Induced pluripotent stem (iPS) cells generated from patients hold great promise for studying diseases that affect the central nervous system, as differentiation into the neuronal lineage creates a limitless supply of affected cells for disease study. Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder primarily caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Due to the inaccessibility of patient neurons, most of what is known about underlying phenotypes has been described using mouse models. iPS cells provide a potential solution, but reprogramming of patient cells is hampered by low efficiency, and early methods of identifying iPS cells involve transgenic techniques that are not translatable to human patient samples. The first part of this thesis describes the generation and characterization of a pluripotency reporter to address this issue. The EOS lentiviral reporter allows real-time observation of pluripotency changes during reprogramming, and is a useful tool for more efficient isolation of reprogrammed cell lines. Further, the EOS selection system can be used in a disease context to reproducibly mark and maintain disease-specific iPS cell lines for future use in disease modelling. Though iPS cells have been used to study RTT in vitro, extensive assessments of neuron function and electrophysiology have not yet been performed. In the second part of this thesis, iPS cell lines generated from a RTT mouse model were tested for their ability to model disease in vitro. Directed differentiation of multiple Mecp2-deficient and wild-type iPS cell lines to glutamatergic neurons revealed neurons that lack Mecp2 have a smaller soma size, diminished sodium currents, and are less excitable, firing fewer, prolonged action potentials that are smaller in magnitude. This deficiency in intrinsic excitability was accompanied by a dysfunction at excitatory glutamatergic synapses, which together recapitulate changes previously observed in the Mecp2-deficient mouse brain. Having accumulated counts and recordings from hundreds of neurons with consistent responses among lines, the iPS cell system is a representative model of the neuronal and synaptic defects in RTT. These results illustrate the requirement of MeCP2 in normal neuronal function, and suggest altered neuronal homeostasis or aberrant network circuitry may underlie RTT pathogenesis.

Pervasive developmental disorders are a group of neurodevelopmental-neuropsychiatric disorders that are characterized by variable and severe pervasive impairments in several areas of child development, notably social interaction, communication and imagination. They all share clinical features but differ in the severity and age of onset of the impairments. Except for Rett Syndrome (RTT), the etiology of these disorders is unknown, but there is strong evidence that genetic factors contribute to their pathogenesis. While no major genes have been linked to theses disorders linkages, association and chromosomal studies suggest that many loci may be involved. / One aim of the present study was to search for genetics variants associated with autism and other related disorders. This study represents the first family-based association study looking at the entire X chromosome using a French-Canadian autistic population, a genetically homogenous group. We found association between autism and markers at two loci. Our results support the existence of a putative gene located on the X chromosome and moreover the founder effect, in the French-Canadian population, may provide greater power to fine map disease genes especially in complex traits. / The second aim of the present thesis was to confirm the involvement of the MECP2 gene in our RTT group of patients. While we confirm the presence of mutations in this gene in our cohort of RTT patients we also demonstrated that clinical stringency greatly influences the mutation detection rate for this disorder.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references.

