Injury and neurodegenerative conditions of the spinal cord can lead to paralysis and loss of sensation. Cell therapeutic approaches can restore sensory innervation of the spinal cord following injury and protect spinal cord cells from degeneration. This thesis primarily focuses on the restoration of deaffarented sensory fibres following injury to the dorsal root and spinal cord. These injuries lead to the formation of a non-permissive glial scar that prevents sensory axons from reinnervating spinal cord targets. It takes advantage of a dorsal root injury model that closely mimics spinal root avulsion injuries occurring in humans. In the first part of the thesis, three different neural progenitor types from human or murine sources are tested for their regenerative properties following their transplantation to the site of dorsal root avulsion injury. In the second part, the ability of murine neural progenitors to protect spinal motor neurons from a neurodegenerative process is tested. In the first original research article, I show that human embryonic stem cell derived neural progenitors are able to restore sensorimotor functions, mediated by the formation of a tissue bridge that allows ingrowth of sensory axons into the spinal cord. In the second research article, I present that murine boundary cap neural crest stem cells, a special type of neural progenitor that governs the entry of sensory axons into the spinal cord during development, are unable to form a permissive tissue bridge. This is possibly caused by the contribution of transplant derived ingrowth non-permissive glial cells. In the third research article, I show that human neural progenitors derived from foetal sources are capable of stimulating sensory ingrowth and that they ameliorate the glial scar. When this approach is combined with the delivery of sensory outgrowth stimulating neurotrophic factors, these cells fail to form a permissive tissue bridge and fail to modify the glial scar. In the final research article, murine boundary cap neural crest stem cells are shown to protect motor neurons, which harbor an amyotrophic lateral sclerosis causing mutation, from oxidative stress. Oxidative stress is a pathological component of amyotrophic lateral sclerosis in human patients. Taken together, this thesis provides first evidence that sensory regeneration following a spinal root avulsion injury can be achieved by transplantation of human neural progenitors. In addition, it introduces murine boundary cap neural crest stem cells as interesting candidates for the cell therapeutic treatment of amyotrophic lateral sclerosis.

Drugs of abuse have a long history in humanity. Currently however, a subject of great interest is the phenylalkylamine family of drugs. Not only is the abuse liability of interest but also the potential therapeutic expansion of the capabilities of this family of drugs by utilizing the unique stereospecific effects of the newly discovered hybrid compounds. Based upon prior data of N-Alkyl 4-MA the enantiomers of N-Methyl, N-Ethyl, and N-Propyl were analyzed in hDAT, hNET, and hSERT. It was found that there was a negative correlation between chain length and potency and dopaminergic component. In agreement with the currently established paradigm it was also found that in almost all cases the S(+) enantiomer was the more potent.

Current clinical practice of resective surgery in focal epilepsy involves electroencephalogram (EEG) brain source imaging to localize irritative brain areas from where Inter-ictal epileptiform discharges (IEDs) emerge, useful to localize the seizures-onset zones. Unfortunately, there are no previous systematic studies to characterize the pathophysiological mechanisms and abnormal cellular substrates in these irritative areas since histological data are available only from the final resective zones. To address this issue we applied a combination of EEG brain imaging described by Bae et al. (2015) using cutting-edge technology for high-density scalp EEG in rodents and histological analysis on a chronic rat model of focal cortical dysplasia. Post-mortem brain sections were stained for anatomical, functional and inflammatory biomarkers. Abnormal anatomical structures and increased expression of inflammatory biomarkers were found in the irritative regions. We conclude that IED-based brain source imaging can help to localize abnormal tissues highly prospective for epileptogenesis.

This thesis describes the development of an optimized method for culturing dissociated, monolayer neuronal networks from murine frontal cortex and midbrain. It is presented as a guidebook for use by cell culture specialists and laboratory personnel who require updated and complete procedures for use with microelectrode array (MEA) recording technology. Specific cell culture protocols, contamination prevention and control, as well common problems encountered within the cell culture facility, are discussed. This volume offers value and utility to the rapidly expanding fields of MEA recording and neuronal cell culture. Due to increasing interest in determining the mechanisms underlying Parkinson's disease, the newly developed procedures for mesencephalon isolation and culture on MEAs are an important research contribution.

