Les sarcomes sont des tumeurs rares (environ 1% des cancers) des tissus mous. Leur rareté et leur hétérogénéité clinique rend la prise en charge des patients très difficile. Ainsi, il est nécessaire d'identifier et de comprendre les mécanismes de l'oncogenèse de ces tumeurs.Pour cela, notre équipe a analysé 106 sarcomes par CGH-array et puces d'expression afin d'identifier des gènes d'intérêt pour expliquer la biologie de ces tumeurs. Grâce à des analyses intégratives bio-informatiques, deux gènes candidats ont été identifiés : les gènes RCBTB1 et DMD, dont la perte génomique dans respectivement 41% et 15% des cas et la diminution d'expression sont significativement associées à l'évolution métastatique. Nos résultats montrent que la délétion de RCBTB1 est pronostique de l'évolution métastatique. Il semblerait que cela soit dû à la sensibilisation des cellules à certaines chimiothérapies, plus particulièrement le docétaxel, lorsque RCBTB1 est surexprimé. En effet, nous avons pu montrer qu'une surexpression de ce gène augmentait le pourcentage de cellules en mitose or, le docétaxel agissant uniquement en mitose, ceci explique la sensibilisation des cellules au docétaxel. Enfin, nous avons vérifié les effets de la sensibilisation au docétaxel in vivo, en étudiant la croissance tumorale après traitement en fonction de l'expression de RCBTB1.Concernant DMD, nos résultats obtenus par RNA-seq montrent que seules deux isoformes sont exprimées dans les tumeurs d'origine myogénique : Dp427 et Dp71. Cependant, dans environ 20% des tumeurs, nous observons une délétion ciblant spécifiquement l'isoforme Dp427. Etant donné que la délétion de Dp427 n'a pas d'effet sur l'agressivité des cellules, j'ai testé l'hypothèse selon laquelle Dp71 aurait un rôle oncogénique. Ainsi, l'inhibition de Dp71 révèle qu'elle est nécessaire à la croissance cellulaire et à la formation de clones, car son inhibition entraîne un blocage du cycle cellulaire en phase G2/M / Sarcomas are rare malignant tumors that form a heterogeneous group with numerous histotypes and molecular subtypes. Among them, sarcomas with complex genomic profiles displaying no specific and recurrent genetic alterations represent 50% of all soft tissue sarcomas, and poorly respond to systemic treatment. It is therefore needed to improve our understanding of the mechanisms involved in response to chemotherapy and ultimately find new therapeutic targets.One hundred and six sarcoma samples were analyzed by CGH and cDNA arrays in order to establish both genomic and transcriptomic data from each sample. This analysis highlighted RCBTB1 and DMD genes which are lost and down-regulated in 41% and 15% of cases respectively. Their lost are significantly associated with metastatic evolution.Our results show that the deletion of RCBTB1 is prognostic of the metastatic evolution. It appears that this is due to the sensitization of the cells to specific chemotherapies, more particularly docetaxel, when RCBTB1 is overexpressed. Indeed, we have showed that overexpression of this gene increased the percentage of cells in mitosis. Docetaxel acting only in mitosis, this explains the sensitization of cells to docetaxel. Finally, we validated the effects of docetaxel sensitization in vivo by studying tumor growth after treatment.Concerning DMD, our results obtained by RNA-sequencing show that only two isoforms are expressed in sarcomas with complex genomics: Dp427 and Dp71. However, in 20% of tumors, we observed a deletion specifically targeting the Dp427 isoform. Since the deletion of Dp427 did not affect aggressiveness of cells, I tested the hypothesis that Dp71 would have an oncogenic role. Thus, inhibition of Dp71 reveals that it is necessary for cell growth and for the formation of clones, because its inhibition results in blocking of the G2 / M phase cell cycle

La dystrophine Dp71, issue du gène DMD impliqué dans la dystrophie musculaire de Duchenne intervient dans le maintien de l'homéostasie rétinienne et de la barrière hémato-rétinienne. Nous avons mis en évidence une localisation rétinienne jusque-là méconnue de la Dp71 au sein des astrocytes rétiniens. Nous avons également étudié l'effet in vivo de l'absence de Dp71, sur le développement vasculaire et observé une plus faible densité et une morphologie astrocytaire différente comparativement aux souris contrôles, à l'origine d'un développement vasculaire retardé. Compte tenu de la spécificité du réseau vasculaire rétinien dont l'étanchéité des parois est maintenue en partie par les pieds des CGM et des astrocytes, nous avons émis l'hypothèse, étant donné l'implication de la Dp71 dans la stabilisation de ces cellules, d'une altération de cette protéine dans les phénomènes de rupture de la BHR. Ainsi dans un modèle