The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer’s disease.

Le syndrome de Usher est une maladie génétique associant surdité congénitale et rétinopathie pigmentaire (RP), auxquelles peuvent s'ajouter des troubles vestibulaires. Les différences phénotypiques, distinguées en 3 types cliniques, s'accompagnent d'une hétérogénéité génétique impliquant au moins 10 gènes. Identifier et caractériser les causes moléculaires grâce aux outils d'analyses génétiques disponibles permet d'améliorer la compréhension des mécanismes physiopathologiques à l'origine des symptômes du syndrome de Usher. Dans ce cadre, nous nous sommes inscrits dans une recherche d'exhaustivité des études moléculaires. Dans un premier temps, nous avons ainsi mis en place l'analyse et défini le spectre mutationnel des gènes minoritairement impliqués dans le type II (GPR98 et DFNB31). Nous avons également développé différents outils, notamment pour l'analyse de variants altérant le mécanisme d'épissage ou touchant les régions promotrices des gènes USH2.Ces travaux permettent d'obtenir un taux de détection des altérations conduisant au syndrome de Usher type 2 de 90 %. Ce taux est maintenant similaire à celui observé pour le type 1, qui constituait jusqu'ici la référence.Nous avons, dans un second temps, développé le séquençage nouvelle génération (NGS) appliqué à l'exome Usher. L'objectif de cette analyse était de tester la faisabilité et l'efficacité de cette approche, en vue de son éventuelle utilisation en diagnostic moléculaire. La définition des critères de qualité et la mise en place de la priorisation des variants ont été réalisées sur un groupe contrôle. L'étude a ensuite été étendue sur une cohorte de patients. Les résultats obtenus montrent qu'une utilisation en diagnostic est possible mais restera dépendante de l'amélioration de la technique du séquençage, de son analyse et des outils bioinformatiques pour interpréter le volume de données ainsi généré. / Usher syndrome is a genetic disorder combining sensorineural hearing loss (HL) and retinitis pigmentosa (RP). Some patients will also exhibit vestibular areflexia (VA). Clinical and genetic heterogeneity is recognized as the 3 clinical subgroups, defined mainly on the degree of HL and VA, can be caused by mutations in one of the 10 known genes. It is important to use all accessible genetic tools to identify and characterize molecular origin in order to improve the knowledge of the physiopathological mechanisms causing Usher Syndrome.In this context, we have developed an exhaustive approach. In a first step, we have implemented the analysis and established the mutational spectrum of the 2 minor USH2 genes (GPR98 and DFNB31). In addition, we have developed several tools, in particular to study variants susceptible to alter splicing or lying in the promoter regions of the USH2 genes.Thanks to this work, the USH2 mutation detection rate has now been raised to 90%, similar to that of USH1.We have then designed a targeted exome of the Usher genes to be sequenced using the GS Junior system (Roche 454). The aim of the study was to test the feasibility of this new technics for a possible transfer to diagnostic facilities. Quality criteria and variant priorization were set up on a control cohort (previously studied in one of the USH gene). The study has then been extended on a patient cohort. Our results indicate that NGS Usher-exome can be used in molecular diagnostics but improvement of the reliability of the sequencing technology, bioinformatics tools and dedicated databases is essential.

Human Usher syndrome is a severe and congenital form of syndromic deafness that affects 1 person in 25,000 people in the world population. Normally the stereocilia, microvillar protrusions of the apical membrane of inner ear hair cells, are organized into coherent bundles. This precise organization is critical for mechanosensing, i.e. for hearing. Mutation in any of the five known Usher syndrome genes is sufficient to alter the precise organization of stereocilia, a condition that results in deafness. To date, however, the molecular mechanisms responsible for the splaying of stereocilia and genesis of the disease are not well understood. Here, I identified Drosophila melanogaster genes related to human Usher syndrome and characterized some of them (Cad99C, DSANS and crinkled) during Drosophila development, in the processes of microvilli morphogenesis in the follicular and wing imaginal disc epithelia. Cadherin Cad99C is a transmembrane protein with putative cell adhesion properties. Similar to its human ortholog Protocadherin 15, Drosophila Cad99C localizes to microvillar protrusions in the follicular epithelium. In this epithelium, Cad99C is required for the proper morphogenesis and organization of microvilli into bundles, similar to human Protocadherin 15. Further, overexpression of the full-length Cad99C or of a deleted version, devoid of the cytoplasmic region, promotes microvilli bundling. This finding suggests that Cad99C establishes adhesive interactions between microvilli via its extracellular region. Interestingly, morphological alteration of follicle cell microvilli associates with defective deposition of the vitelline membrane, an extracellular matrix that protects the embryo from osmotic stresses. These findings suggest that microvilli are normally required for the even deposition of the extracellular matrix. In order to test whether Cad99C is involved in microvilli morphogenesis and bundling in other tissues, I analyzed the function of Cad99C in a larval