Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-25T20:54:16Z No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-26T21:40:31Z (GMT) No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) / Made available in DSpace on 2016-08-26T21:40:31Z (GMT). No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) Previous issue date: 2016-02-29 / Recently, the field of cellular reprogramming has been revolutionized by works showing the potential to directly lineage-reprogram somatic cells into neurons upon overexpression of specific transcription factors. This technique offers a promising strategy to study the molecular mechanisms of neuronal specification, identify potential therapeutic targets for neurological diseases and eventually repair the central nervous system damaged by neurological conditions. Notably, studies with cortical astroglia revealed the high potential of these cells to reprogram into neurons using a single neuronal transcription factor. However, it remains unknown whether astroglia isolated from different regions of the central nervous system have the same neurogenic potential and generate induced neurons (iN) with similar phenotypes. Similarly, little is known about the fate that iNs could adopt after transplantation in the brain of host animals. In this study we compare the potential to reprogram astroglial cells isolated from the postnatal cerebral cortex and cerebellum into iNs both in vitro and in vivo using the proneural transcription factors Neurogenin-2 (Neurog2) and Achaete scute homolog-1 (Ascl1). Our results indicate cerebellar astroglia can be reprogrammed into induced neurons (iNs) with similar efficiencies to cerebral cortex astroglia. Notably however, while iNs in vitro adopt fates reminiscent of cortical or cerebellar neurons depending on the astroglial population used for reprogramming, in situ, after transplantation in the postnatal and adult mouse brain, iNs adopt fates compatible with the region of integration. Thus, our data suggest that the origin of the astroglial population used for lineage-reprogramming affects the fate of iNs in vitro, but this imprinting can be overridden by environmental cues after grafting.

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Le cervelet, composé d'un cortex et de noyaux, est responsable du contrôle moteur fin des mouvements et de la posture. En combinant une approche génétique (basée sur l'utilisation de lignées de souris transgéniques) avec des traçages anatomiques, des marquages immunohistochimiques et des expériences d'électrophysiologie et d'optogénétique, nous établissons les caractères distinctifs des neurones inhibiteurs des noyaux cérébelleux et en détaillons la connectivité ainsi que les fonctions dans le circuit cérébelleux. Les neurones inhibiteurs glycinergiques des noyaux profonds constituent une population de neurones distincts des autres types cellulaires identifiables par leur phénotype inhibiteur mixte GABAergique/glycinergique. Ces neurones se distinguent également par leur plexus axonal qui comporte une arborisation locale dans les noyaux cérébelleux où ils contactent les neurones principaux et une projection vers le cortex cérébelleux où ils contactent les cellules de Golgi. Ces neurones inhibiteurs reçoivent également des afférences inhibitrices des cellules de Purkinje et pourraient être contactés par les fibres moussues ou les fibres grimpantes.Nous apportons ainsi la première étude d'une transmission mixte fonctionnelle par les neurones inhibiteurs des noyaux cérébelleux, projetant à la fois dans les noyaux et le cortex cérébelleux. L'ensemble de nos données établissent les neurones inhibiteurs mixtes des noyaux cérébelleux comme la troisième composante cellulaire des noyaux profonds. Leur importance dans l'organisation modulaire du cervelet, ainsi que leur impact sur l'intégration sensori-motrice, devront être confirmés par des études optogénétiques in vivo. / The cerebellum is composed of a three-layered cortex and of nuclei and is responsible for the learned fine control of posture and movements. I combined a genetic approach (based on the use of transgenic mouse lines) with anatomical tracings, immunohistochemical stainings, electrophysiological recordings and optogenetic stimulations to establish the distinctive characteristics of the inhibitory neurons of the cerebellar nuclei and to detail their connectivity and their role in the cerebellar circuitry.We showed that the glycinergic inhibitory neurons of the cerebellar nuclei constitute a distinct neuronal population and are characterized by their mixed inhibitory GABAergic/glycinergic phenotype. Those inhibitory neurons are also distinguished by their axonal plexus which includes a local arborization with the cerebellar nuclei where they contact principal output neurons and a projection to the granular layer of the cerebellar cortex where they end onto Golgi cells dendrites. Finally, the inhibitory neurons of the cerebellar nuclei receive inhibitory afferents from Purkinje cells and may be contacted by mossy fibers or climbing fibers.We provided the first evidence of functional mixed transmission in the cerebellar nuclei and the first demonstration of a mixed inhibitory nucleo-cortical projection. Overall, our data establish the inhibitory neurons as the third cellular component of the cerebellar nuclei. Their importance in the modular organization of the cerebellum and their impact on sensory-motor integration need to be confirmed by optogenetic experiments in vivo.

