miótomo acabam por diferenciar-se em miócitos. O miótomo cresce nos estádios subsequentes com a adição progressiva de células estaminais musculares que diferenciam. Com o início da dissociação do dermamiótomo a E10.5, os progenitores que não se diferenciam acabam por migrar para as massas constituídas por miócitos. Entre E11.5 e E14.5, alguns destes progenitores diferenciam-se em mioblastos primários que fundem com os miócitos para formar as fibras primárias- miogénese primária. Durante os estádios subsequentes até ao nascimento, outra porção de células estaminais que se diferencia em mioblastos, desta vez secundários, que se fundem entre si para formas as fibras secundárias, mas também fundem com as fibras primárias. Esta fase é responsável pelo aumento do tamanho das massas musculares, quer em número de fibras quer no tamanho das mesmas. O sistema de inervação do músculo, mais especificamente a formação das junções neuromusculares (NMJs), sinapses especializadas que se formam entre o músculo e o nervo, desenvolve-se em paralelo com a miogénese. O primeiro contacto entre músculo e nervo antecede o início da miogénese secundária. Por esta altura, já existe uma pré-padronização da distribuição dos receptores de acetilcolina (AChR) no músculo, que será posteriormente remodelada. Dado que a miogénese e a inervação são processos interdependentes para o correcto funcionamento do músculo, estes processos requerem uma comunicação estruturada entre o músculo e o nervo. Durante a miogénese secundária (por volta de E16.5) as células musculares estaminais, positivas para Pax7, migram para o espaço existente entre a fibra muscular e a membrana basal. Esta localização é mantida pelas células estaminais musculares que não se diferenciam durante a miogénese in utero e que constituem a população de células satélite, as células estaminais musculares adultas. Dado que estas se encontram em contacto directo com a membrana basal, a matriz extracelular adquire um papel crucial na regulação do comportamento destas células. Os diferentes elementos que constituem a membrana basal, tais como colagénio, perlecano e laminina permitem que as células estaminais musculares reconheçam o microambiente que as envolve. Além do microambiente que providencia à fibra e às células estaminais musculares durante o desenvolvimento do músculo esquelético, a membrana basal é um componente essencial no desenvolvimento das NMJs. De entre os vários componentes da membrana basal, as lamininas são dos componentes mais bem estudados. As lamininas são trímeros, que apresentam uma estrutura cruciforme ou em T com três cadeias: alpha (α), beta (β) e gamma (γ). Actualmente são conhecidas 16 isoformas diferentes denominadas com base na sua constituição. Por exemplo, a laminina 211 é constituída pelas cadeias α2, β1 e γ1. As lamininas ligam-se principalmente a dois tipos de receptores no músculo: (1) integrinas, receptores transmembranar compostos por duas sub-unidades alpha (α) e beta (β); (2) distroglicano, que se liga intracelularmente à distrofina. Durante a miogénese secundária, as principais isoformas presentes no músculo e nas NMJ são, respectivamente, 211, 411, 511 e 221, 421 e 521. Porém, no músculo adulto, a isoforma que permanece a volta das miofibras é a 211, enquanto nas NMJs adultas continuam presentes as isoformas 221, 421, 521, todas elas cruciais para o desenvolvimento e correcto funcionamento do sistema neuromuscular. As lamininas são determinantes desde cedo no desenvolvimento do músculo-esquelético durante a formação do miótomo através do controlo do balanço entre proliferação e diferenciação das células do dermamiótomo. Em estádios mais tardios do desenvolvimento fetal, as lamininas são parte integrante do microambiente das fibras e das células estaminais musculares que parece ser determinante para o crescimento normal das massas musculares. Em paralelo, as lamininas desempenham papel igualmente preponderante durante o desenvolvimento das junções neuromusculares As células estaminais musculares localizadas entre a membrana basal e a fibra representam no músculo adulto a principal fonte da capacidade regenerativa. Para que a reserva de células estaminais musculares não se esgote é necessário garantir que exista um equilíbrio entre a proporção de células que se mantêm quiescentes, as células que são activadas e as células que se diferenciam no momento da regeneração. A membrana basal que hospeda estas células representa um elemento determinante em distintas vias de sinalização que operam no sentido de instruir as células a manterem-se quiescentes, a activar, a proliferar ou diferenciar. A sinalização Notch destaca-se como reguladora deste processo. Quando abolida, as células estaminais musculares diferenciam-se precocemente sem a necessária proliferação que permite manter a população e desta forma a população acaba por esgotar-se. A Distrofia muscular congénita merosina negativa (MDC1A) é um tipo de distrofia causado por mutações no gene LAMA2 que levam à perda das lamininas 211 e 221 da membrana basal das fibras e junções neuromusculares, respectivamente. Esta doença é caracterizada por fraqueza muscular, neuropatia, dificuldades respiratórias, entre outros sintomas. Neste estudo, usámos o modelo de ratinho dyW como modelo de estudo para a MDC1A. Estudos recentes do nosso laboratório demonstraram que no ratinho, o desenvolvimento da MDC1A inicia-se in utero entre E17.5 e E18.5. O início desta distrofia é demarcado por uma diminuição significativa no número de células positivas para Pax7, em paralelo com uma diminuição do crescimento do músculo fetal. O trabalho realizado nesta tese teve como objectivo compreender melhor como é que as células musculares constroiém o seu microambiente e de que forma alterações no microambiente tanto das células musculares como das junções neuromusculares influencia o crescimento do músculo fetal. Numa primeira abordagem, avaliámos a capacidade das células musculares, tanto as células estaminais musculares como as fibras, de produzirem e montarem as matrizes de laminina. Os nossos