tissue, the wing imaginal disc. Cad99C overexpression, but not Cad99C removal, is sufficient to alter microvilli morphology and organization in the columnar epithelium of the wing imaginal disc. Likely, other molecules can compensate for Cad99C loss of function in this tissue. To possibly get some insights on the molecular function of other Usher syndrome proteins, I analyzed the function of Drosophila SANS and crinkled in the follicular epithelium, where both these genes are expressed. crinkled is the ortholog of myosinVIIa, that encodes a motor protein of the actin cytoskeleton. DSANS is related to human SANS and encodes a cytoplasmic protein of unknown function. It has been puzzling how removal of SANS, a cytoplasmic protein, could impair adhesion and bundling of stereocilia. To study the function of DSANS, I generated null mutant flies and observed that, in the absence of DSANS, delivery of Cad99C to microvilli is impaired. Cad99C localization is however unperturbed in crinkled mutant follicle cells. By immunostaining, DSANS immunoreactivity was detected diffusively in the cytoplasm and in dot-like structures, possibly corresponding to vesicles. In conclusion, DSANS is a cytoplasmic protein that is required for the efficient delivery of Cad99C to microvilli protrusions. Taken together, the analysis that I here performed of Drosophila Usher syndrome related genes indicates two novel molecular mechanisms of function for the corresponding human Usher syndrome proteins. First, human Protocadherin 15, like Drosophila Cad99C, could be involved in establishing adhesive interactions between microvilli protrusions of the inner ear (stereocilia). Removal of Protocadherin 15 would then cause splaying of stereocilia due to lack of inter-stereocilia adhesive links. Second, the analysis here performed suggests that SANS is involved in the efficient delivery of Protocadherin 15 to stereocilia. Mutations in SANS would then lead to splaying of stereocilia and deafness due to poor localization of Protocadherin 15 to stereocilia.

Visual conditions such as retinitis pigmentosa and Usher syndrome can gradually cause tunnel vision. Patients with these conditions usually face difficulties with navigation, avoiding obstacles, and performing visual search. Loss of mobility can affect patients' independence and quality of life. One of the rehabilitation strategies for patients with tunnel vision is the use of optical aids to enhance mobility performance. The main method used to evaluate the usefulness of optical aids is the patient’s subjective report after extended wear. In order to evaluate optical aid effectiveness in the clinic, a new test based on the visual search paradigm was designed to assess the patient's visual awareness. This was named the assessment of visual awareness (AVA) test. The main aim of this study was to develop the AVA test, establish its sensitivity, validity and repeatability, and then use it to investigate the efficacy of optical aids in this group of people. The AVA test consists of 32 peripheral targets presented at four different locations: 1st annulus (at 5° from the central fixation), 2nd annulus (10°), 3rd annulus (20°) and 4th annulus (30°)). In this study, the peripheral targets were presented singly against a spatial noise background in a presentation area of 81° H × 62° V. Participants were allowed to use head and eye movements and were asked to search for and locate each target. The detection time (DT) was recorded. A new, sensitive and easy to set up indoor mobility course was also designed and validated prior to its use in validating the AVA test. A total of 50 normally sighted participants with simulated tunnel vision (TV) (5° to 20°, in 5° steps) and 20 patients with TV (retained field 4° to 21°) were tested. The AVA test was found to be responsive to the change in field of view (FoV) and to the target locations in both groups of participants. In the simulated group, a significant relationship was found between FoV and DT at each annulus (r ranging from -0.55 to -0.77, p < 0.0001). A significant relationship was found between target location and DT within each FoV size (20°, 15°, 10° and 5°) (r ranging from 0.53 to 0.84, p < 0.0001). In the TV patients, a statistically significant relationship was found between FoV and DT at each annulus (r range from -0.40 to -0.60, p < 0.05). The target location was shown to have a significant relationship with the DT within each FoV size (r ranging from 0.50 to 0.60, p < 0.05). Finally, the AVA test was found to be significantly related to the simulated TV participants' performance on the indoor mobility course. The AVA test was used to assess the efficacy of three optical aids: the partial aperture prism (10 patients), the Tri-field prism (10 patients) and the reverse telescope (4 patients). The AVA test showed no significant improvement in DT with either of the prisms and the participants did not find these aids helpful. DT with the reverse telescope improved, but none of the participants were willing to use these on extended trial. The AVA test gave clear indications of the efficacy of each aid, a result which could affirm the importance of the AVA test. In conclusion, the AVA test was found to be sensitive, valid and repeatable. DT did not improve in either of the optical aids which were found to be unsuccessful, suggesting that the AVA could be a promising clinical test. However the aids which showed improved DT were not evaluated over the longer term, and therefore did not allow full evaluation of the AVA test.