transitoire de rupture de la BHR in vivo relativement " pur ", sans ischémie ni injection de VEGF, nous avons observé une diminution de l'expression de la dystrophine Dp71 ainsi que du canal aqueux AQP4 et une délocalisation du canal potassique Kir4.1 dans les CGM. L'injection intra-vitréenne de Dexaméthasone dans ce modèle a permis de prévenir les modifications d'expression et de localisation de ces protéines. / Dp71, a dystrophin produced from DMD gene, is involved in retinal homeostasis and maintenance of blood retinal barrier. We have previously shown that Dp71, part of a complex called Dystrophin associated protein, is involved in the localization of aqueous and potassic channels in Muller glial cell (MGC), particularly around blood vessels, allowing maintenance of retinal homeostasis. Based on the knowledge that growing retinal vessels migrate on an astrocyte template during development, we highlighted the expression of Dp71 in retinal astrocytes. In absence of Dp71, in vivo, we observed a lower density and a different morphology of retinal astrocytes compared to control retina in mice, responsible for a delayed vascular development. Due to the role of barrier of the retinal vascular network, insured also by astrocytes, we studied a model of post surgical BRB breakdown and found a decreased of Dp71 protein expression associated with Kir4.1 delocalization and AQP4 decrease in MGC. Intravitreal Dexamethasone prevents these protein expression changes. We suggest here that the membrane associated cytoskeletal protein, Dp71, expressed in astrocytes is involved in the maintenance of astrocytes density and morphology necessary as a template for retinal vascular development. This protein insure also a key role in retinal homeostasis by localizing and maintaining aqueous and potassic channels in MGC. Moreover when this protein is altered, Dexamethasone seems to be capable to promote Dp71 expression which could have wide clinical implications in retinal diseases treatment and the target for retinal neuroprotection under pathological conditions seems to be the macroglial cells.

Muscle stem cells self-renew to maintain the long-term capacity for skeletal muscles to regenerate. However, the homeostatic regulation of muscle stem cell self-renewal is poorly understood. By utilizing high-throughput screening and transcriptomic approaches, we identify the critical function of dystrophin, the epidermal growth factor receptor (EGFR), and fibronectin in the establishment of cell polarity and in determining symmetric and asymmetric modes of muscle stem cell self-renewal. These findings reveal an orchestrated network of paracrine signaling that regulate muscle stem cell homeostasis during regeneration and have profound implications for the pathogenesis and development of therapies for Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is an x-linked neuromuscular disease that is caused by an absence of dystrophin protein, rendering skeletal muscle more susceptible to contraction-induced damage. One therapeutic strategy focuses on increasing the expression of endogenous utrophin A, a dystrophin homologue. Interestingly, slow muscle is more resistant to the dystrophic pathology and has increased utrophin A expression (Webster 1998; Gramolini 2001b). These observations led researchers to explore the therapeutic potential of stimulating the slow, oxidative myogenic program (SOMP) in the mdx context. Beneficial adaptations were seen with pharmacological activation of PPARδ and AMPK. We treated mdx mice with resveratrol (~100mg/kg/day), a putative SIRT1 activator, for 6-7 weeks and evaluated the activity of phenotypic modifiers that are known to influence the SOMP. SIRT1 activity and protein levels increased significantly, as well as downstream PGC-1α activity. There was evidence of a fibre type conversion as the treated mice had a higher proportion of the slow myosin heavy chain isoforms in both the EDL and Soleus skeletal muscles. Utrophin A protein levels showed modest, but consistent increases with resveratrol treatment. Finally, histological analysis revealed improvements in central nucleation and fibre size variability. These findings were promising, but raised the question of whether modifying the treatment regimen may result in greater therapeutic benefits. Surprisingly, we discovered that an elevated dose of 500mg/kg/day was ineffective in its promotion of the SOMP. SIRT1 was not activated and there was no change in utrophin A levels with resveratrol treatment. Taken together, this study demonstrates that resveratrol has the ability to promote the SOMP through SIRT1 and PGC-1α activation. It also highlights the importance of selecting an appropriate dose of resveratrol to maximize its effectiveness.

