Abstract: Molecular and cellular insights into IKAP and Elongator functions As the first step in the complex process of gene expression, the transcription of genes from DNA to RNA by RNA polymerase II is subject to a multiplicity of controls and is thereby the endpoint of multiple cell regulatory pathways. We focused here on the molecular and cellular functions of IKAP and by extension of Elongator complex, initially found associated with the hyperphosphorylated RNA polymerase II during the elongation stage of transcription. IKAP is required for the assembly of Elongator subunits into a functional complex. Elongator has a histone acetyltransferase (HAT) activity associated with one of its subunits, named hELP3. In agreement with a potential role in transcript elongation, Elongator is associated with nascent RNA emanating from the elongating RNA polymerase II along the transcribed region of several yeast genes and chromatin immunoprecipitation experiments have also demonstrated an association of Elongator with genes in human cells. Different mutations in the human IKBKAP gene, encoding IKAP/hELP1, cause familial dysautonomia, a severe neurodevelopmental disease with complex clinical characteristics. Affected individuals are born with the disease and abnormally low numbers of neurons in peripheral nervous ganglions. To gain insight into the role played by IKAP and the Elongator complex in the transcription of genes and concomitantly learn about the molecular defects underlying the FD, an RNA interference approach was used to deplete the IKAP protein in human cells. In yeast, disruption of ELP1 (yeast homolog of human IKAP) is known to destabilize the ELP3 catalytic subunit, which leads to loss of Elongator integrity. Our experiments performed in human cells revealed that the levels of hELP3 protein is also affected by IKAP depletion after RNAi. We took advantage of this cellular loss-of-function model to identify genes whose transcription requires IKAP, by microarray experiments. Among the identified candidates, several were previously described to be involved in cell motility, or actin cytoskeleton remodelling. Because cell motility is of crucial importance for the developing nervous system, and therefore of obvious relevance to FD, the potential role of IKAP in cell motility was characterized at the cellular level. Several cell motility/migration assays demonstrated that the IKAP depletion has functional consequences so that IKAP-depleted cells showed defects in migration. Particularly, the reduced cell motility of neuronal-derived cell lines may be highly relevant to the neurodevelopmental disorder that affects FD patients. Whether or not the defects in cell migration resulted of impaired transcriptional elongation of the IKAP-dependent genes was investigated by chromatin immuno-precipitation technique. These experiments indicated that IKAP depletion leads to a decreased histone H3 acetylation in the transcribed region of its target genes in the context of Elongator complex. These acetylation defects are correlated with a decrease of the RNA polymerase II recruitment through the transcribed region of target genes, whereas the recruitment on the promoter is mostly unaffected. These results indicate that Elongator affects transcript elongation in vivo, but not the recruitment of the RNA polymerase II to the promoter. These very specific effects of IKAP/hELP1 depletion on histone acetylation and RNA polymerase II density across target genes are consistent with a direct effect of Elongator on transcriptional elongation in vivo and point to a function for Elongator in histone acetylation during transcript elongation. Résumé: Caractérisation des rôles biologiques de la protéine IKAP et du complexe Elongator La transcription des gènes de lADN en ARN est fondamentale pour lexpression des protéines et la capacité de nos cellules à sadapter à leur environnement. Ce processus finement régulé est catalysé par un enzyme, lARN polymérase II, vers lequel convergent une multitude de voies de signalisation. Dans le cadre de ce travail, nous nous sommes intéressés aux fonctions moléculaires et cellulaires de la protéine IKAP et du complexe Elongator. IKAP est la protéine qui assemble les sous unités dElongator en un complexe fonctionnel. Le complexe Elongator est associé à lARN polymérase II hyper-phosphorylée pendant létape délongation de la transcription et possède une activité histone acétyltransferase associée à une de ses sous unités, appelée ELP3. Chez la levure, Elongator est recruté an niveau des ARNs naissants, qui émanent directement de lARN polymérase II au niveau de la région transcrite des gènes étudiés. De plus, des expériences dimmunoprécipitation de la chromatine ont mis en évidence la présence du complexe Elongator au niveau de plusieurs gènes humains. Différentes mutations au niveau du gène IKBKAP, codant pour la protéine IKAP, sont responsables de la dysautonomie familiale, une maladie génétique qui affecte le développement du système nerveux périphérique. En effet, les individus affectés présentent une diminution de la densité de neurones au niveau des ganglions nerveux périphériques. Lobjectif de nos travaux est de comprendre davantage le rôle de la protéine IKAP et du complexe Elongator dans la transcription des gènes et ainsi, dinvestiguer les mécanismes moléculaires responsables dans la physiopathologie de la dysautonomie familiale. Un modèle de perte