Dissertation submitted in partial compliance with the requirements for the Masterà ¢ s Degree in Technology: Chiropractic, Durban University of Technology, 2008. / Objectives: To determine the short-term effect of an inhaled, short-acting 2-agonist bronchodilator on chest wall expansion (cm) and lung function parameters (FEV1, FVC and FEV1/FVC%) in chronic moderate asthmatics. To determine the short-term effect of spinal manipulation (SMT) and ribcage mobilisation on chest wall expansion (cm) and lung function parameters (FEV1, FVC and FEV1/FVC%) in chronic moderate asthmatics. To determine the short-term effect of a combination of SMT, ribcage mobilisation and an inhaled, short-acting 2-agonist bronchodilator on chest wall expansion (cm) and lung function parameters (FEV1, FVC and FEV1/FVC%) in chronic moderate asthmatics. Methods: Forty-five chronic moderate asthmatics who met all the inclusion criteria of the study were divided into three groups of fifteen each. Group A received a short-acting 2-agonist bronchodilator, Group B received SMT and ribcage mobilisation and Group C received a combination of SMT, ribcage mobilisation and a short-acting 2-agonist bronchodilator. Baseline measurements and testing included chest wall expansion and the lung function parameters FEV1, FVC and FEV1/FVC%. These measurements were repeated 15 minutes post-intervention. Data was analyzed using SPSS version 15.0. Results: There were no statistically significant changes between pre- and post-intervention in the short-acting 2-agonist bronchodilator group with respect to any of the chest wall expansion measurements. There was a statisticallly significant increase in FEV1 between pre- and post-intervention in the short-acting 2-agonist bronchodilator group (p = 0.008). There was a statistically significant increase in the mean pre- and post-intervention axillary chest wall expansion (p = 0.014) as well as the mean of the half-way measurement (p = 0.014) and the overall mean chest wall expansion value (p = 0.001) following SMT and ribcage mobilisation. There were no statistically significant changes in any of the lung function parameter values following SMT and ribcage mobilisation. There was a significant increase for the half-way measurement in chest wall expansion (p = 0.018) in the combination of SMT, ribcage mobilisation and the inhaled, short-acting 2-agonist bronchodilator group. There were no statistically significant changes in any of the lung function parameter values in the combination of SMT, ribcage mobilisation and an inhaled, short-acting 2-agonist bronchodilator. For FEV1, the effect in the short-acting 2-agonist bronchodilator group vs. the SMT and ribcage mobilisation group was statistically significant (p = 0.018). There was no statistical difference in any of the chest wall expansion measurements and FVC and FEV1/FVC% parameters between all three groups. Conclusions The results did not point specifically to one intervention over another for all outcomes. SMT and rib mobilisation had no effect on the lung function parameters, at least in the short term. There was a statisticallly significant increase in FEV1 between pre- and post-intervention in the short-acting 2-agonist bronchodilator group.

Chronic hypersensitive pain states can become established following sustained, repeated or earlier noxious stimuli and are notably difficult to treat, especially in cases where nerve injury contributes to the trauma. A key underlying reason is that a variety of plastic changes occur in the central nervous system (CNS) at spinal and potentially also supraspinal levels to upregulate functional activity in pain processing pathways. A major component of these changes is the enhanced function of excitatory amino acid receptors and related signalling pathways. Here we utilised rodent models of neuropathic and inflammatory pain to investigate whether evidence could be found for lasting hypersensitivity following neonatal (or adult) noxious stimuli, in terms of programming hyper-responsiveness to subsequent noxious stimuli, and whether we could identify underlying biochemical mechanisms. We found that neonatal (postnatal day 8, P8) nerve injury induced either long lasting mechanical allodynia or shorter lasting allodynia that nonetheless was associated with hyper-responsiveness to a subsequent noxious formalin stimulus at P42 despite recovery of normal mechanical thresholds. By developing a new micro-scale method for preparation of postsynaptic densities (PSD) from appropriate spinal cord quadrants we were able to show increased formalin-induced trafficking of GluA1- containing AMPA receptors into the PSD of animals that had received (and apparently recovered from) nerve injury at P8. This was associated with increased activation of ERK MAP kinase (a known mediator of GluA1 translocation) and increased expression of the ERK pathway regulator, Sos-1. Synaptic insertion of GluA1, as well as its interaction with a key partner protein 4.1N, was also seen in adults during a nerve injury-induced hypersensitive pain state. Further experiments were carried out to develop and optimise a new technological platform enabling fluorometric assessment of Ca2+ and membrane potential responses of acutely isolated CNS tissue; 30-100 μm tissue segments, synaptoneurosomes (synaptic entities comprising sealed and apposed pre- and postsynaptic elements) and 150 × 150 μm microslices. After extensive trials, specialised conditions were found that produced viable preparations, which could consistently deliver dynamic functional responses. Responsiveness of these new preparations to metabotropic and ionotropic receptor stimuli as well as nociceptive afferent stimulant agents was characterised in frontal cortex and spinal cord. These studies have provided new opportunities for assessment of plasticity in pain processing (and other) pathways in the CNS at the interface of in vivo and in vitro techniques. They allow for the first time, valuable approaches such as microscale measurement of synaptic insertion of GluA1 AMPA receptor subunits and ex vivo assessment of dynamic receptor-mediated Ca2+ and membrane potential responses.