Dans la rétine, la protéine dystrophine Dp71 est principalement exprimée dans les cellules gliales de Müller (CGM), qui contribuent à la stabilisation de la barrière hémato-rétinienne (BHR). Les CGM sont aussi les principales sources de facteurs inflammatoires. Ainsi, nous avons étudié les effets de l’absence de Dp71 sur l’homéostasie potassique et aqueuse, ainsi que sur l’expression de médiateurs de l’inflammation et la perméabilité vasculaire rétinienne.L'absence de Dp71 diminue l'expression de la protéine AQP4 et induit la redistribution de Kir4.1 tout le long des CGM. Par ailleurs, nous avons également constaté que le décollement expérimental de la rétine chez les souris WT induit une diminution de Dp71 associée à une délocalisation de Kir4.1, une régulation à la baisse de la protéine AQP4 dans les CGM.Nos données montrent clairement que l'absence de la Dp71 entraîne une augmentation de l'expression du VEGF, d’ICAM-1, une augmentation du nombre de leucocytes adhérents rétiniens, une dégénérescence accrue des capillaires associée à une forte perméabilité vasculaire chez les souris Dp71-null.L’ensemble de nos résultats a mis en évidence le rôle de la Dp71 dans les mécanismes visant à réguler l'homéostasie rétinienne et à assurer la stabilisation de la BHR. Nous apportons la preuve que la perte de Dp71 favorise l'inflammation vasculaire rétinienne et la dégénérescence des capillaires associée à une perméabilité vasculaire. Ensemble, ces observations suggèrent que la souris Dp71-null serait un modèle approprié pour étudier les pathologies vasculaires rétiniennes telles que la rétinopathie diabétique, l’uvéite rétinienne et l’occlusion veineuse rétinienne. / In the retina, the Dp71 dystrophin protein is mainly expressed in Müller glial cells (MGC), which contribute to the stabilization of the blood-retinal barrier (BRB). MGC are also the main sources of inflammatory factors. Thus, in our thesis project we studied the effects of the absence of the Dp71 protein on potassium and water homeostasis, as well as the expression of inflammatory mediators and retinal vascular permeability.The absence of the Dp71 protein decreased the expression of AQP4 protein and induces the redistribution of Kir4.1, initially restricted to the end-feet of MGC and around vessels, all along the cell membrane. Moreover, we have also shown that the experimental retinal detachment in WT mice induces a reduction of Dp71 which is associated with Kir4.1 mislocation, a down regulation of AQP4 protein in MGC.Our data clearly demonstrate that the absence of the Dp71 leads to increased retinal VEGF and ICAM-1 expression in Dp71-null mouse compared to WT mouse strain. There is also an increase of the number of retinal adherent leukocytes, capillary degeneration associated with high BRB permeability observed in Dp71-null mice.Our findings highlight Dp71 as an important component in the mechanisms leading to the regulation of retinal homeostasis; and to the maintaining of the BRB stabilization. We provide evidence that deficiency of Dp71 promotes retinal vascular inflammation and significantly exacerbated degeneration of capillaries and BRB breakdown. Together these results suggest that the Dp71-null mouse could be a good model to study retinal vascular diseases such as diabetic retinopathy, retinal uveitis and retinal vein occlusion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19229号 / 医博第4028号 / 新制||医||1011(附属図書館) / 32228 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 妻木 範行, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The dystrophin-associated protein complex (DAPC) provides a mechanical link between the intracellular cytoskeleton and extracellular matrix, serving as a mechanosensor and signal transducer across the sarcolemma. Pharmacological stimulation of AMP-activated protein kinase (AMPK) induces the expression of DAPC components in skeletal muscle, whereas physiological reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast glycolytic extensor digitorum longus (EDL) and slow oxidative soleus (SOL) muscles from wild-type (WT) mice, as well as from littermates deficient in both isoforms of the AMPK-β subunit in skeletal muscle (MKO) were analyzed. DAPC mRNA levels, as well as protein