de fonction pour la protéine IKAP a dabord été généré par interférence dARN. Des travaux réalisés chez la levure indiquent que la protéine ELP1 (homologue de IKAP chez la levure) est essentielle pour la stabilité de la sous unité catalytique du complexe, la protéine ELP3. Les expériences réalisées sur notre modèle humain démontrent que le taux de la protéine ELP3 est également affecté par la déplétion dIKAP causée par linterférence dARN. Ce modèle de perte de fonction a été utilisé afin détablir la liste des gènes dont lexpression est contrôlée par la protéine IKAP, par des expériences de microarrays. Parmi les candidats identifiés, plusieurs ont été décrits comme impliqués dans la migration cellulaire et le remodelage du cytosquelette dactine. Le processus de migration des cellules est fondamental au cours du développement du système nerveux et par conséquent particulièrement relevant dans le contexte de la dysautonomie familiale. Limplication dIKAP dans la migration cellulaire a été investigué par différents tests de fonction qui montrent que la diminution dIKAP dans différentes lignées cellulaires entraîne une réduction significative de leur capacité migratoire. Ces résultats suggèrent que la diminution du nombre de neurones observée dans les ganglions périphériques des patients atteints de la dysautonomie familiale pourrait résulter dune altération de leur capacité à migrer au cours du développement. Enfin, des expériences dimmunoprécipitation de la chromatine ont été menées en utilisant notre modèle afin de déterminer dans quelle mesure le déficit de migration observé en labsence dIKAP serait la conséquence dun défaut de la fonction dElongator au niveau de lélongation de la transcription des gènes. Les résultats nous ont montré que la diminution dexpression dIKAP entraîne une réduction de lacétylation des histones H3 dans la région transcrite de ses gènes cibles. De plus, ce déficit dacétylation est directement corrélé avec un désengagement progressif de lARN polymérase II le long de la région transcrite de ces gènes. Par conséquent, ces résultats démontrent que le complexe Elongator affecte lélongation des transcrits in vivo, mais pas le recrutement de lARN polymérase II au niveau du promoteur. Ces effets très spécifiques de labsence dIKAP sur lacétylation des histones et lengagement de la polymérase II dans la transcription des gènes cibles montrent quElongator exerce un rôle direct au niveau de lélongation de la transcription de ces gènes. De plus, ces résultats suggèrent que la fonction dElongator serait dacétyler les histones au cours de lélongation transcriptionnelle in vivo.

This mixed-media memoir uses a variety of forms from short epigrammatic essays to straightforward stories and graphic narratives to explore the author’s coming-of-age experiences augmented by chronic illness. Trying to succeed in the film industry, romance, and family situations, the young female narrator navigates the often unexpected or disappointing consequences of having an autonomic nervous system disorder. Relationships between conflicting identities emerge—between healthy versus sick self, projected/envisioned versus actual self, and tough versus vulnerable self—as the narrator journeys toward a more complete and accepting self-understanding.

Made available in DSpace on 2014-07-29T15:25:14Z (GMT). No. of bitstreams: 1 Tese de doutorado Luiza Cristina Lacerda Jacomini.pdf: 1113139 bytes, checksum: 6f7dbdd64a41cff3100ce05df4ddcf61 (MD5) Previous issue date: 2010-08-05 / Lacerda Jacomini, LC. Investigation on dysautonomia, epinephrine and norepinephrine-evoked pain, and effect of beta-blocker in fibromyalgia and systemic lupus erythematosus. 2010, 169 p. Doctoral thesis - Universidade Federal de Goiás, Goiânia. Dysautonomia is a condition in which an altered autonomic function affects the health in an adverse way. The present study aims: to search for the presence of epinephrine and norepinephrine-evoked pain; to evaluate the cardiovascular autonomic function and the effect of propranolol in women with fibromyalgia (FM), systemic lupus erythematosus (SLE) and controls (CTR). For each objective a separate research was developed, including a clinical trial. Epinephrine and norepinephrine-evoked pain were diagnosed when the subcutaneous injections containing these substances (10 micrograms/ 0.1 mL saline solution) induced greater pain than the saline solution did (n=7). Autonomic function was assessed through the standard Ewing tests battery, through heart rate responses to Valsalva maneuver, deep breathing, standing and blood pressure responses to ortostatism and to hand grip (n=7). Functional symptoms related to autonomic manifestations were checked. In a randomized, double-blind, placebo-controlled, crossover clinical trial with 6 women with FM, SLE and CTR, propranolol (80 mg/day po/4 weeks) was added to the usual schedule of prescribed medicines and its effect was examined regarding: pain, fatigue, tender points, blood pressure, heart rate, health related quality of life (SF-36) and fibromyalgia impact questionnaire. Epinephrine-evoked pain was diagnosed in SLE and FM groups and norepinephrine-evoked pain was diagnosed in FM group. Epinephrine and norepinephrine-evoked pain intensity has a trend to be greater in FM patients when compared to healthy CTR. FM and SLE patients had an elevated number of functional symptoms related to autonomic manifestations. Cardiovascular