The purpose of this thesis project was to elucidate the immune transcriptomic changes in the diaphragm of mdx mice treated with microdystrophin gene therapy with and without running wheel activity. Mdx mice are a model of Duchenne Muscular Dystrophy (DMD). Similar to DMD, mdx pathophysiology is associated with chronic inflammation due to sarcolemma fragility and cellular membrane leakage. Immune modulation has not yet been described when endurance exercise and AAV-microdystrophin gene therapy have been combined in mdx mice. An increase of physical activity in DMD individuals is a potential outcome of current clinical studies investigating microdystrophin treatment; therefore, understanding the impacts of physical activity on the immune system, particularly for the diaphragm, may be important to minimize risk. Recently, the Grange lab published the endurance and contractile property outcomes of combined microdystrophin gene therapy and running wheel activity in mdx mice.1 Diaphragm RNA-seq transcriptomic data were also collected from this study for gene expression analysis. Using this dataset, I tested the hypothesis that relative to mdxGT (mdx mice treated with gene therapy), transcripts related to the immune response such as immune cell recruitment, activation, and downstream signals that promote fibrosis deposition were unchanged or downregulated in mdxRGT (mdx mice treated with gene therapy and access to running wheel). DEGs (differentially expressed genes) were analyzed with Microsoft Excel, R, and bioinformatic tools such as KEGG and DAVID to explain immune system adaptations in response to combined microdystrophin treatment and running in mdx mice. Two major inflammatory signaling pathways, the IL-6/JAK/STAT and NF-kB signaling pathways translationally relevant to DMD patients were rescued by gene therapy towards WT expression levels. Although running maintained the majority of the rescued transcriptome profile (691 of 724 genes), some immune response-related gene expressions (33 of 724 genes) were modulated including genes related to chemotaxis and cellular migration. These changes suggested potential signaling for angiogenesis and a fast to slow fiber type shift; however, unbiased analysis with bioinformatic tools did not confirm either of these possibilities. The data from this study revealed inflammatory and fibrotic signaling pathways commonly observed in DMD patients and mdx mice were rescued by the AAV microdystrophin gene therapy and were maintained by voluntary wheel running / Master of Science / Duchenne Muscular Dystrophy (DMD) is an X chromosome-linked muscular dystrophy, a genetic disease that affects around 1 in 14,000 boys globally. DMD is lethal and currently there is no cure. Mutations in the DMD gene results in the absence of the protein dystrophin. The dystrophin protein and other proteins associated with it provide structural support to the skeletal muscle membrane. Without it, muscles are more easily damaged during contraction. This damage promotes recruitment of immune cells which initiates the first stage of muscle repair. Under normal circumstances, this inflammatory reaction caused by immune cells restores the skeletal muscles. However, in DMD patients, repeated breakdown and regeneration of skeletal muscles leads to abnormal inflammation which promotes negative outcomes such as increased fibrosis. Fibrosis impairs muscle function, especially the diaphragm . Hamm et al., 2021 from the Grange lab investigated the effects of microdystrophin gene therapy and increased physical activity in mdx mice, a mouse model of DMD, with the idea that some of the negative changes with muscular dystrophy could be improved. The results showed a positive increase of endurance capacity in mdx mice treated with gene therapy alone (mdxGT group) and a greater increase if the mice also used a running wheel (mdxRGT group) compared to untreated mdx mice (mdx group). These findings suggested that gene therapy can increase a DMD patient's ability to become more physically active. However, the effects of running and microdystrophin gene therapy on the damaging inflammatory response in the diaphragm were not reported. To address this question, gene expression data from diaphragm muscles of all treatment groups were collected in the Hamm et al., 2021 study for later analysis. In my study, these diaphragm gene expression data were used to compare inflammatory signals between the various treatment groups. Indicators of skeletal muscle damage, immune cell accumulation and fibrosis deposition were rescued (i.e., returned to healthy mice levels) by microdystrophin gene therapy (mdxGT group). Running did not exert any negative effects on the majority of genes rescued by the microdystrophin therapy (mdxRGT group). These results indicated that voluntary wheel running could maintain the reduced inflammatory signals due to the microdystrophin gene therapy in mdx mice. If the function of the skeletal muscle of dystrophic boys was similarly improved by microdystrophin gene therapy and exercise did not interfere with its positive effects, DMD boys could potentially be physically active similar to normal boys of their age.