Made available in DSpace on 2016-03-15T19:40:41Z (GMT). No. of bitstreams: 1 CRISTIANE_AMOROSINO_DIST.pdf: 3413258 bytes, checksum: ca798f17b25ae97d4c59d537efcfb5b1 (MD5) Previous issue date: 2006-08-01 / Fundo Mackenzie de Pesquisa / The Syndrome of Rett (SR.) was described for the first time for Andreas Rett(1966). It is characterized for a neurobiologic clutter that affects the femile sex preferentially. Its cause is due to a genetic alteration to the chromosome X. Due to the complex scenario its verbal comunication and gestual are damaged in, many times when inexistent, it causes serious difficulties the social development, cognitive and emotional these girls. Even thought the high number of investigation in this area, the comunicative potential is still a doubt and is still a big challenge for education professionals relatives and health professional. The objective of this study is to prove the comunicative intensional look and evoluate receptive vocabulary. The were 14 patients studied that belong to the classic form of Rett Syndrome classified in III & IV stage of evolution of the syndrome and filedit at Associação Brasileira de Síndrome de Rett de São Paulo (ABRE-TE/SP). The equipament used on it was Eyegaze®, that evaluates and registers the visual scanning of an individual facing a visual stimulous at the screen projeited. The instruments of evolution where: Vocabulary Test per Peabody images TVIP computorized and its variation of a receptive vocabulary test with colorful pictures TVRF colorful. One Way ANOVAS reveled significant differences in the numbers of correct itens at TVIP (F]5,33]= 44,761; p<0,0000) and at TVRF (F]5,33]= 28,3131; p<0,0000) according to the age. Analyses post hoc Bonferroni has shown that there are no diference between girls with SR and the controls of 2 and 3 years old; even thougth the average age were significantly bigger than those normal children that have obtained close assertivy results in those two version of the vocabulary test. The Rett gerts who have obtaind the worst perforface at TVIP were exactly the youngest. Besides that the number of correct itens at TVRF were bigger than observed at TVIP. Such data to suggest the usage of pictures to those girls have better effect to evoluate the vocabulary of those simple drawing, such as from TVIP. The evidenced the glance with communicative intention and also demonstrated tha thouse girls receptive vocabulary can be evaluates twrough the instrument computerized TVIP and its variation of TVRF. / A Síndrome de Rett (SR) foi descrita pela primeira vez por Andreas Rett (1966). Caracteriza-se por uma desordem neurobiológica que afeta preferentemente o sexo feminino. Sua causa é decorrente de uma alteração genética ligada ao cromossomo X. Devido ao complexo quadro, sua comunicação verbal e gestual encontram-se prejudicadas e, muitas vezes, inexistentes, o que acarreta sérias dificuldades no desenvolvimento social, cognitivo e emocional dessas meninas. Apesar do crescente número de investigações nesta área, o potencial comunicativo ainda é uma incógnita e um grande desafio para educadores, familiares e profissionais da saúde. O objetivo desse estudo foi comprovar o olhar com intenção comunicativa e avaliar o vocabulário receptivo. Foram estudadas 14 pacientes pertencentes a forma clássica da Síndrome de Rett, classificadas nos estágio III e IV de evolução da síndrome e cadastradas na Associação Brasileira de Síndrome de Rett de São Paulo (ABRE-TE/SP). Foi utilizado o equipamento Eyegaze®, que avalia e registra a varredura visual de um sujeito frente a um estimulo visual projetado na tela. Os instrumentos de avaliação utilizados foram: o Teste de Vocabulário por Imagens Peabody - TVIP computadorizado e sua variação o Teste de Vocabulário Receptivo com fotos coloridas - TVRF colorido. Resultados: ANOVAS Unifatorial revelaram diferença significativa no número de ítens corretos no TVIP (F]5,33]= 44,761; p<0,000) e no TVRF( F]5,33] = 28,3131; p< 0,000) em função da idade . Análise post hoc Bonferroni mostrou não haver diferença entre meninas com SR e os controles de 2 e 3 anos de idade; embora a média de idade fosse significativamente maior do que aquelas crianças normais, elas obtiveram resultados semelhantes de acertos nas duas versões do teste de vocabulário. As meninas Rett que obtiveram pior desempenho no TVIP foram exatamente as mais novas. No entanto, o número de ítens corretos no TVRF foi maior do que o observado no TVIP. Tais dados parecem sugerir que o uso de fotos para estas meninas tem efeito melhor para a avaliação de vocabulário do que de desenhos simples, tal como o do TVIP. Os resultados da análise dos dados evidenciaram o olhar com intenção comunicativa e demonstraram que o vocabulário receptivo destas meninas pode ser avaliado através do instrumento TVIP computadorizado e sua variação, o TVRF.

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Although the central nervous system was long perceived as the ivory tower without immune entities, there is growing evidence that the immune and nervous systems are intimated connected. These two systems have been shown to communicate both cellularly and molecularly under physiological and pathological conditions. Despite our increasing understanding of the interplay between these two systems, there are still numerous open questions. In this thesis, I address such unanswered questions related to: the role of microglia and their mechanism in contributing to pathologies in Rett syndrome; the beneficial effects of T-cell secreted cytokines in supporting social brain function; the evolutionary link of the interactions between the nervous and immune systems; the transcription regulation of a subset of microglia population in common neurodegenerative diseases. Collectively, the current thesis is focused on the joint frontier of bioinformatics and experimental work in neuroimmunology. A multifaceted approach, that includes transcriptomics, genomics and other biomolecular modules, was implemented to unearth signaling pathways and mechanisms underlying the presenting biological phenomena. The findings of this thesis can be summarized as follows: 1) MeCP2 acts as a master regulator in the transcriptional repression of inflammatory stimuli in macrophages; 2) T-cell secreted IFN-γ supports social brain function through an evolutionally conserved interaction between the immune and nervous systems; 3) The APOE-TREM2 pathway regulates the microglia phenotype switch in neurodegenerative diseases. Provided that recent technologies allow for readily manipulating the immune system, the findings presented herein may create new vistas for therapeutic interventions in various neurological disorders.

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