For many dominant diseases, conventional treatment options are limited. This makes them attractive candidates for gene therapy, which may be directed to specifically silence a disease-causing allele. However, many mutations are not easily amenable to this technique, including nucleotide repeat expansions, which cause numerous neurodegenerative diseases such as Huntington’s disease and several Spinocerebellar Ataxias. Combined gene knockdown and replacement (K&R) may present a more practical approach for such conditions, whereby the gene of interest is subject to mutation-independent non-allele-specific silencing and concurrently replaced with a functional copy. Artificial mimics of naturally occurring intronic microRNAs are theoretically ideal for this purpose, since they can be nested within the replacement gene. This thesis investigates the development of mimics of two different intronic miRNA systems (mirtron miR-1224 and the miR-106b cluster) to silence Ataxin-7 and to be incorporated into novel single K&R constructs for testing in vitro. Artificial mirtrons and intronic miRNAs were successfully developed and shown to silence Ataxin-7 mRNA in numerous cell lines. An RNAi-resistant gene was developed and mirtrons could be successfully incorporated as introns. Patient-derived fibroblasts and iPSC-derived neuronal cells were investigated as models for testing of gene silencing therapies. This work suggests that mirtron-based K&R is achievable, and warrants further investigation.

La dysautonomie familiale (FD) est une neuropathie héréditaire provoquée par des mutations au sein du gène IKBKAP, la plus commune d'entre elles induisant un épissage alternatif de l'exon 20 au sein de du pré-ARNm de façon tissu-spécifique. L'épissage aberrant est particulièrement prononcé dans les tissus nerveux, conduisant à la dégénerescence progressive des neurones sensoriels et autonomes. La spécificité de la perte des cellules nerveuses dans la FD est mal comprise, par manque d'un modèle approprié. Afin de mieux comprendre les mécanismes moléculaires de l'épissage des ARNm d'IKBKAP, nous avons utilisé un modèle original : les cellules souches olfactives ecto-mesenchymateuses (hOE-MSC) de patients FD. Les hOE-MSC sont pluripotentes et ont la capacité de se différencier en diverses lignées cellulaires, y compris les neurones et les cellules gliales.Nous avons confirmé la présence du transcrit exempt de l'exon 20 d'IKBKAP dans les hOE-MSC de FD et nous avons observé une expression significativement inférieure de la somme des transcrits IKBKAP chez ces patients, du fait de la dégradation d'une partie des isoforme aberrants. Cette réduction est correlée avec une réduction d'expression de la protéine traduite à partir du transcrit d’IKBKAP possèdant l’exon 20, IKAP/hELP1. Nous avons localisé IKAP/hELP1 dans différents compartiments cellulaires, y compris le noyau, ce qui soutient des rôles multiples de cette protéine. Nous avons confirmé que la kinétine, une cytokinine, améliorait le taux de transcrit incluant l'exon 20 et rétablissait des niveaux normaux d'IKAP/hELP1 dans les hOE-MSC de FD. Par ailleurs, nous avons pu modifier le rapport d'épissage d'IKBKAP en augmentant ou en réduisant le ratio WT (inclusion de l'exon 20) : MU (saut de l'exon 20) respectivement, en produisant des sphères flottantes, ou en engageant les cellules vers une différentiation neurale. Les sphères et les cellules différenciées ont été étudiées au niveau pan-génomique, ce qui a permis d'identifier le développement du système nerveux comme étant le processus le plus affecté chez les FD. De plus, nous soulignons le rôle de la kinétine comme un probable régulateur de facteurs d'épissage contribuant à la restauration d'un épissage correct d'IKBKAP.Les hOE-MSC isolées de patients FD représentent une nouvelle approche pour modéliser la pathologie et mieux comprendre l'expression génétique et les approches thérapeutiques possibles de la FD. En outre, elles offrent une application originale à la compréhension d'autres maladies génétiques neurologiques. / Familial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSCs) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells.We confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and observed a significant lower expression of both IKBKAP transcripts and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation. Spheres forming cells and lineage neuroglial progenitors were investigated at the genome-wide level, and we confirmed that nervous system development was the most altered process in FD. More, we highlight kinetin role as a putative regulator of splicing factors which contribute to restore a correct splicing of IKBKAP.hOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases.