expression and localization were similar between genotypes, with the exception of nNOS, which displayed a compensatory sarcolemmal enrichment in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC, such as PGC-1α and KSRP, were also not affected by the loss of AMPK. However, MyoD and myogenin expression was significantly diminished in MKO muscles, which is consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO SOL muscles, despite an adaptive accumulation of PGC-1α at the sarcolemmal compartment. Collectively the evidence indicates that AMPK is sufficient, but not essential for the maintenance of DAPC expression in skeletal muscle. However, AMPK is required for preserving extrasynaptic utrophin levels in slow, oxidative muscles, which underscores the role of AMPK in the gene expression of this disease modifying protein. / Thesis / Master of Science (MSc) / The dystrophin-associated protein complex (DAPC) connects the interior and exterior of muscle cells. Activation of AMP-activated protein kinase (AMPK) increases the expression of the DAPC in skeletal muscle. We sought to determine whether AMPK was necessary for DAPC expression in skeletal muscle. Fast and slow muscles from normal mice, as well as from those deficient in skeletal muscle AMPK (MKO) were analyzed. We found DAPC levels and localization were similar between both groups, with the exception of nNOS, which was enriched at the muscle membrane in MKO muscles. Regulators of the DAPC were also not affected by the loss of AMPK. However, genes important for the production of muscle were significantly diminished in MKO muscles. Furthermore, we observed decrements in utrophin at the muscle membrane selectively in slow MKO muscles. Our work indicates that AMPK is not essential for the DAPC expression in skeletal muscle, however it is required for preserving utrophin levels in slow, oxidative muscles.

Background: Snoring and obstructive sleep apnea (OSA) are both common disorders with a number of negative health effects. The safety and efficacy of treating snoring and OSA surgically have been questioned and there has been a lack of studies in the field. Aims: 1) To investigate the frequency of serious complications, including death, after surgery for the treatment of snoring and sleep apnea; 2) to evaluate the effect on daytime sleepiness after radiofrequency surgery of the soft palate in snoring men with mild or no OSA; 3) to evaluate the effect of tonsillectomy on sleep apnea in adults with OSA and tonsillar hypertrophy; 4) to investigate the morphology and cytoarchitecture of muscle fibers in human soft palatal muscles with immunohistochemical and morphological techniques. Methods and results: In paper 1, a retrospective database study. All Swedish adults who were treated surgically because of snoring or OSA from January 1997 to December 2005 were identified in the National Patient Register. None of the surgically treated patients died in the peri- and postoperative period. Severe complications were recorded in 37.1 of 1,000 patients treated with uvulopalatopharyngoplasty (UPPP), in 5.6 of 1,000 patients after uvulopalatoplasty (UPP) and in 8.8 of 1,000 patients after nasal surgery. In paper 2, the study was designed as a randomized, controlled trial. 35 snoring men with mild or no OSA were randomized to either radiofrequency or sham surgery of the soft palate. Radiofrequency surgery was not found to be effective since there was no significant difference between the two groups in relation to the Epworth Sleepiness Scale (ESS) or apnea-hypopnea index (AHI) at follow-up. Paper 3 was a prospective study, including 28 patients with an AHI of >10 and with large tonsils. In these patients, tonsillectomy was an effective treatment for OSA; the mean AHI was reduced from 40 units/h to 7 units/h (p<0.001), and the mean ESS was reduced from 10.1 to 6.0 (p<0.001) at the six-month follow-up after surgery. Minor and moderate swallowing dysfunction was found in seven of eight patients investigated before surgery and the swallowing function improved in 5 of them after surgery, while no one deteriorated. In paper 4, we investigated the morphology and cytoarchitecture in normal soft palate muscles. Human limb muscles were used as