In the field of spinal-cord neurophysiology, the nature of and the rules which govern the strength of functional connections between muscle afferents and motoneurons supplying the same muscle are important to delineate. This study addressed a facet of this issue by testing the possibility that the strength of the spindle Ia-motoneuronal connections is stronger (as demonstrated by the differing amplitudes of the mean maximum composite Ia EPSPs) if both neurons supply the same sub-volume of the muscle, providing the various sub-volumes of the muscle are capable of independent action. Intracellular recordings were made of the Ia EPSP responses of semimembranosus (SM) and lateral gastrocnemius (LG) motoneurons in anesthetized low-spinal cats to electrical stimulation (Group I range) of nerve branches supplying different parts of the homonymous muscle, as well as different heteronymous muscles. For study of SM motoneurons, stimulated nerve branches included those supplying the anterior (SMa) and posterior (SMp) heads of the SM muscle and three providing heteronymous input from the anterior (BFa) and posterior (BFp) parts of biceps femoris and the distal part of the semitendinosus (STd) muscle. Ia EPSPs were partitioned such that stimulation of the SMa nerve branch produced significantly larger EPSPs in SMa motoneurons than in SMp cells; likewise, stimulation of the SMp nerve branch produced larger EPSPs in SMp motoneurons than in SMa cells. Study of the differences in the strength of heteronymous Ia input (i.e., from BFa, BFp and STd) between the SMa and SMp cell groups correlates with the different actions reported previously for the two heads of the SM muscle. For study of LG motoneurons, the stimulated nerve branches were those supplying the four neuromuscular compartments of the LG muscle (LG1, LG2, LG3 and LGm) and the nerve to a heteronymous muscle, soleus (SOL). In all five instances, partitioned Ia effects were evident. An association is suggested between the present results and previous electromyographic studies. The previous studies have shown that the muscle heads (SM) or neuromuscular compartments (LG) under consideration in this study are capable of somewhat separate actions. The present study also included assessment of the relative extent to which the partitioned Ia effects could be attributed, in part, to one or two developmental factors, topographic specificity and species specificity. The analysis suggested that both factors were potentially implicated, with species specificity somewhat predominant over topographic specificity.