Depuis sa première description par James Parkinson dans son essai sur la paralysie agitante, la maladie de Parkinson (PD) a été reconnue comme une maladie du système moteur identifié par une tétrade de symptômes, à savoir : akinésie, rigidité musculaire, tremblement au repos et instabilité posturale. Ces symptômes sont liés à la perte de la dopamine (DA) dans le striatum après la dégénérescence neuronale dans la substance noire (SN). Il est de plus en plus reconnu que les symptômes non moteurs et peut-être non dopaminergiques inévitablement émergent et s'aggravent au cours de la progression de la maladie. Les perturbations du sommeil sont parmi les principaux symptômes non moteurs et ont été reconnus comme marqueurs précliniques de la maladie. Les modèles de régulation du sommeil ont insisté sur deux processus distincts : un mécanisme de contrôle du sommeil, ou homéostat sommeil, et un oscillateur circadien. L'oscillateur circadien, basé dans le noyau suprachiasmatique (NSC) est responsable de la tendance à dormir pendant certaines phases du cycle de 24 heures et la consolidation du sommeil et de réveil en épisodes distincts. L'homéostat sommeil est chargé de surveiller et de réagir à la nécessité pour le sommeil, provoquant l'envie de dormir à dépendre sur les montants avant du sommeil ou de l'éveil. Alors que les perturbations dans les circuits et les processus homéostatiques impliqués dans la régulation du sommeil-éveil comportement sont documenté dans la maladie de Parkinson, l'implication potentielle des altérations du système circadien n'ont pas été étudiés en détail. Le but de ma thèse est d'étudier les modifications dans le système circadien en utilisant deux modèles animaux de PD : la souris et le primate non-humain / Since the first description by James Parkinson in his essay on the shaking palsy, Parkinson’s disease (PD) was recognized as a motor disease identified by a tetrad of symptoms, namely; akinesia, muscular rigidity, resting tremor and postural instability. These symptoms are known to be related to loss of dopamine (DA) in the striatum following neural degeneration in the substantia nigra (SN). It is increasingly recognized that non-motor and perhaps non-dopaminergic related symptoms inevitably emerge and worsen during disease progression. Sleep disruption is one of the major non-motor symptoms and has been suggested as a preclinical marker of the disease. Models of sleep regulation have emphasized two distinct processes: a sleep-control mechanism, or sleep homeostat, and a circadian oscillator. The circadian oscillator, based in the suprachiasmatic nucleus (SCN), is responsible for the tendency to sleep during certain phases of the 24-hour cycle and the consolidation of sleep and wake into distinct episodes. The sleep homeostat is responsible for monitoring and reacting to the need for sleep, causing the urge to sleep to depend on prior amounts of sleep or wakefulness. While disruptions in the circuitry and the homeostatic processes involved in the regulation of sleep-wake behaviour is will documented in PD, the potential involvement of alterations of the circadian system have not been studied in detail. The aim of my thesis is to investigate alterations in the circadian timing system using two animal models of PD: the mouse and the non-human primate. Taken together, the studies show that disturbances of circadian functions occur after MPTP treatment in the non-human primate but not in the mouse model of PD. These results emphasize the limitations of the MPTP-treated mouse model of PD for the study of non-motor symptoms, and reinforce previous studies that question the adequacy of this model to replicate cardinal motor features of the disease. In contrast, results in the non-human primate model stress the importance of dopaminergic degeneration in the circadian organisation of behavioral sleep wake cycle in PD