reference. The findings showed that the soft palate muscle fibers have a cytoskeletal architecture and cellmembrane complex that differs from that of the limb muscles. Conclusions No case of death related to surgery was found among 4,876 patients treated with UPPP, UPP or nasal surgery for snoring or OSA in Sweden between 1997 and 2005. Radiofrequency surgery of the soft palate has no effect on daytime sleepiness, snoring or apnea frequency in snoring men with mild or no OSA. Tonsillectomy can be an effective treatment for OSA in adults with large tonsils. A subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles. / Snarkning och obstruktiv sömnapné (OSA) är idag en global folksjukdom. Snarkning är det ”oljud” som uppstår när luftvägen under sömn förminskas och vävnaden börjar vibrera under andning. Vid obstruktiv sömnapné faller vävnaden samman och blockerar luftflödet till lungorna. Ett andningsuppehåll, en s.k. apné inträffar. Ett andningsuppehåll kan pågå allt ifrån några sekunder till mer än en minut och kan uppstå hundratals gånger per natt. För att klassificeras som en patologisk apné enligt internationell standard måste andningsuppehållet vara längre än 10 sek. Snarksjukdomen förvärras sannolikt över tid och övergår succesivt i obstruktiv sömnapné med ökande antal andningsuppehåll under sömn. Detta leder till ett stresspåslag för kroppen med oftast uttalad dagtrötthet och en mängd negativa hälsoeffekter. Snarksjukdom och sömnapné ökar risken för bl.a. högt blodtryck och hjärt-kärlsjukdom samt också för att den drabbade ska orsaka trafikolyckor på grund av försämrad koncentrationsförmåga och trötthet. En del av den negativa utvecklingen från snarkning till sömnapné anses bero på att snarkvibrationer kan ge neuromuskulära skador i gom och svalg. Dessa vävnadsskador anses också vara orsaken till att personer som snarkat länge ofta uppvisar störd sväljningsfunktion i form av felsväljning, där maten i uttalade fall hamnar i luftstrupen istället för i matstrupen. I dagsläget är förstahandsbehandling vid sömnapné CPAP, en mask som placeras över näsa och mun och som skapar ett övertryck i luftvägen vilket förhindrar att luftvägen faller samman och att andningsstopp uppstår. CPAP har enligt flera studier den bästa effekten mot andningsuppehåll. En annan vanlig behandling är en bettskena som för underkäken nedåt och framåt så att luftvägen bli mer öppen. Bettskenan är en vanlig och effektiv behandlingsmetod för personer utan kraftig övervikt vid vanemässig snarkning eller måttlig sömnapné. För ett tjugotal år sedan var kirurgi förstahandsmetoden vid behandling av snarkning och måttlig sömnapné. Man utförde då ofta operationer i svalg och gomm, s.k. gomplastiker. Bruket av kirurgisk behandling har dock minskat med tiden, dels p.g.a. biverkningar men också för att det saknades vetenskapliga studier som bevisade att kirurgin gav önskad och långsiktig effekt. Kirurgi utgör dock fortfarande ett komplement till behandling av snarkning och sömnapné när CPAP eller bettskena av olika skäl inte fungerar eller kan tolereras av patienten. 8 Även barn kan lida av snarkning och sömnapné men behandlingsprinciperna för barn skiljer sig från dem hos vuxna och berörs inte i avhandlingen. I denna avhandling studeras: i) biverkningsfrekvenser efter olika typer av snarkkirurgi, ii) effekten av radiovågsbehandling i mjuka gommen på vuxna män med snarkning, iii) effekten av att operera bort halsmandlarna på vuxna med sömnapné och stora halsmandlar, iv) muskelvävnadens struktur och molekylära uppbyggnad i mjuka gommen hos friska personer som inte snarkar. Avhandlingen består av fyra delstudier: 1. En registerstudie med kartläggning av svåra biverkningar efter kirurgi i form av uvulopalatopharyngoplastik, uvulupalatoplastik samt näskirurgi för behandling av sömnapné och snarkning och utfört i Sverige mellan åren 1997-2005. Studien omfattade 4 876 patienter. Inga dödsfall noterades. Komplikationsrisken var störst vid operationer där man tog bort delar av mjuka gommen samt halsmandlarna, där i snitt 37 av 1000 opererade fick biverkningar, framförallt p.g.a. infektioner eller blödningar. 