After traumatic injuries to the brachial plexus there is a risk that one or more of the spinal roots are torn from the spinal cord, known as avulsion injury. This often leads to paralysis and chronic pain, notoriously difficult to treat with current pharmacotherapy. Surgical treatment may improve motor function but sensory recovery is usually poor as sensory axons fail to establish functional connections inside the spinal cord. The aims of this thesis were to develop a model for dorsal root avulsion in rodents in order to investigate the potentials of stem cell therapy for enhancing sensory regeneration after spinal root avulsion. Two different types of stem cells, embryonic and neural crest stem cells, have been transplanted to the avulsion model and analysed using immunohistochemical methods. The results indicate that stem cells survive after transplantation to the avulsed dorsal root and associate with regenerating axons. Furthermore, the different stem cells display different phenotypes after transplantation where embryonic stem cells give rise to neurons located outside the spinal cord that could serve as projection neurons whereas the neural crest stem cells form elongated tubes outlining the avulsed dorsal root and are associated with regenerating neuronal fibers. We have also discovered that the neural crest stem cells migrate into the damaged spinal cord as single cells. The neural crest stem cells also display a diversity in generating both neuronal and glial cells that may have different beneficial effects in neural repair following dorsal root avulsion. To improve the survival of stem cell transplants, the potentials of co-transplanting embryonic stem cells together with nanoparticle delivered growth factor mimetics has been investigated. The results indicate that nanoparticle delivered growth factors improve both transplant survival and maturation in comparison to untreated controls and may be a promising strategy in stem cell transplantation.

Artificial olfaction has the potential to revolutionise medical diagnosis, speed up threat detection, and provide long-term data collection on atmospheric pollution. Current technology provides a means to detect some compounds, but detection speed is slow, and accuracy is often balanced against cost and reusability of devices. Insects have long been the inspiration for artificial olfaction and are able to detect low concentrations of compounds over vast areas and navigate towards these targets. A deeper understanding of how this is achieved could inform the design of better artificial olfactory devices. What features of the insect olfactory system allow the rapid detection of miniscule concentrations of a wide variety of compounds? How does an insect discriminate odours using a limited number of olfactory receptor types?Using UAS/GAL4 technology, Drosophila melanogaster larvae expressing only a single functional olfactory sensory neuron (OSN), of their full repertoire of 21, were used to explore peripheral olfactory responses. Three different fly lines, which each expressed different olfactory receptor (OR) types, were used to record the electrophysiological responses of the peripheral OSNs to a panel of biologically significant odours, at differing concentrations. These responses were compared to those from a leading electronic nose (metal oxide sensors) for the same odour conditions, and the features of the responses were characterised. A novel odour delivery system was also developed to accurately deliver these odours and concentrations repeatably, and was validated using a commercially available photoionisation detector. The ability to correctly select the odour from which a response was recorded, out of a choice of five odours, at two concentrations each, was scrutinised using a classifier algorithm. The three OSN types achieved 53%, 62% and 75% accuracy, respectively. This is the first instance that Drosophila larvae have been conclusively shown to be able to discriminate odour concentrations using only a single peripheral OSN. The two metal oxide sensors achieved 92% and 95% accuracy under the same conditions. Whilst the features of the responses used to discriminate odours differed between the biological and electronic systems, the time frame required for correct classification of sensor data was only, in some cases, three seconds longer than in OSNs (~0.5 seconds), indicating that metal oxide sensors may have a useful role to play in biologically inspired artificial olfaction.

The mammalian neocortex contains diverse neuronal and glial cell types. Among them lies an important subclass, the subcerebral projection neurons (SCPN) that project to distant targets like the spinal cord. Aiming at identifying molecular controls over the postnatal development of SCPN, I focus my investigations on the role of Ppargc1á because its function remains relatively unknown in the brain while it is important for metabolism and survival in other tissue systems.

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The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.

Sonic Hedgehog (SHH) is a secreted morphogen that is an essential regulator of patterning and growth. The SHH protein requires cleavage of its full-length precursor (SHHFL) for secretion of biologically active SHH (SHHNp). Mutations in SHH that affect SHH processing are associated with human disease, which highlights the importance of processing for patterning in vivo. We identified Sortilin (SORT1), a member of the VPS10P receptor family, as a novel SHH interacting protein. SORT1 preferentially associates with SHHFL and SORT1 levels correlate inversely with cleavage of SHHFL. Consistent with an antagonistic relationship between SORT1 and SHH processing, loss of SORT1 results in an increase in SHH levels in axons and a partial rescue of Hedgehog-associated patterning defects in a mouse model of deficient SHH processing. Finally, we demonstrate a functional requirement for SORT1-mediated trafficking on SHH-dependent signaling from axons in the developing visual system in vivo. Our findings identify a novel role for SORT1 in the regulation of SHH processing and trafficking.