Eine Gemeinsamkeit verschiedener neurodegenerativer Erkrankungen ist die abnormale Ansammlung von Proteinen im Gehirn, wie z. B. von alpha-Synuclein (Syn)-Aggregaten bei der Parkinson''schen Krankheit (PD) oder von Huntingtin (Htt)-Aggregaten bei Chorea Huntington (HD). Am Anfang dieser Studie wurde eine Bibliothek von ca. 5000 natürlichen Substanzen nach Inhibitoren der Htt-Aggregation durchsucht. Eine der wirksamen Substanzen war (-)-Epigallocatechingallat (EGCG), eine Verbindung, die in grünem und schwarzem Tee vorkommt. Die antioxidativen Eigenschaften von EGCG wurden bereits mit einer neuroprotektiven Wirkung in Verbindung gebracht, was EGCG zu einem vielversprechenden Kandidaten für die Entwicklung einer neuen Behandlungsmethode macht. Eine inhibierende Wirkung auf Proteinaggregation wurde jedoch bis jetzt noch nicht nachgewiesen. Diese Studie zeigt, dass EGCG die Aggregation von Htt und Syn hemmt, indem es dosisabhängig eine oligomere Proteinkonformation stabilisiert. Diese Oligomere wirken jedoch nicht als Keime in Aggregationsreaktionen. Zusätzlich verändert EGCG die Exposition bestimmter Epitope, die von konformationsspezifischen Antikörpern im Laufe der Aggregation erkannt werden. Daher könnte die Substanz Proteine, die zur Aggregation neigen, auf einen alternativen Faltungspfad in der Missfaltungskaskade führen. Weiterhin legen die Ergebnisse nahe, dass eine direkte Wechselwirkung zwischen EGCG und Proteinen in einer ungefalteten Konformation stattfindet. In verschiedenen Zellkultur-Modellsystemen verringerte EGCG die Toxizität, die von missgefalteten Proteinen ausgeht, was nahelegt, dass die neu geformten oligomeren Spezies nicht toxisch sind. EGCG könnte daher ein chemisches Chaperon darstellen, das die Missfaltung und Toxizität von Proteinen, die mit neurodegenerativen Krankheiten assoziiert sind, verringert. Die Substanz könnte daher die Basis zur Entwicklung einer neuen Therapie für diese unheilbaren Krankheiten darstellen. / A common feature of neurodegenerative disorders is the abnormal accumulation of aggregated protein the brain, such as alpha-Synuclein (Syn) aggregates in Parkinson''s disease (PD) and Huntingtin (Htt) aggregates in Huntington''s disease (HD). In this study, a library of approximately 5000 natural compounds was screened for inhibitors of Htt aggregation. One of the hits was (-)- Epigallocatechin gallate (EGCG), a compound present in green and black tea. The antioxidant properties of this substance have been linked to neuroprotection before, making it a promising candidate for the development of a treatment for neurodegenerative diseases. Inhibition of protein aggregation by EGCG, however, has not been demonstrated so far. This study shows that EGCG inhibits the aggregation of Htt and Syn by stabilizing an oligomeric conformation of the respective proteins in a dose-dependent manner. These oligomers do not seed the aggregation of Htt and Syn. Also, EGCG modifies the exposure of different epitopes recognized by conformation-specific antibodies during the aggregation process. The compound might therefore lead aggregation-prone proteins on an alternative folding pathway in the misfolding cascade. The results furthermore suggest that direct interaction occurs between EGCG and proteins in an unfolded conformation. EGCG also reduces toxicity caused by misfolded Htt or Syn in cell culture model systems, suggesting that the oligomeric protein species formed in the presence of EGCG are not toxic to living cells. EGCG might therefore represent a chemical chaperone that can modulate misfolding and toxicity of proteins associated with neurodegenerative diseases and could provide the basis for the development of a novel pharmacotherapy for these fatal disorders.