Salamanders have a remarkable ability to regenerate complex body parts including the limb, tail, and central nervous system. Although salamander regeneration has been studied for several hundred years, molecular-level studies have been limited to a relatively few important transcription factors and signaling molecules that are highly conserved among animals. Physiological genomic approaches were used here to investigate spinal cord and limb regeneration. Chapter 2 reports that hundreds of gene expression changes were identified during spinal cord regeneration, showing that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. Chapter 3 presents results that identify the salamander ortholog of mammalian Nogo-A, a gene known to inhibit mammalian nerve axon regeneration. Nogo-A gene expression was characterized during salamander development and adulthood in order to address the roles of Nogo-A in the nervous system. Chapters 4 and 5 use physiological genomic approaches to examine limb regeneration and why this process is dependent upon an intact nerve supply. Results presented in Chapter 4 showed that many processes regulated during early limb regeneration do not depend upon nerve-derived factors, but striking differences arise between innervated and denervated limbs by 14 days after amputation. Chapter 5 identified genes associated with peripheral nerve axon regeneration and identified gene candidates that may be secreted by nerves to support limb regeneration. Lastly, chapter 6 characterizes the expression of a developmentally important family of genes, matrix metalloproteinases, during tail regeneration. These results suggest that matrix metalloproteinases play multiple roles throughout the regeneration process. Primarily, this dissertation presents data from the first genomic studies of salamander regeneration. The results suggest genes such as matrix metalloproteinases, and molecular pathways such as the Wnt and FGF signaling pathways that can be exploited to enhance regenerative ability in humans.

Lower motor neuron diseases (LMNDs) include a large spectrum of clinically and genetically heterogeneous disorders, characterized by progressive anterior horn cell degeneration. The aims of this thesis were on the one hand to refine the phenotypic description and the clinical classification of hereditary LMNDs, and on the other hand to improve our knowledge of the genetic bases of these disorders. This work was performed in collaboration with the Centre of Human Genetics of the Necker-Enfants-Malades Hospital in Paris. We focused our researches on autosomal recessive variants of LMNDs. First, we selected patients with Spinal Muscular Atrophy with Respiratory Distress (SMARD or d-HMN VI). This severe variant of autosomal recessive LMND is characterized by neurogenic muscular atrophy associated with early life-threatening respiratory failure due to diaphragmatic dysfunction. SMARD type 1 has been ascribed to mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene on chromosome 11q13-q21. We reported the identification of 9 novel IGHMBP2 mutations in five SMARD1 patients, Seven of them occurred at highly conserved residues of the putative DNA helicase domain, suggesting that this particular domain plays a major role in the SMARD1 disease causing mechanism (Hum Mutat. 2004; 23(5):525-6). Then, we collected families and sporadic patients affected by chronic distal spinal muscular atrophy (d-HMN III/IV), an autosomal recessive variant of LMND characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of chronic dSMA gene had been mapped to a 10.3 cM interval on chromosome 11q13. By linkage analysis in 12 European chronic dSMA families, we reduced the genetic interval to a 2.6cM region on chromosome 11q13.3 and showed partial linkage disequilibrium between 3 rare alleles and the mutant chromosome in European patients, suggesting that most chronic dSMA chromosomes are derived from a single ancestor (Eur J Hum Genet. 2004;12(6):483-8). Additional experiments are now in progress at Necker-Enfants-Malades Hospital, in order to identify the disease-causing gene. At last, we described the clinical features of a novel variant of autosomal recessive LMND, characterized by childhood onset, generalized muscle involvement, and severe outcome. Studying a large inbred African family, we mapped the disease gene to a 3.9-cM interval on chromosome 1p36 (Neurology.2006;67(1):120-4). We identified a homozygous missense mutation of the PLEKHG5 gene and performed in vitro experiments to clarify the pathogenic function of this mutation. In transiently transfected HEK293 and MCF10A cell lines, we found that wild-type PLEKHG5 activated the NFkB signaling pathway and that both the stability and the intracellular location of mutant PLEKHG5 protein were altered, severely impairing the NFkB transduction pathway. Moreover, we observed aggregates in transiently transfected NSC34 murine motor neurons overexpressing the mutant PLEKHG5 protein. In conclusion, we showed that both loss of PLEKHG5 function and aggregate formation might contribute to neurotoxicity in this novel form of LMND (Am J Hum Genet. 