Absence epilepsy is a neurological disorder that commonly occurs in children. Some studies have shown that absence seizures predominantly originate either in the thalamus or the cerebral cortex. Some cerebellar nuclei (CN) neurons project to these brain areas, as explained further in Fig. 2.6 in Chapter 2. Also, some CN neurons have been observed to show modulation during the absence seizures. This indicates that they somehow participate in the seizure and hence are referred to as "participating neurons" in this thesis. In this research, I demonstrate how machine learning techniques and computer simulations can be applied to investigate the properties and the input conditions present in these participating neurons. My investigation found a sub-group of CN neurons, with similar interictal spiking activity, spiking activity between the seizures, that are most likely to participate in seizures. To investigate the input conditions present in the CN neurons that produce this type of interictal activity, I used a morphologically realistic conductance based model of an excitatory CN projection neuron [66] and optimised the input parameters to this model using an Evolutionary Algorithm (EA). The results of the EA revealed that these participating CN neurons receive a synchronous and bursting input from Purkinje cells and bursting input with long intervals(approx. 500ms) from mossy fibre. The same interictal activity can also be produced when the Purkinje cell input to the CN neuron is asynchronous. The excitatory input in this case also had long interburst intervals but there is a decrease in excitatory and inhibitory synaptic weight. Surprisingly, a slight change in these input parameters can change the interictal spiking pattern to an ictal spiking pattern, the spiking pattern observed during absence seizures. I also discovered that it is possible to prevent a participating CN neuron from taking part in the seizures by blocking the Purkinje cell input.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1(-/-) mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1(nmf164) for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1(nmf164)/Npc1(nmf164) pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1(nmf164) homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-beta-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs. These findings indicate that in Npc1(nmf164) homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.

Simple circuits perform simple tasks. Complex circuits can perform more complicated tasks. This is true for artificial circuits and for brain circuits. As is known from artificial networks, a complexity that makes circuits substantially more powerful is distributing learning across multiple layers. In fact, most brain circuits in vertebrate systems are multi-layer circuits (but for few that perform simple reflexes) in which learning is distributed across layers. Despite the crucial contribution of learning in middle layer neurons to the output of the circuits they are embedded in, there is little understanding of the principles defining this contribution. A very common feature in brain circuits is that middle layer neurons generate two types of signals, known as spikes. These middle layer neurons commonly have long dendrites where they generate dendritic spikes. As well, like most neurons, they generate axonal spikes near the cell body. Neurons exhibiting these two spike types include pyramidal cells in the neo-cortex and the hippocampus, the Purkinje cells in the cerebellum and many more. In this thesis I study another circuit that contains middle layer neurons, the electrosensory lateral lobe (ELL) of the mormyrid fish. The ELL is a tractable brain circuit in which the middle layer neurons generate dendritic and axonal spikes. In this thesis I show that these spike types are not two different expressions of the same inputs. Rather, they have a symbiotic relationship. Instead of all inputs triggering both spikes, some inputs can selectively drive dendritic spikes. The dendritic spikes in return modify the synaptic strength of another set of inputs. The modified inputs are then transmitted to downstream neurons via the axonal spikes, which contributes a desired signal to the output of the circuits. Effectively there is a separation of learning and signaling in the middle layer neurons through the two spike types. Having two types of spikes in the same neuron doing different computations enormously expands the computational power of the neuron. But, being in the same neuron means the separation of function is constrained and needs to be supported by biophysical principles. I have thus built a biophysical model to understand the biophysical principles underlying the separation of function. I show that in the middle layer neurons of the ELL, the axonal spikes are strongly reduced in amplitude as they backpropagate to the apical dendrites, yet they remain crucial in driving dendritic spikes. Critically, modulation of inhibitory inputs can selectively dial up or down the ability of the backpropagating axonal spikes to drive dendritic spikes. Thus, a set of inhibitory modulating inputs can selectively modulate dendritic spikes. Having learning in different layers contributing to the outcome of the circuit, naturally leads to asking how the work is divided across layers and neuron types within the circuit. In this thesis I answer this question in the context of the outcome of the ELL circuit. Finally, another signature of a complex circuit is the ability to integrate many different inputs, usually in middle layer neurons, to generate sophisticated outputs. A goal for scientists studying systems neuroscience is to understand how this integration works. In this thesis I provide a coherent model of a learning behavior called vestibulo occular reflex (VOR) adaptation, that depends on the integration of separate inputs to yield a learned behavior. The VOR is a simple reflex generated in the brain stem. Inputs from the brain stem are also sent to an area in the cerebellar cortex called the flocculus. The flocculus also receives another set of inputs that generate a different behavior, called smooth-pursuit. The integration of VOR inputs with smooth-pursuit inputs in the flocculus generate VOR adaptation. Understanding complex circuits is one of the greatest challenges for today's neuroscientists. In this thesis I tackle two such circuits and hope that through a better understandings of these circuits we gain principles that apply to other circuits and thereby advance our understanding of the brain.