Les similitudes entre les mécanismes neurotoxiques responsables des encéphalopathies spongiformes Transmissibles (EST) et des synucléinoapthies, ainsi que la présence concomitante des formes pathologiques de la protéine prion et de l’α-synucléine au sein d’une même maladie neurodégénérative sont deux observations qui nous ont conduits à étudier les relations existant potentiellement entre les altérations moléculaires de l’α-synucléine et les maladies à prion. Après avoir développé des anticorps monoclonaux en immunisant avec de l’α-synucléine recombinante humaine des souris n’exprimant pas de façon endogène cet immunogène, nous avons caractérisé les altérations moléculaires de l’α-synucléine apparaissant conjointement à une symptomatologie motrice sévère lors du vieillissement de souris transgéniques (TgM83) surexprimant l’α-synucléine humaine mutée en A53T. Les essais d’inoculation intracérébrale de souris TgM83 par différentes souches de prion ont mis en évidence que la transmission de l’encéphalopathie spongiforme bovine de type H permet de déclencher chez ces animaux une maladie à prion de façon concomitante au développement d’altérations moléculaires de l’α-synucléine. Enfin, l’importante accélération de la pathologie liée a l’α-synucléine observée chez des souris TgM83 ayant été inoculées par des tissus contenant des formes altérées de l’α-synucléine, constitue un résultat soutenant le fait que la pathologie liée a l’α-synucléine serait capable de se propager expérimentalement de proche en proche, comme la protéine prion pathologique au cours des EST / The overlap of neurotoxic mecanisms involved in prion diseases and synucleinopathies, and the concomitant detection of pathological forms of prion and α-synuclein in a same neurodegenerative disease, raise questions about the existence of potential relationship between α‐synuclein molecular alteration and prion diseases. First, we developed monoclonal antibodies by immunizing mice presenting a spontaneous deletion of the α-synuclein gene with human recombinant α‐synuclein. Then, we characterized the molecular alterations appearing jointly to clinical signs during the aging of a transgenic mouse model of synucleinopathies (TgM83), overexpressing human A53T α‐synuclein. Then, an approach routinely done in the field of prion was used to trigger a synucleinopathy alongside a prion disease. For this purpose, TgM83 mice were inoculated intracerebrally by three different prion strains : transmission of H-type bovine spongiform encephalopathy allows the onset of a prion disease concomitantly to the α‐synuclein pathology developed by the TgM83 mouse model. Finally, intracerebral inoculation of TgM83 mice with brain homogenates from symptomatic mice affected by a synucleinopathy triggers an important acceleration of the α‐synuclein pathology, resulting in the early onset of motor clinical signs associated with molecular alterations of α-synuclein. These data suggest that α-synuclein alterations can be experimentally transmitted from one mouse to another, supporting the idea that, far from being confined to the transmissible spongiform encephalopathies, the « prion-like » propagation of misfolded neuronal proteins might occur in synucleinopathies

Ce travail porte sur l’étude fonctionnelle de deux gènes préférentiellement exprimés dans deux régions du cerveau touchées par des pathologies neurodégénératives : Capucine, un marqueur du striatum, structure qui dégénère au cours de la maladie de Huntington et Agpat4, un marqueur de l’aire tegmentaire ventrale et de la substance noire compacte, dont les neurones dopaminergiques sont sélectivement atteints lors de la maladie de Parkinson. Des lignées de souris invalidées pour ces gènes ont été générées au laboratoire et au cours de ma thèse j’ai procédé à leur caractérisation. L’expression striatale du gène de la Capucine étant significativement diminuée dans des modèles murins de la maladie de Huntington, nous avons souhaité évaluer son rôle éventuel dans la pathogenèse de cette maladie. Pour ce faire, nous avons examiné, dans le cadre d’une collaboration, l’effet du knock-out et de la surexpression du gène de la Capucine sur la vulnérabilité des neurones striataux à un fragment de la Huntingtine mutée dans un modèle murin de la maladie de Huntington. Les données montrent que la Capucine n’a pas d’effet significatif sur la toxicité du fragment de la Huntingtine mutée dans le modèle étudié.La protéine Agpat4 présente des homologies de séquence avec des acyltransférases impliquées dans le métabolisme des phosphoglycérides. J’ai réalisé des études d’expression par différentes techniques de biologie moléculaire qui montrent que le gène d’Agpat4 est exprimé dans la plupart des