2. I en prospektiv, randomiserad placebostudie utvärderades effekten av radiovågsbehandling i mjuka gommen vid snarkning och lindrig sömnapne. Trettiotvå patienter lottades till att få radiovågsbehandling eller placebo behandling. Patienterna visste inte vilken grupp de tillhörde. Vid uppföljning efter 12 månader var det inga statistiska belägg för att radiovågsbehandling minskade vare sig antal andningsuppehåll eller dagtrötthet. 3. Effekten av att ta bort halsmandlarna på patienter med stora halsmandlar och olika grad av sömnapné utvärderades i denna studie. Totalt deltog 28 patienter. Vid uppföljning 6 månader efter operationen hade antalet andningsuppehåll sjunkit drastiskt, från i snitt 40 till 7 andningsuppehåll per timme nattsömn. Inga allvarliga biverkningar uppstod. Dessa fynd talar för att man som förstahandsmetod ska erbjuda patienter med sömnapné och stora halsmandlar att ta bort halsmandlarna. 4. I detta projekt undersökte vi utseendet och uppbyggnaden av cellskelettet i två normala muskler i mjuka gommen hos friska personer utan känd snarkning och sömnapné. Muskler från armar och ben användes som referens. Fynden i studien visar att de normala muskelfibrernas uppbyggnad i mjuka gomen skiljer sig från jämförade muskler i armar och ben. Detta kan vara ett uttryck för en evolutionär utveckling för att möjligöra de komplexa funktioner som krävs av svalgets muskulatur. 9 Sammanfattningsvis kan vi konstatera: Att inga dödsfall har skett i Sverige efter operationer i gom, svalg eller näsa, utförda för att behandla snarkning och sömnapné under åren 1997 till 2005. Att radiovågsbehandling av mjuka gommen hos snarkande män med lindrig sömnapné inte har någon effekt på dagtrötthet, snarkning eller andningsuppehåll vid uppföljning efter 12 månader. Metoden kan därför inte rekommenderas. Att när man opererar bort stora halsmandlar på personer med andningsuppehåll så leder detta ofta till att andningsuppehållen minskar drastiskt. Metoden kan därför oftast rekommenderas som en förstahandsbehandling för denna patientgrupp. Att mjuka gommens muskelfibrer är uppbyggda på ett unikt sätt indikerar att deras specifika biomekaniska egenskaper skiljer sig från referens muskler i armar och ben.

La formation et l’intégrité de la barrière hémato-rétinienne (BHR) sont nécessaires au maintien d’une bonne vision et la violation de cette barrière contribue à l’apparition d’un grand nombre de pathologies rétiniennes tel que la rétinopathie diabétique (RD) ou l’occlusion de la veine centrale de la rétine (OVCR). La dystrophine Dp71 est une protéine du cytosquelette associée à la membrane qui s’exprime majoritairement dans les cellules gliales de Müller. Son absence a été associée à une augmentation de la perméabilité vasculaire liée à la délocalisation et à la diminution de l’expression des canaux AQP4 et Kir4.1. La souris Dp71-null est donc un excellent modèle d’étude des pathologies rétiniennes présentant une rupture de la BHR. L’ensemble de nos résultats démontrent que chez la souris déficiente en Dp71 ayant une rupture de la BHR, il est possible de restaurer une perméabilité et une homéostasie rétinienne normale grâce à la surexpression de la Dp71 via les virus adéno-associés. Cette étude est à la base du développement de nouvelles stratégies thérapeutiques dans le traitement de pathologies associées à une rupture de la BHR et à un œdème maculaire, comme la RD ou l’OVCR. / Formation and maintenance of the blood-retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as diabetic retinopathy (DR) or Central retinal vein occlusion (CRVO). Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller glial cells, its absence has been related to BRB permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels. Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing blood-retinal barrier permeability. Our results collectively demonstrated that in Dp71 deficient mouse with compromised barriers, normal BRB permeability and retinal homeostasis can be restored through over-expression of Dp71 via adeno-associated virus. This study is the basis for development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular edema such as DR or CRVO.

A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença / The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient\'s life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease