The aggressive scaling of CMOS technology, to reduce device size while also increasing device performance, has reached a point where continuing improvement is becoming increasingly problematic and alternative routes for the development of future memory and processing devices may be necessary; in this thesis the use of phase-change and carbon based materials as one such alternative route is investigated. As pointed out by Ovshinsky [1, 2] some phase-change material should be capable of non-binary arithmetic processing, multi-value logic and biological (neuromorphic) type processing. In this thesis, generic, nanometre-sized, phase-change pseudodevices were fabricated and utilised to perform various types of computational operations for the first time, including addition, subtraction, division, parallel factorization and logic using a novel resistive switching accumulator-type regime in the electrical domain. The same accumulator response is also shown to provide an electronic mimic of an integrate-and-fire type neuron. The accumulator-type regime uses fast electrical pulses to gradually crystallize a phase-change device in a finite number of steps and does not require a multilevel detection scheme. The phase-change materials used in this study were protected by a capping layer of sputtered amorphous carbon. It was found that this amorphous carbon layer also underwent a form of resistive switching when subjected to electrical pulses. In particular, sputtered amorphous carbon layers were found to switch from an initially high resistivity state to a low resistivity state when a voltage pulse was locally applied using a Conductive Atomic Force Microscope (CAFM) tip. Further experiments on amorphous carbon vertical pseudo-devices and lithographically defined planar devices showed that it has potential as a new material for Resistive Random Access Memory (ReRam) applications. The switching mechanism was identified as clustering of the sp2 hybridized carbon sites induced by Joule heating. It was not possible to reset the devices back to their initial high resistivity state presumably due to the highly conductive nature of sputtered amorphous carbon.

No description available.

Throughout this work several thin film resists have been studied with substantial focus on HafSOx and SU-8. The study of HafSOx has granted more insight into how inorganic, spin coated films form and react under the electron beam. These films have been shown to form a thin dense crust at the surface that could have interesting implications in the interaction of the electrons. Continuing to further understand the electron interactions within the resist, low voltage patterns were created allowing the accelerating voltage to be matched to the film. With this general knowledge, higher resolution films can be constructed with shorter patterning times. Both resists complement each other in that HafSOx produces incredibly thin, dense structures to be formed with features below 10 nm in all dimensions. SU-8 allows micron thick features to be created over several millimeters. This flexibility in feature size enabled the creation of large fractals that could improve neuron binding to artificial retina down to the smallest fractals reported that are interesting for their applications as antennas. The final facet of this work involved looking at other methods of making structures. This was done through adding differing salts to organic molecules that stack into unique crystals. This dissertation includes previously published co-authored material.

The adaptation of standard integrated circuit (IC) technology for biosensors in drug discovery pharmacology, neural interface systems, environmental sensors and electrophysiology requires electrodes to be electrochemically stable, biocompatible and affordable. Unfortunately, the ubiquitous IC technology, complementary metal oxide semiconductor (CMOS), does not meet the first of these requirements. For devices intended only for research, modification of CMOS by post-processing using cleanroom facilities has been achieved by others. However, to enable adoption of CMOS as a basis for commercial biosensors, the economies of scale of CMOS fabrication must be maintained by using only low-cost post-processing techniques. The scope of this work was to develop post-processing methods that meet the electrochemical and biocompatibility requirements but within the low-cost constraint. Several approaches were appraised with the two most promising designs taken forward for further investigation. Firstly, a process was developed whereby the corrodible aluminium is anodised to form nanoporous alumina and further processed to optimise its impedance. A second design included a noble metal in the alumina pores to enhance further the electrical characteristics of the electrode. Experiments demonstrated for the first time the ability to anodise CMOS metallisation to form the desired electrodes. Tests showed the electrode addressed the problems of corrosion and presented a surface that was biocompatible with the NG108-15 neuronal cell line. Difficulties in assessing the influence of alumina porosity led to the development of a novel cell adhesion assay that showed for the first time neuronal cells adhere preferentially to large pores rather than small pores or planar aluminium. It was also demonstrated that porosity can be manipulated at room temperature by modifying the anodising electrolyte with polyethylene glycol. CMOS ICs were designed as multiple electrode arrays and optimised for neuronal recordings. This utilised the design incorporating a noble metal deposited into the porous alumina. Deposition of platinum was only partially successful, with better results using gold. This provided an electrode surface suitable for electric cell-substrate impedance sensors (ECIS) and many other sensor applications. Further processing deposited platinum black to improve signal-to-noise ratio for neuronal recordings. The developed processes require no specialised semiconductor fabrication equipment and can process CMOS ICs on laboratory or factory bench tops in less than one hour. During the course of electrode development, new methods for biosensor packaging were assessed: firstly, a biocompatible polyethylene glycol mould process was developed for improved prototype assembly. Secondly, a commercial ‘partial encapsulation’ process (Quik-Pak, U.S.) was assessed for biocompatibility. Cell vitality tests showed both methods were biocompatible and therefore suitable for use in cell-based biosensors. The post-processed CMOS electrode arrays were demonstrated by successfully recording neuronal cell electrical activity (action potentials) and by ECIS with a human epithelial cell line (Caco2). It is evident that these developments may provide a missing link that can enable commercialisation of CMOS biosensors. Further work is being planned to demonstrate the technology in context for specific markets.