tissus catécholaminergiques. Pour déterminer l’activité endogène d’Agpat4 et son rôle physiologique dans les tissus où elle est exprimée, j’ai comparé le métabolome de tissus de souris invalidées pour le gène d’Agpat4 et sauvages par chromatographie en phase liquide couplée à la spectrométrie de masse. Mes résultats indiquent que l’invalidation du gène d’Agpat4 perturbe le métabolisme non seulement de différentes classes de lipides, notamment les lysophosphatidyléthanolamines, mais aussi celui des catécholamines. / This work concerns the functional study of two genes preferentially expressed in two brain regions affected by neurodegenerative diseases: Capucine, a marker of the striatum, a structure that degenerates in Huntington's disease and Agpat4, a marker of the ventral tegmental area and the substantia nigra pars compacta, whose dopaminergic neurons are selectively affected in Parkinson's disease. Mouse lines deficient for Capucine and Agpat4 have been generated in the laboratory and during my PhD thesis I carried out their characterization.As the striatal gene expression of Capucine is significantly reduced in mouse models of Huntington's disease, we wished to evaluate its possible role in the pathogenesis of this disease. In a collaborative work, we examined the effect of the knockout and overexpression of the Capucine gene on the vulnerability of striatal neurons to a mutant Huntingtin fragment in a mouse model of Huntington’s disease. The data show that Capucine has no significant effect on the toxicity of the mutant Huntingtin fragment in the considered model.The Agpat4 protein has sequence homologies with acyltransferases involved in the metabolism of phosphoglycerides. I conducted expression studies using different molecular biology techniques, which showed that the Agpat4 gene is expressed in most catecholaminergic tissues. To determine the endogenous activity of Agpat4 and its physiological role in the tissues where it is expressed, I compared the metabolomes of Agpat4-deficient and wild-type mice tissues by liquid chromatography coupled with mass spectrometry. My results indicate that Agpat4 deficiency alters not only the metabolism of different lipid classes, in particular lysophosphatidylethanolamines, but also the metabolism of catecholamines.

Neurons connect to their remote targets via axons, which usually survive for the lifetime of an organism. Spatiotemporal regulation of the axonal proteome by local protein synthesis (LPS) plays a critical role in neuronal wiring and axon survival, raising the intriguing possibility that some neurological disorders involve LPS dysfunction. To visualise LPS in situ, I optimised multiple imaging techniques to investigate Netrin-1-induced translation in cultured retinal axons. Total axonal protein synthesis measured by metabolic and puromycin labelling indicates axons experience stage-dependent alterations in translation rate upon Netrin-1 stimulation. Remarkably, Netrin-1 triggers a burst of β-actin synthesis starting within 20 seconds of cue application at multiple non-repetitive sites visualised by single molecule translation imaging, an approach that allows direct visualisation of translation dynamics in response to external stimuli. Further studies have shown that local translation can occur on Rab7a-associated late endosomes, where mRNA recruitment and translation are coordinately regulated. Notably, mRNAs encoding mitochondria-related proteins are found translating on late endosomes docking in the vicinity of mitochondria, suggesting late endosomes act as ‘platforms’ for the localised synthesis of mitochondrial proteins necessary for maintaining mitochondrial integrity. Moreover, this process is affected in axons expressing the Charcot-Marie-Tooth disease type 2B (CMT2B)-related Rab7a mutants, leading to abnormal mitochondrial biogenesis and activity and compromised axon survival. Finally, attenuated de novo protein synthesis is observed in axons expressing amyotrophic lateral sclerosis (ALS)-associated fused in sarcoma (FUS) mutants and hypomethylated wild-type FUS. Live imaging reveals mislocalised mutant or hypomethylated FUS granules are transported along axons and accumulate at growth cones, possibly irreversibly trapping RNA molecules, resulting in reduced distance travelled by RNA granules in axons. Furthermore, mutant FUS expression results in defective retinal projections in vivo, highlighting the importance of RNA metabolism and local translation in axonal homeostatic mechanisms. In conclusion, aberrant translational activity in axons leads to prominent axonopathy, which recapitulates features of early stages of neurological diseases, providing the basis for novel therapeutic strategies.