Submitted by Rosa Assis (rosa_assis@yahoo.com.br) on 2017-09-21T15:16:11Z No. of bitstreams: 2 FABIANA APARECIDA RODRIGUES.pdf: 13689288 bytes, checksum: dcec7a1cd26a4ef822b626be4130a613 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Paola Damato (repositorio@mackenzie.br) on 2017-09-22T13:33:31Z (GMT) No. of bitstreams: 2 FABIANA APARECIDA RODRIGUES.pdf: 13689288 bytes, checksum: dcec7a1cd26a4ef822b626be4130a613 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-22T13:33:32Z (GMT). No. of bitstreams: 2 FABIANA APARECIDA RODRIGUES.pdf: 13689288 bytes, checksum: dcec7a1cd26a4ef822b626be4130a613 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-24 / The mechanisms for managing evaluation and analysis of writing are strategies that may help to follow the tracks of information generated throughout e-learning courses. In this research, we detail the course of techniques application and presentation of results by means of reports. Such presentation was conditioned to the set of themes and to the evaluation criterion of Discussion Forum, which was restricted to the development of a set of analysis techniques about data generated in on-line courses interactions of a school institution, in order to enable the visualization and provide conditions to evaluate the quality of posts, as professors/tutors face di culties in following the discussions due to the quantity of interactions. For this reason, this study adopted as strategy a qualitative and quantitative approach that aimed to analyze both quantity of posts of 841 participants of 18 classes within the discussion forums universe where we may nd problems, and quality of students comments during the analysis, by checking if they were suitable and if they met the objective proposed. The objective of this research was extracting comments and having relevant information about the posts. To achieve this purpose, it was developed a set of techniques in an R Markdown le to present reports with results of the evaluation based on Data Mining (DM), speci cally Text Mining (TM) and exploratory analysis, whose investigations were summative and formative. In this way, we hope that the action developed in this research may support managers to analyze students contributions and follow the work of the professional in charge of class learning. The methodology showed and the indexes used may be applied in any type of text database. / Os mecanismos voltados para controle de avaliação e análise da produção textual são estratégias que podem auxiliar o acompanhamento de informacões geradas ao longo dos cursos na modalidade a distância. Nesta pesquisa, descreve-se o percurso de aplicação de técnicas e demonstração de resultados por meio de relatórios. Tal demonstração esteve condicionada á temática e aos critérios de avaliação de Fórum de Discussão, restrita ao desenvolvimento de conjunto de técnicas de análise, relacionadas aos dados gerados na interação de cursos on-line de uma instituição de ensino, a fim de facilitar a visualização e oferecer condições de avaliação da qualidade das postagens, vito que professores/tutores têm di ficuldade de seguir as discussões dado o número de participações. Por isso, este estudo teve como estratégia a abordagem quali-quantitativa que visou analisar tanto a quantidade de postagens de 841 participantes, de 18 salas, no universo dos fóruns em que residem problemas detidos, quanto a qualidade dos comentários dos estudantes durante a análise, veri cando se foram satisfatórios e atingiram o objetivo proposto. O objetivo desta pesquisa foi extrair comentários e obter informações relevantes sobre as postagens. Para alcançá-lo, foi elaborado um conjunto de técnicas em R Markdown para apresentar, por meio de relatórios, os resultados da avaliação baseada na linha da Mineração de Dados (MD), em especial a Mineração de Textos (MT) e análises exploratórias, cujas investigacões foram do tipo somativa e formativa. Dessa forma, espera-se que a ação desenvolvida nesta pesquisa possa auxiliar gestores e professores na tarefa de examinar as contribuições dos alunos e acompanhar o trabalho do pro fissional responsável pelo ensino da turma. A metodologia demonstrada e os indicadores utilizados podem ser aplicados para qualquer tipo de base de dados textual.

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