2007;81(1):67-76). Further experiments should now be planned, in particular to understand the role of aggregates in neurodegeneration, and to precise the links between the PLEKHG5 gene and the other LMNDs-causing genes. In conclusion, we hope that this work, contributing to a better understanding of the molecular mechanisms involved in motor neuron degeneration, will open the way to new therapeutic strategies. / Les amyotrophies spinales (SMA) se caractérisent par une dégénérescence des motoneurones des cornes antérieures de la moelle épinière ou des noyaux du tronc cérébral. Elles comprennent de nombreuses entités, très hétérogènes tant sur le plan clinique que sur le plan génétique. Cette thèse vise à préciser les caractéristiques phénotypiques et génétiques des différentes formes d’amyotrophie spinale, en particulier celles des variants de transmission autosomique récessive. En premier lieu, nous avons sélectionné une cohorte de patients dont le tableau clinique était compatible avec la variante SMARD d’amyotrophie spinale (pour Spinal Muscular Atrophy with Respiratory Distress). Il s’agit d’une forme très sévère d’amyotrophie spinale autosomique récessive, qui associe une faiblesse musculaire à prédominance distale et une détresse respiratoire précoce secondaire à une paralysie diaphragmatique. Nous avons décrit 9 nouvelles mutations au sein du gène IGHMBP2 (pour immunoglobulin µ-binding protein 2) chez 5 patients atteints et confirmé ainsi le rôle pathogène de ce gène. Sept des 9 mutations décrites concernent des acides aminés conservés dans les espèces et localisés dans le domaine hélicase. Ce domaine pourrait donc jouer un rôle essentiel dans la physiopathologie de la maladie (Hum Mutat. 2004; 23(5):525-6). Deuxièmement, nous avons rassemblé des patients atteints d’amyotrophie spinale chronique distale (d-HMN III/IV), de transmission autosomique récessive. Cette affection se définit par une amyotrophie et une faiblesse musculaire progressive qui prédomine au niveau des pieds et des mains. Grâce à des analyses de liaison réalisées dans 12 familles européennes, nous avons restreint la localisation génétique sur le chromosome 11 (en 11q13.3) à un intervalle de 2.6 cM. Nous avons également mis en évidence un déséquilibre de liaison entre 3 allèles rares et le locus génétique, ce qui suggère un phénomène d’effet fondateur dans la population caucasienne (Eur J Hum Genet. 2004;12(6):483-8). Des études complémentaires sont actuellement en cours à l’Hôpital Necker-Enfants-malades à Paris pour identifier le gène responsable de cette forme chronique d’amyotrophie spinale distale. Troisièment, nous avons décrit un nouveau variant d’amyotrophie spinale de transmission autosomique récessive. L’amyotrophie et la faiblesse musculaire débutent vers l’âge de 3 ans et concernent rapidement l’ensemble de la musculature. Le pronostic est sévère, avec perte de la marche durant l’enfance et altération de l’autonomie respiratoire à l’adolescence. L’étude d’une grande famille Malienne consanguine nous a permis de localiser le gène responsable de la maladie dans un intervalle de 3.9 cM sur le chromosome 1, en 1p36 (Neurology. 2006;67(1):120-4). Nous avons ensuite mis en évidence une mutation faux-sens à l’état homozygote dans le gène PLEKHG5 chez les patients atteints et avons prouvé le caractère pathogène de cette mutation grâce à une série d’études fonctionnelles. Nous avons montré que la protéine PLEKHG5 sauvage a une distribution cytoplasmique homogène dans des cellules rénales (HEK293) et mammaires (MCF10A) humaines transfectées et qu’elle y active la voie de signalisation NF-kappaB. La protéine PLEKHG5 mutée est quant à elle instable, ce qui entraîne une perte de sa fonction activatrice sur NF-kappaB. De plus, grâce à des études de transfection transitoire de motoneurones murins (cellules NSC34), nous avons montré que la protéine PLEKHG5 mutée entraîne la formation d’importants agrégats cytoplasmiques. Dans cette nouvelle forme d’amyotrophie spinale, la perte de la fonction activatrice de la voie de signalisation NF-kappaB et la formation d’agrégats pourraient toutes deux contribuer à la neurotoxicité de la protéine PLEKHG5 mutée et conduire ainsi à la dégénérescence des motoneurones (Am J Hum Genet. 2007;81(1):67-76). En conclusion, nous espérons que ces résultats, qui contribuent à améliorer la connaissance des mécanismes physiopathologiques responsables de la dégénérescence des motoneurones, ouvriront à l’avenir la voie vers de nouvelles perspectives thérapeutiques.

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