abstract: The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics. / Dissertation/Thesis / Masters Thesis Bioengineering 2016

La microscopie non-linéaire résolue en polarisation est un outil puissant pour accéder à des informations structurelles dans les assemblages biomoléculaires. Les interactions non-linéaires entre matière et lumière induisent des processus complexes où des champs électromagnétiques cohérents interagissent avec les dipôles de transitions moléculaires. Le contrôle de la polarisation des champs électromagnétiques excitateurs et l’étude des réponses non-linéaires induites procurent de riches informations sur la distribution angulaire des molécules présentes dans le volume focal de l’objectif du microscope. Dans cette thèse, nous appliquons cette sensibilité à la polarisation à plusieurs modalités de microscopie cohérentes sans marquage (diffusion cohérente Raman anti-Stokes (CARS), diffusion Cohérente stimulée (SRS)) et à la fluorescence à deux photons (2PEF) afin d’obtenir des informations quantitatives sur la forme de la distribution moléculaire et l’orientation des lipides dans les membranes artificielles, ainsi que dans les membranes biologiques telles que la myéline des tissus de la moelle épinière. Avec cette technique, nous adressons une question fondamentale sur le comportement des ensembles lipidiques dans les membranes et sur l’effet d’autres molécules telles que le cholestérol et les marqueurs fluorescents. Nous démontrons que le CARS résolu en polarisation permet d’accéder à de fines informations sur l’organisation des lipides dans les membranes de la myéline, en deçà de la limite de diffraction. / Polarization resolved nonlinear microscopy is a powerful tool to image structural information in biomolecular assemblies. Nonlinear interaction between light and matter lead to complex processes where coherent combinations of optical fields couple to assemblies of molecular transition dipoles. Controlling polarized optical fields and monitoring nonlinear induced signals in a medium can nevertheless bring rich information on molecular orientational organization within the focal spot of a microscope objective. In this PhD thesis we apply this polarization sensitivity to different label-free optical coherent techniques (coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS)) and to two-photon fluorescence (2PEF) to retrieve quantitative information on the static molecular distribution shape and orientation of lipids in model membranes and biological membranes such as myelin sheaths in spinal cord tissues. With this technique, we address fundamental questions about lipid packing behavior in membranes, and how it can be affected by other molecules such as cholesterol and the insertion of fluorescent lipid probes. We demonstrate that polarization resolved CARS give access to fine details on lipids arrangement in myelin sheaths, at a sub-diffraction scale. In the context of experimental autoimmune encephalomyelitis disease (EAE) we show, that even at the stage of disruption of the myelin envelope during the demyelination process, lipids multilayers reveal strong capability to preserve their macroscopic self-assembly into highly organized structures, with a degree of disorganization occurring only at the molecular scale.

The onset of a progressive, fatal illness such as Motor Neuron Disease (MND) inevitably results in physical and communication disabilities that impinge on the individuals’ ability to remain functionally independent. The loss of speech as a result of dysarthria, a motor speech disorder, is one of the most profound changes that the person with MND will experience. The decline in the individuals’ speech intelligibility, that negatively influences communication effectiveness, implies that in 80% of cases alternative and augmentative communication (AAC) strategies are required to support the daily communication needs of individuals with MND. The dyadic nature of chronic illness implies that multiple aspects of one of the most important adult relationships, marriage, will be affected. Roles and responsibilities performed by each member of the couple will continually change as the disease progresses. The emotional trauma of adjusting to the unavoidable alteration in their relationship elicits strong emotions such as guilt, anger and frustration. Communication is one of the most constructive ways of dealing with these emotions. The ability of spouses to convey their innermost thoughts, feelings and intimacy through communicative interaction is vitally important in marital communication. The aim of this study was to compare how persons with MND and their spouses perceive changes in their marital communication in relation to the deteriorating speech of persons with MND. Fourteen couples divided into two participant groups, persons with MND and spouses, participated in this non-experimental correlational research study. Data was collected during three visits at six-monthly intervals over a 12 month period. At each of these visits both participant groups completed a variety of objective and subjective measures, of which twenty percent were interrated by independent raters. Results confirmed the inevitable decline in speech intelligibility of persons with MND across the disease progression. The persons with MND did not report a change in their perception of marital communication although their spouses indicated a statistically significant decrease between the first and last visits. Interestingly, there was no statistically significant relationship between the deteriorating speech of persons with MND and the couples’ perception of marital communication, confirming that marital communication was not influenced by decreased speech intelligibility. / Thesis (PhD)--University of Pretoria, 2010. / Centre for Augmentative and Alternative Communication (CAAC) / unrestricted