autonomic function was altered in FM and SLE groups. Parasympathetic cardiovascular autonomic function tests were mainly abnormal in SLE patients while in FM patients both, parasympathetic and sympathetic tests were abnormal. Propranolol reduced tender points count and the number of symptoms related to autonomic manifestations in FM group. Four in six patients presented significant improvement in health related quality of life evaluated by SF-36. These results suggest that FM belong to the group of sympathetically maintained pain syndromes. The study demonstrates that FM and SLE patients have cardiovascular autonomic function alterations which can be detected by simple, standardized, non-invasive and inexpensively methodology and that propranolol has a potential benefit in FM treatment. / Lacerda Jacomini, LC. Pesquisa de disautonomia, dor evocada por adrenalina e noradrenalina e efeito de beta-bloqueador na fibromialgia e no lupus eritematoso sistêmico. 2010, 169 p. Tese de doutorado - Universidade Federal de Goiás, Goiânia. Disautonomia é uma condição na qual a função autonômica alterada afeta a saúde de modo adverso. Os objetivos do presente estudo foram: pesquisar a presença de dor evocada por adrenalina (AD) e por noradrenalina (NA), avaliar a função autonômica cardiovascular e o efeito do propranolol, em mulheres com fibromialgia (FM), lupus eritematoso sistêmico (LES) e controles (CTR). Para cada objetivo foi desenvolvida uma etapa de estudo, sendo incluído um ensaio clínico. A dor evocada por AD ou NA foi diagnosticada quando estas substâncias produziram dor maior que soro fisiológico quando injetadas (10 microgramas/ 0,1 mL de soro fisiológico), via subcutânea (n=7). A função autonômica foi testada usando-se a bateria de testes de Ewing (respostas da frequência cardíaca à manobra de Valsalva, respiração profunda e ao ortostatismo e respostas da pressão arterial ao ortostatismo e à preensão sustentada) (n=7). Foram pesquisados sintomas funcionais relacionados às manifestações autonômicas. No ensaio-clínico randomizado, duplo-cego, placebo controlado e cruzado em grupos de 6 mulheres com LES, FM e CTR, o propranolol (80 mg/dia, via oral/4 semanas) foi adicionado ao esquema terapêutico das pacientes e seu efeito avaliado segundo as variáveis: dor, fadiga, tender points, pressão arterial, frequência cardíaca, qualidade de vida e questionário do impacto da FM. A dor evocada por AD ocorreu nos grupos FM e LES comparada ao CTR e, por NA ocorreu no grupo FM. Os escores de dor evocada por AD e por NA, no grupo FM, tiveram uma tendência a serem maiores do que os do grupo CTR. Pacientes com FM e com LES apresentaram elevado número de sintomas funcionais relacionados a manifestações autonômicas. A função autonômica cardiovascular estava alterada no LES e na FM. No grupo LES, os testes de função parassimpática tiveram frequência maior de respostas anormais e no grupo FM estavam alterados tanto os da função parassimpática como da simpática. O propranolol reduziu o número de tender points e de sintomas funcionais relacionados a manifestações autonômicas no grupo FM. Quatro em 6 pacientes do grupo FM apresentaram melhora significativa na qualidade de vida avaliada pelo SF-36. Os resultados sugerem que a FM faz parte do grupo de doenças com dor simpaticamente mantida. O estudo demonstrou que as pacientes com LES e FM têm alterações da função autonômica cardiovascular detectáveis por metodologia simples, padronizada, não invasiva e de baixo custo e que o propranolol tem um benefício potencial no tratamento da FM.

“Dysautonomia” or a dysfunction of the autonomic nervous system, affects approximately 70 million children and adults worldwide. Despite this, a small fraction of studies focus on the experiences of children and teenagers' living with conditions related to dysautonomia. The aim of this study is thus to explore the experiences and perspective of teenagers living with dysautonomia, by focusing on how they describe their lives in relation to being chronically ill and how they view the relationships among themselves, their doctors and their peers. The study entails a social constructionist and an interpretative phenomenological approach, which focuses on the lived experiences of the teenagers. To collect the data, an email questionnaire1 was conducted with 16 teenagers from different parts of the world. The data was analyzed with the help of a thematic analysis. Seven themes were identified under my areas of interest; a regular day, relationships with peers and relationships with doctors. The results indicated that dysautonomia, similar to other chronic illnesses, was reducing life quality. The results also indicated that most days were spent trying to distract themselves from their illnesses. The participants described feelings related to uncertainty, fear, loneliness and grief. Difficulties remaining socially active and maintaining friendships were described by some of the participants. Relationships with peers were described as both supportive and unsupportive and were often described in terms of healthy and sick peer groups. The doctor-patient relationship was described in terms of good and bad qualities. Although all participants described feeling dismissed, belittled and accused of making their symptoms and illnesses up in their heads, some also described the opposite - the good and respectful listener, who valued the autonomy of the participants.