Injury and degenerative disease of the central nervous system (CNS) are among the major causes for disabilities in humans. They result in permanent damage that is not repaired by regenerative processes. In contrast, anamniotes like fish and amphibia display a striking potential for successful regeneration in the CNS. The zebrafish (Danio rerio) has been established as a model for successful regeneration after spinal cord injury. However, it is yet unknown which factors are involved in regeneration after spinal lesions and other insults to the CNS. Focusing on motor neurons, I asked whether regeneration can also be observed in larval zebrafish. This would allow to take advantage of their accessibility to live imaging, pharmacological and genetic manipulation. It is unknown, whether the loss of a specific cell type in the absence of injury, which is reminiscent of the pathological change observed in neurodegenerative diseases, would be sufficient to induce regeneration. Comparing the regenerative response after spinal lesion to that after selective neuronal cell loss would allow to identify factors that act as a trigger for regeneration, e.g. mechanical injury signals, the extent of cell death or microglia activation. To address these questions, an experimental paradigm in which motor neurons can be selectively ablated without the need to inflict tissue damage would prove useful. Key findings of this work are: · Motor neuron generation ceases during early larval developmental stages. · The Nitroreductase system can be used for successful ablation of motor neurons in the larval spinal cord. · New motor neurons are generated in a regenerative response to both targeted ablation of motor neurons and spinal lesion in larval zebrafish after cessation of developmental generation of MNs. To test whether larval zebrafish can be used to analyse motor neuron regeneration, I carried out a birthdating study to establish a developmental time line for motor neuron generation in the spinal cord. The end of developmental motor neuron generation at an early time point, at around 54 hours post fertilisation, allows for the use of larval zebrafish to assess the regenerative response after insults to the spinal cord. In addition, I could show a time dependent role for Hedgehog signalling during the generation of a motor neuron subpopulation. The influence of Hedgehog is diminished before the end of motor neurogenesis. Utilizing the Gal4/UAS system to combine the Nitroreductase‐mCherry fusion protein expressing Tg(UAS:nfsB‐mCherry) with the motor neuron specific driver Tg(hb9:Gal4), I generated a new transgenic zebrafish line for the genetically targeted ablation of motor neurons. In the resulting transgenic fish, the administration of the prodrug Metronidazole induces apoptotic cell death in ~25% of spinal motor neurons leading to impaired motor performance and increased numbers of microglia in the spinal cord. My work shows that larval animals subjected to motor neuron ablation or spinal lesion display a regenerative response detected by increased numbers of newborn motor neurons. Importantly, this happens after developmental production of motor neurons has ceased, suggesting that progenitor cells are reverting to the generation of motor neurons. The data presented shows that in larval zebrafish, the selective loss of motor neurons is sufficient to induce a regenerative response in the spinal cord. The increased numbers of microglial profiles in the spinal cord after both spinal lesion and targeted cell ablation indicates a role for the immune system in mediating a regenerative response. This new targeted cell ablation paradigm in larval zebrafish will allow to identify and characterize the progenitor cell population forming new motor neurons. One can then further investigate how specific loss of motor neurons is sensed and which factors contribute to the activation of the endogenous stem cell populations. Using larval zebrafish has many benefits, as they are accessible to pharmacological testing with small molecules and live imaging. Moreover, the combination of additional transgenic reporter lines will allow for the investigation of single cell behaviour during regeneration.