The aim of this study is to contribute to the standardization of the new diagnostic tools assessing the motor manifestations of laterality in adults and children aged 8 to 10 years, both in terms of determining the theoretical concept and the selection of appropriate items, and the verification of structural hypotheses concerning the design of acceptable models, including the diagnostic quality of individual parts of the test battery. Moreover in this study we try to suggest new approach in assessing of motor laterality manifestation by means of relationship between cerebellar dominance and hand performance. The first part of this thesis deals with the concept of laterality, its manifestations and meaning in non-living systems and living organisms. As a human characteristic, laterality is manifested in a variety of functional and structural asymmetries. This part also discusses ways of diagnosing motor manifestations of laterality and the issue of cerebellar dominance, including its reflection in the form of asymmetry of the extinction physiological syndrome of upper limbs. The second part focuses on the process of the standardization study, the statistical method of structural equation modelling, and the actual design of test battery construction. The last part of this thesis presents the results...

The prevalence of carriers of three genetic disorders, severe combined immunodeficiency, lavender foal syndrome and cerebellar abiotrophy, in registered, purebred Arabians in South Africa was assessed. Genotyping for the three disorders was performed on individuals randomly selected from two populations: purebred Arabian horses born in South Africa during the intervals 1 August 2004 to 31 July 2005 and 1 August 2009 to 31 July 2010, in line with physiological breeding seasons. This permitted an estimation of the change in prevalence of each disorder between 2004 and 2009, during which time compulsory testing for SCID, and selective breeding based on the results thereof, was performed. The prevalence of lavender foal syndrome and cerebellar abiotrophy for the 2009 breeding season was found to be 11.7% (95% confidence interval [CI] 7.80 – 16.37) and 5.1% (95% CI 2.56 – 8.69) respectively, with no statistically significant change in the prevalence of these disorders between 2004 and 2009. Utilizing a larger sample size, the prevalence of severe combined immunodeficiency was found to have decreased significantly from 6.4% (95% CI 6.05 – 6.78) in 2004 to 3.4% (95% CI 2.67 – 4.23) in 2009 (P < 0.0001). These results will encourage the genetic screening of Arabian horses intended for breeding purposes in order to prevent the birth of clinically affected individuals. This study also highlights the usefulness of genetic testing as a tool to decrease the prevalence of specific genetic disorders within animal populations. / Dissertation (MSc)--University of Pretoria, 2012. / Production Animal Studies / unrestricted

The overall architecture and cell types are highly conserved from mammals to teleost fish. The rapid transparent ex utero development in zebrafish allows direct access and precise visualization of all the major events in cerebellar development. The superficial position of the cerebellar primoridum and cerebellum further facilitates in vivo imaging of cerebellar structures and developmental events at cell resolution. Furthermore, zebrafish model have a comprehensive genetic toolbox that allow forward and reverse genetic approaches to study and manipulate gene function. Consequently, zebrafish is emerging as an excellent vertebrate model for studies of molecular, cellular and physiological mechanisms involved in cerebellar development and function at gene, cell and circuit level.

The aim of this study is to contribute to the standardization of the new diagnostic tools assessing the motor manifestations of laterality in adults and children aged 8 to 10 years, both in terms of determining the theoretical concept and the selection of appropriate items, and the verification of structural hypotheses concerning the design of acceptable models, including the diagnostic quality of individual parts of the test battery. Moreover in this study we try to suggest new approach in assessing of motor laterality manifestation by means of relationship between cerebellar dominance and hand performance. The first part of this thesis deals with the concept of laterality, its manifestations and meaning in non-living systems and living organisms. As a human characteristic, laterality is manifested in a variety of functional and structural asymmetries. This part also discusses ways of diagnosing motor manifestations of laterality and the issue of cerebellar dominance, including its reflection in the form of asymmetry of the extinction physiological syndrome of upper limbs. The second part focuses on the process of the standardization study, the statistical method of structural equation modelling, and the actual design of test battery construction. The last part of this thesis presents the results...