The present work investigates the applicability of Genetic Algorithms (GA) to the problem of signal propagation in Native Neuronal Networks (NNNs). These networks are comprised of neurons, some of which receive input signals. The signals propagate though the network by transmission between neurons. The research focuses on the regeneration of the output signal of the network without knowing the original input signal. The computational complexity of the problem is prohibitive for the exact computation. We propose to use a heuristic approach called Genetic Algorithm. Three algorithms are developed, based on the GA technique. The developed algorithms are tested on two different networks with varying input signals. The results obtained from the testing indicate significantly better performance of the developed algorithms compared to the Uniform Random Search (URS) technique, which is used as a control group. The importance of the research is in the demonstration of the ability of GA-based algorithms to successfully solve the problem at hand.

Au cours du processus neurodégénératif lié à la Sclérose Latérale Amyotrophique (SLA), les motoneurones innervant les muscles des membres sont très atteints tandis que ceux innervant les muscles axiaux sont relativement préservés. Les motoneurones diffèrent aussi dans leur réponse à plusieurs facteurs neurotrophiques (NTF). Je me suis intéressée aux mécanismes moléculaires déterminant la morphologie distincte et les réponses aux NTF de sous-populations de motoneurones pertinentes dans la SLA. J'ai démontré que les sous-populations de motoneurones murins innervant les muscles axiaux (MN-MMC) ou les muscles des membres (MN-LMC) ont de fortes différences morphologiques. Ces différences sont dues à une régulation différentielle de gènes codant pour la Péripherine et la Diacylglycérol kinase beta (DGK-β) qui sont régulés par les facteurs de transcription LIM-HD FoxP1/HB9. J'ai montré que les MN-LMC et les MN-MMC répondaient distinctement à trois facteurs neurotrophiques: le HGF (Hepatocyte Growth Factor), l'Artémine et le CNTF (Ciliary Neurotrophic Factor). J'ai étudié les motoneurones lombaires dans deux situations pathologiques: 1) les altérations de l'appareil de Golgi et du trafic axonal suite à la perte de TBCE (Tubulin Binding Cofactor E), 2) l'effet de l'augmentation de KCC2 à la membrane plasmique des motoneurones sur la transmission synaptique inhibitrice après lésion.Enfin, j'ai développé une nouvelle méthode de purification de motoneurones dérivés d'iPS (induced Pluripotent Stem Cell) humains en utilisant un vecteur rapporteur lentiviral Hb9::GFP et un anticorps dirigé contre le récepteur de faible affinité des neurotrophines, p75. / Muscles are highly vulnerable whereas motor neurons innervating axial muscles are relatively resistant. Motor neurons also seem to differ in their response to several neurotrophic factors (NTF). I investigated the molecular mechanisms determining the distinct morphology and the differential NTF response of ALS-relevant motor neuron subsets. First, I demonstrated that mouse lumbar motor neurons innervating either axial muscles (MMC-MN) or hindlimb muscles (LMC-MN) display remarkable morphological differences. These differences involve a differential regulation of genes coding for Peripherin and Diacylglycerol kinase-b (DGK-β) which are regulated by the transcription factors FoxP1/HB9. Second, I showed that LMC-MN and MMC-MN respond differentially to the three neurotrophic factors HGF (Hepatocyte Growth Factor), Artemin and CNTF (Ciliary NeuroTrophic Factor). Their differential survival is explained by the corresponding receptor gene expression in specific pools of MMC-MN and LMC-MN. Third, I studied lumbar motor neurons in two pathological conditions: 1) alteration of Golgi apparatus and axonal trafficking induced by loss of TBCE (Tubulin Binding Cofactor E) 2) the effect of KCC2 increase at motor neuron plasma membrane on inhibitory synaptic transmission after trauma.Finally, I developed a new FACS-based method for isolating human iPS (induced Pluripotent Stem Cell)-derived motor neurons with both an HB9::GFP reporter lentivirus and an antibody directed against the low-affinity neurotrophin receptor p75.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22340号 / 医博第4581号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 影山 龍一郎, 教授 井上 治久, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Improved knowledge of human nerve function and recruitment would enable innovation in the Biomedical Engineering field. Better understanding holds the potential for greater integration between devices and the nervous system as well as the ability to develop therapeutic devices to treat conditions affecting the nervous system. This work presents a three-dimensional volume conductor model of the human arm for coupling with code describing nerve membrane characteristics. The model utilizes an inhomogeneous medium composed of bone, muscle, skin, nerve, artery, and vein. Dielectric properties of each tissue were collected from the literature and applied to corresponding material subdomains. Both a fully anatomical version and a simplified version are presented. The computational model for this study was developed in COMSOL and formatted to be coupled with SPICE netlist code. Limitations to this model due to computational power as well as future work are discussed. The final model incorporated both anatomically correct geometries and simplified geometries to enhance computational power. A stationary study was performed implementing a boundary current source through the surface of a conventionally placed electrode. Results from the volume conductor study are presented and validated through previous studies.