The hallmark of an adult mammalian central nervous system is the inability to regenerate after an injury. Zebrafish, on the other hand, have an astounding regenerative capacity. After a spinal cord lesion, zebrafish can re-establish the damaged neuronal network and regain their swimming ability within weeks. This is partly due to the presence of the ependymal radial glia (ERGs), which line the wall of the central canal and act as the stem/progenitor cells of the spinal cord. Under homeostatic conditions the ERGs are largely quiescent, however, the lesion triggers them to proliferate and replace cells that have been lost due to the damage. Previous studies have shown that the regeneration of the motor neurons is affected by the signalling pathways similar to those governing the first development of these cells during embryogenesis, such as Sonic hedgehog, Notch and dopamine signalling. Serotonin (5-HT), similar to dopamine, is a monoaminergic neurotransmitter with a wide range of physiological and behavioural functions. It has also been shown to play a role during development of the nervous system. In this doctoral thesis I address the hypothesis that 5-HT has a positive effect on the development and adult regeneration of motor neurons. In addition, I expand on the previously discovered augmenting effect of dopamine on motor neuron development, by analysing the downstream pathways of its action. I show that during the development, incubating embryos in 5-HT increases the proliferation of the motor neuron progenitor (pMN) cells, which leads to augmented motor neuron production. RT-PCR on FAC sorted pMN cells highlights a number of serotonergic receptors that might be responsible for this effect. Although the downstream pathways are still unknown, 5-HT appears not to act on the sonic hedgehog canonical pathway, as shown by the unchanged expression of the hedgehog effector gene, patched2 after 5-HT treatment. I show that 5-HT does not affect the generation of vsx1+ or pax2a+ interneurons, suggesting that it has a predominant effect on motor neuron production. In the intact spinal cord of an adult zebrafish, the pMN-like ERGs express serotonergic receptors, indicating they are responsive to 5-HT stimulation. After a lesion, 5-HT administration enhances the proliferation of the pMN-like ERGs caudal to the lesion site resulting in an increase in the number of newborn motor neurons. Rostral to the lesion site, administration of exogenous 5-HT does not have an effect on the ERG proliferation, possibly due to the fact that the endogenous source of 5-HT, in the form of the descending axons, is still present and might already elicit a maximal response of the progenitor cells. 5-HT does not have an effect on the proliferation of the progenitor cells dorsal or ventral to the pMNlike domain, nor does it affect the regeneration of the serotonergic interneurons. These results suggest that 5-HT from the brain preferentially contributes to the regeneration of the motor neurons. Dopamine is another monoamine shown to enhance motor neuron production during the development and regeneration. To investigate the downstream pathways of dopamine signalling on motor neuron production during embryogenesis, RNA-sequencing was performed on FAC sorted pMN cells after a treatment with a dopamine agonist, pergolide. The results yielded 14 differentially expressed genes (FDR < 0.05) with diverse functions in the cell, indicating that dopamine might act on multiple targets to promote motor neuron production. Taken together, these results demonstrate the positive effect of monoaminergic stimulation on motor neuron development and regeneration. They provide an insight into the pathways that govern the proliferation of stem/progenitor cells in the embryonic and adult spinal cord, which might contribute to the research working on enhancing adult neurogenesis in mammals.