resultados demonstram que numa fase inicial da miogénese secundária, as células estaminais musculares são as principais produtoras de laminina no músculo e montam as suas matrizes de laminina mesmo na ausência das fibras. Durante fases mais tardias da miogénese secundária, as fibras passam a expressar os diferentes genes de laminina e a sua presença parece ser importante para que as matrizes de laminina sejam mantidas no microambiente das células estaminais musculares. Desta forma, este trabalho revela que as células musculares desempenham papéis diferentes na construção das matrizes de laminina em fases distintas da miogénese secundária e que as células estaminais e as fibras são interdependentes na construção das matrizes de laminina. Numa segunda abordagem, esta tese teve como objectivo compreender em maior detalhe o papel da laminina 221 durante a formação das NMJs e compreeender a sua influência no início/progressão da MDC1A. Para tal, estudámos o desenvolvimento das NMJs durante a miogénese secundária em ratinhos dyW. Os nossos resultados revelam que enquanto as lamininas α2 e α4 não aparentam ter um contacto directo com sinapse a E15.5, as lamininas α5 apresentam uma proximidade com a sinapse. Esta dinâmica não parece estar alterada na ausência da laminina α2 (211/221). Contudo, a nossa análise do desenvolvimento das NMJ na ausência de laminina 221, ainda que preliminar, sugere que a distribuição dos receptores de acetilcolina está alterada e que há uma tendência para que os receptores se encontrem mais dispersos ao longo do músculo na ausência de lamininas α2. Estes resultados apontam para um papel das lamininas na agregação dos receptores junto da fenda sináptica. Em suma, o trabalho desenvolvido ao longo desta tese realça a complexidade das dinâmicas de produção e construção das matrizes de laminina durante a miogénese secundária. Os dados desta tese exemplificam igualmente a diversidade de microambientes aos quais as células estaminais estão sujeitas durante diferentes fases da miogénse secundária. Esta tese analisa em particular o papel das lamininas α2 durante o desenvolvimento das NMJs e fornece novas evidências acerca da influência da inervação do músculo no início da MDC1A.
MDC1A is a crippling neuromuscular disease caused by the absence of the α2-chain of laminins 211/221, major components of basement membranes. The onset of this disease during development in utero is marked by impaired muscle growth which correlates with a reduction in the number of mononucleated muscle cells in the fetal muscle masses (Nunes et al., 2017). Skeletal muscle development starts during early embryogenesis, when the dermomyotomal Pax3- and/or Pax7-positive muscle precursors cells are induced to enter the myogenic program and subsequently delaminate from the dermomyotome to form the myotome. Later on, when the dermomyotome dissociates, Pax3- and/or Pax7-positive muscle stem cells are released, some of which differentiate into myoblasts and fuse with myotomal cells or with each other, forming the primary myofibers during primary myogenesis that occurs between E11.5 and E14.5. The primary myofibers later serve as a scaffold for the formation of secondary myofibers and secondary myoblasts fuse with both primary and secondary myofibers to increase their size. Motor axons enter the muscle masses in parallel with primary myogenesis, but it is during secondary myogenesis (between E14.5 until birth) that the nerve contacts the muscle, and proper innervation is essential for normal fetal muscle development. During mid-secondary myogenesis, the Pax7-positive muscle stem cells become closely associated with the myofibers and their basement membrane. Laminin 211 and 221 are assembled around the adult myofiber and synaptic endplate, respectively, and are known to play important roles both in myofiber and neuromuscular junction development. In this thesis we aimed to contribute to the study of the fetal myogenesis defect in dyW mice in two ways: First, we asked what cell types produce laminins during fetal myogenesis. We performed a detailed analysis of laminin production and assembly in fetal muscles at stages preceding the onset of MDC1A. We found that mononucleated cells, including Pax7-positive cells, are a major source of laminins at the beginning of secondary myogenesis, but during later stages of secondary myogenesis, myofibers also express laminin genes. This suggests that Pax7-positive muscle stem cells play a major role in constructing the laminin microenvironment in the fetal muscles. We then used the Myf5cre-NICD mouse model (Mourikis et al., 2012) where Pax7-positive cells are unable to differentiate and thus do not form muscle fibers. We found that Pax7-positive cells at E14.5 produce and assemble laminins 211, 411 and 511, but at E17.5, the capacity to produce laminins is greatly diminished as they only produce laminin 511. These results indicate that Pax7-positive cells at E17.5 require the presence of myofibers to produce and assemble laminins 211 and 411. Second, since it is known that fetal myogenesis depends on innervation, we used the dyW mouse model, which has a mutation in the Lama2 gene, to assess the role of laminin 211/221 during neuromuscular junction development. We found that laminin 521 is closely assembled around the synapse, while laminins 421 is present, but does not seem to be in direct contact with the synapse. In the wildtype, laminin 221 has a distribution like laminin 421. However, our spatial analysis showed that α2-laminin deficient synapses tend to have a less clustered organization compared to wildtype ones and display a deficient AChR patterning. Based on these results, we hypothesize that laminin 221 might play a crucial role in NMJ development and that this may contribute to the onset of the fetal myogenesis defect in dyW mice. Together, our results provide new insights into laminin production and assembly during fetal muscle development and provide new indications into the mechanism underlying disease onset during development in utero in a mouse model for MDC1A.