Orientadores: José Antônio Brum, Marcus Aloizio Martinez de Aguiar / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-29T15:40:55Z (GMT). No. of bitstreams: 1 Reis_ElohimFonsecados_M.pdf: 2277988 bytes, checksum: 08f2c3b84a391217d575c0f425159fca (MD5) Previous issue date: 2015 / Resumo: Este trabalho é dividido em duas partes. Na primeira parte, uma rede de correlação é construída baseada em um modelo de Ising em diferentes temperaturas, crítica, subcrítica e supercrítica, usando um algorítimo de Metropolis Monte-Carlo com dinâmica de \textit{single-spin-flip}. Este modelo teórico é comparado com uma rede do cérebro construída a partir de correlações das séries temporais do sinal BOLD de fMRI de regiões do cérebro. Medidas de rede, como coeficiente de aglomeração, mínimo caminho médio e distribuição de grau são analisadas. As mesmas medidas de rede são calculadas para a rede obtida pelas correlações das séries temporais dos spins no modelo de Ising. Os resultados da rede cerebral são melhor explicados pelo modelo teórico na temperatura crítica, sugerindo aspectos de criticalidade na dinâmica cerebral. Na segunda parte, é estudada a dinâmica temporal da atividade de um população neural, ou seja, a atividade de células ganglionares da retina gravadas em uma matriz de multi-eletrodos. Vários estudos têm focado em descrever a atividade de redes neurais usando modelos de Ising com desordem, não dando atenção à estrutura dinâmica. Tratando o tempo como uma dimensão extra do sistema, a dinâmica temporal da atividade da população neural é modelada. O princípio de máxima entropia é usado para construir um modelo de Ising com interação entre pares das atividades de diferentes neurônios em tempos diferentes. O ajuste do modelo é feito com uma combinação de amostragem de Monte-Carlo e método do gradiente descendente. O sistema é caracterizado pelos parâmetros aprendidos, questões como balanço detalhado e reversibilidade temporal são analisadas e variáveis termodinâmicas, como o calor específico, podem ser calculadas para estudar aspectos de criticalidade / Abstract: This work is divided in two parts. In the first part, a correlation network is build based on an Ising model at different temperatures, critical, subcritical and supercritical, using a Metropolis Monte-Carlo algorithm with single-spin-flip dynamics. This theoretical model is compared with a brain network built from the correlations of BOLD fMRI temporal series of brain regions activity. Network measures, such as clustering coefficient, average shortest path length and degree distributions are analysed. The same network measures are calculated to the network obtained from the time series correlations of the spins in the Ising model. The results from the brain network are better explained by the theoretical model at the critical temperature, suggesting critical aspects in the brain dynamics. In the second part, the temporal dynamics of the activity of a neuron population, that is, the activity of retinal ganglion cells recorded in a multi-electrode array was studied. Many studies have focused on describing the activity of neural networks using disordered Ising models, with no regard to the dynamic nature. Treating time as an extra dimension of the system, the temporal dynamics of the activity of the neuron population is modeled. The maximum entropy principle approach is used to build an Ising model with pairwise interactions between the activities of different neurons at different times. Model fitting is performed by a combination of Metropolis Monte Carlo sampling with gradient descent methods. The system is characterized by the learned parameters, questions like detailed balance and time reversibility are analysed and thermodynamic variables, such as specific heat, can be calculated to study critical aspects / Mestrado / Física / Mestre em Física / 2013/25361-6 / FAPESP

Self-injurious behavior (SIB) is a complex phenotype that occurs with an increasing prevalence of about 7-34% in humans and 10-12% in non-human primates (NHPs). This study evaluated the efficacy of probiotic Lactobacillus reuteri as a treatment for self-injurious behavior (SIB) and sleep disruption in rhesus macaques. The treatment was proposed to alleviate mild self-biting, sleep disruption, and reduce chronically elevated hypothalamic-pituitary-adrenocortical (HPA) axis activity, all hallmark features of monkeys with this condition. The probiotic preparation included two strains of L. reuteri (L. reuteri ATCC PTA 6475 & L. reuteri DSM 17938) containing on average 200 million colony forming units per chewable tablet. The study was conducted on 14 rhesus macaque monkeys (9 males) housed at the University of Massachusetts Amherst. To our knowledge this is the first time that a Lactobacillus strain has been used as a treatment for SIB in rhesus macaques. This study utilizes motion-activated infrared camera technology, modified enzyme-immunosorbent-assays (EIAs) techniques to measure hair cortisol concentrations, and daily behavioral observations to provide an overall assessment of the behavioral, physiological, and sleep associated implications of probiotic treatment on SIB and control non-human primates (NHPs). Administration of L reuteri modestly decreased biting behavior in monkeys with SIB (F(2,12) = 5.64, p= 0.02) and showed overall decrease in nighttime activity across all subjects but did not normalize SIB to nonSIB values. Hair cortisol values are pending. These findings and the findings of previous work further strengthen the argument for probiotics as an efficacious treatment for SIB behavior.

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Age-related macular degeneration (AMD) is the leading cause for visual impairment in the elderly. The etiology of AMD remains unclear. Clinical and histopathological studies suggest that photoreceptors play a role in disease progression. Here, we found that photoreceptors of AMD patients show adaptive changes in gene expression, suggestive of a nutrient shortage. To study the effect of these changes, we mimicked the metabolic alteration in mouse photoreceptors, by disruption of the Tuberous Sclerosis Complex (TSC). This led to AMD hallmarks, including the advanced stages of geographic atrophy (GA) and choroidal neovascularization (CNV). Furthermore, we found that disease onset requires the activity of the mammalian target of rapamycin complex 1 (mTORC1). To study the contribution of photoreceptors to disease, we profiled retinal phospholipids as photoreceptors are rich in phospholipids. We found a reduction in two docosahexaenoic acid (DHA)-containing phospholipids. Feeding DHA to mutant mice, alleviated most AMD-associated hallmarks. To study the inflammatory complications seen with current anti-vascular endothelial growth factor (VEGF) treatments for CNV we used rAAV-mediated gene transfer to overexpress an anti-VEGF protein. We found that inhibition of VEGF can promote retinal inflammation. The data suggests that targeting photoreceptor metabolism may provide novel therapies to treat AMD.

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