Le cerveau de drosophile est constitué entre autres des mushroom body, siège de la mémoire et de l’apprentissage. Cette structure est composée de différents types de neurones, parmi lesquels les neurones /. Ces neurones se présentent sous une forme orthogonale, avec l’axone qui se divise en une branche dorsale : la branche et une branche médiale : la branche . Le but de cette étude est de comprendre les mécanismes et voies de signalisation mis en jeu lors du développement de ces neurones.Chez la drosophile, la protéine APPL (Amyloïd Precursor Protein-Like) est l’homologue de la protéine APP humaine, connue pour son implication dans la maladie d’Alzheimer chez l’homme. Cette pathologie est caractérisée par une dégénérescence neuronale entraînant des défauts cognitifs et mnésiques. Malgré les nombreuses études focalisées sur la fonction pathologique d’APP durant les dernières décennies, peu de choses sont actuellement connues sur les fonctions physiologiques de cette protéine et notamment pendant le développement. C’est dans cette optique que nous avons étudié la fonction d’APPL et son interaction avec différentes protéines lors du développement des mushroom body. La protéine APPL a été identifiée comme étant un co-récepteur de la voie PCP (Planar Cell Polarity), permettant la régulation de la croissance axonale. Lors du développement, APPL permet le recrutement et l’activation de la protéine ABL (Abelson Tyrosine Kinase), qui phosphoryle DSH (dishevelled) et ainsi active la voie de signalisation permettant la croissance axonale.Le premier volet de cette thèse porte sur la régulation de l’activité ABL lors du développement des neurones /. S’il est établi qu’APPL permet une régulation positive de l’activité kinase d’ABL, je montre ici que la protéine HTT (huntingtine) permet de réguler négativement cette activité. Cette protéine HTT est impliquée dans la maladie de Huntington chez l’homme, une autre pathologie neurodégénérative. Ces travaux démontrent qu’HTT régule le niveau de phosphorylation d’ABL et par conséquent son activité. Le deuxième volet de cette thèse porte sur l’interaction d’APPL avec la protéine ARM (armadillo), homologue de la -caténine, lors du développement des neurones /. Je démontre que cette interaction est indépendante du rôle d’APPL dans la voie PCP. Je démontre aussi que cette interaction entre APPL et ARM est dépendante uniquement de la fonction d’ARM dans la dynamique du cytosquelette d’actine.Enfin le troisième volet de cette thèse porte sur la création de nouveaux allèles mutants pour Appl grâce à la technique du CRISPR-CAS9. La production de ces allèles permet d’avancer d’une part un possible rôle du gène voisin vnd (ventral nervous system defective) dans le développement des mushroom body, et d’autre part une interaction génétique entre Appl et vnd. / In the drosophila brain, mushroom bodies are involved in olfactory memory and learning. This structure is composed of different types of / neurons. These neurons form an orthogonal structure, with the branch projecting dorsally and the branch projecting medially. The aim of this study is to understand mechanisms and pathways involved during the development of these neurons.The drosophila APPL protein (Amyloïd Precursor Protein-Like) is the homologue of the human APP, known to be involved in Alzheimer’s disease. This pathology is characterized by neuronal degeneration inducing cognitive and memory defects. In spite of the numerous studies focused on the pathological function of APP during the last decades, few things are known on the physiological functions of this protein and more particularly during the development. This is from this perspective that we studied the APPL function and its interaction with proteins during the mushroom bodies development.The APPL protein was identified as a co-receptor of the PCP pathway (Planar Cell Polarity), involved in the axonal growth regulation. During the development, APPL allows the recruitment and the activation of the ABL protein (Abelson Tyrosine Kinase), which phosphorylates DSH (dishevelled) and so activates the axonal growth pathway.The first part investigates the regulation of ABL activity during the / neuron development. If it’s already established that APPL regulates positively the kinase activity of ABL, I show here that the HTT protein (Huntingtin) allows a negative regulation of ABL activity. In human, HTT is involved in the Huntington’s disease, another neurodegenerative disorder. This thesis work shows that HTT regulates the phosphorylation level of ABL, and therefore its activity.The second part investigates the interaction between APPL and ARM (armadillo), the homologue of the human -catenin, during the development of the / neurons. I show that this interaction is independent of the APPL function in the PCP pathway. Moreover, this interaction between APPL and ARM involves the actin cytoskeleton dynamic function of ARM, and not its Wnt pathway function.The third and last part presents new mutant alleles of APPL obtained with the CRISPR-CAS9 technique. The creation and analysis of these new alleles lead us to propose that vnd (ventral nervous system defective), neighbor gene of Appl, is also involved in / neurons development, and can interact genetically with Appl.

The structure and functions of brain show sexual dimorphism in vertebrates. Brain sexual differentiation is resulted from the neural development. The neural development is determined not only by the genetic regulation, but also by the extrinsic environmental influences. Serotonin (5-hydroxytryptamine, 5-HT ) functions as a neurotransmitter or/and neuromodulator in the central nervous system. Serotonin plays a role in the neural development via serotonin receptors. The sexual differentiation of tilapia is influenced by water temperature. The lower temperature induces a higher proportion of female while the elevated temperature induces a higher proportion of male in tilapia. In the present study, the influence of temperature on the proliferation of the neurons was investigated. These results show that the proliferation of neurons are varied with the temperature. The elevated temperature influences the proliferation of neurons via central serotonin system. Serotonin 1A receptor is involved in the serotonin-induced proliferation of neuron.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.

Transient Receptor Potential ‘Melastatin’ 7 (TRPM7) is a ubiquitously expressed, non-selective divalent cation channel implicated in diverse cellular functions including actomyosin cytoskeletal remodeling, magnesium homeostasis, and anoxic neuronal death. The present study investigates the role of TRPM7 in modulating neuronal morphology and regulating neuronal cytoskeletal dynamics after anoxia. Overexpression of GFP-tagged TRPM7 in neuronal cultures caused a stunted morphology with fewer neurite branches than controls, suggesting that TRPM7 regulates the neuronal cytoskeleton during dendritic outgrowth. I have discovered that TRPM7 may regulate morphology via activation of cofilin-1 (an actin binding protein). I found that TRPM7-dependent cofilin activation during anoxia mediated neuronal death. Overall my work reveals a novel link between anoxia-induced TRPM7 activity and cofilin activation, which likely contributes to neurodegeneration after ischemia.