The present thesis belongs to the research area disability research and deal with specific aspects of cognition in individuals with Usher syndrome type 1 and 2. The subject has been investigated and is discussed within an interdisciplinary framework, though the theories applied and described are derived from the area of cognitive psychology. Usher syndrome is a rare genetic condition causing a combination of visual and hearing impairment: deafblindness. There is a congenital hearing loss that is profound in type 1 and moderate to severe in type 2. During mid-childhood symptoms of visual impairment, e.g. light sensitivity, emerge and a progressive loss of visual field follows as a result of the genetically caused eye disease Retinitis Pigmentosa. The syndrome has previously been well described with respect to the genetical and medical aspects, but there has been very little research with a cognitive perspective on the population. Studies 1 and 2 in the present thesis focused on children with Usher syndrome type 1 with cochlear implants and investigated phonological skills, lexical access, working memory and reading skill in the group. Studies 3 &amp; 4 investigated the same cognitive abilities and theory of mind in adults with Usher syndrome type 2. In study 4 the performance on theory of mind in the adults with Usher syndrome type 2 was also compared to that of another group with genetically caused deafblindness: individuals with Alström syndrome. The results were that both the children and adults with Usher syndrome had significantly poorer phonological processing than the control groups with normal hearing. There was a large variation on performance on lexical access, especially in the group of children, however several individuals performed at the same level as the control group. Reading skill was found to be at level with the control groups’. There was also great variation in performance on ToM, however the majority of individuals performed similar to the control group with normal hearing and vision. The present project has resulted in some new knowledge on cognitive performance in  individuals with Usher syndrome type 1 and type 2. Performance in the participants with Usher syndrome can to a large extent can be understood by application of the models developed in previous research on populations with hearing impairment or deafness for understanding the impact of hearing with a hearing aid or cochlear implant. However, individuals with Usher syndrome experience additional difficulties in accessing information due to the progressive visual loss and the impact this has on performance is still largely unknown. Hence, the present project would recommend that interventions and support would be designed specifically to each individuals’ needs, with consideration of both the visual impairment and the hearing impairment. / Föreliggande avhandling tillhör ämnet handikappvetenskap och beskriver specifika kognitiva förmågor hos personer med Ushers syndrom typ 1 och 2. Avhandlingens ämne har undersökts utifrån ett tvärvetenskapligt perspektiv, även om de teorier som tillämpas och beskrivs huvudsakligen härrör inom området kognitiv psykologi. Ushers syndrom är en ovanlig genetisk åkomma som leder till kombinationen av syn- och hörselnedsättning: dövblindhet. Individer med typ 1 av syndromet har medfödd dövhet medan individer med typ 2 har en medfödd måttlig till grav hörselnedsättning. Någon gång i åldrarna 6-10 år börjar de första symptomen, till exempel nedsatt mörkerseende, på den genetiskt betingade progressiva synnedsättningen Retinitis Pigmentosa att framträda. Syndromet är väl beskrivet i forskningen med avseende på genetiska och medicinska aspekter, men det finns extremt lite tidigare forskning med kognitivt perspektiv om populationen. Studierna 1 och 2 i föreliggande avhandling fokuserade på barn med Ushers syndrom typ 1 och cochleaimplantat. Dessa studier undersökte fonologisk förmåga, lexikal access, arbetsminne och läsning i gruppen. Studie 3 undersökte samma kognitiva förmågor hos vuxna med typ 2 av syndromet. I studie 4 undersöktes även den sammansatta förmågan Theory of Mind hos de vuxna med typ 2 och deras prestation jämfördes både mot en kontroll grupp med normal hörsel och syn och en kontrollgrupp med annan typ av dövblindhet; Alström syndrom. Resultaten visade att både barnen och de vuxna med Ushers syndrom hade signifikant sämre fonologisk förmåga än kontrollgruppen med normal hörsel. Nivån på prestation varierade stort inom grupperna, särskilt mellan barnen med typ 1, och flera av individerna (barn och vuxna) presterade trots hörselnedsättningen på samma nivå som de normalhörande. Läsfärdigheten befanns vara i nivå med kontrollgrupperna. I den vuxna gruppen var det stor variation i prestation även på Theory of Mind, men de flesta av individerna presterade liknande som kontrollgruppen med normal hörsel och syn. Föreliggande projekt har resulterat i lite mer kunskap om kognitiva färdigheter hos individer med Ushers syndrom typ 1 och 2. De resultat som individerna med Ushers syndrome presterade kan till stor del förstås och tolkas genom tillämpning av teorier och modeller utvecklade för att den inverkan på kognitiva förmågor det har att ha nedsatt hörsel och höra med hjälp av hörselapparat eller cochleaimplantat. Dock tyder fynden i detta projekt även på att individer med Ushers syndrom på grund av den allvarliga synnedsättningen har ytterligare svårigheter att få tillgodogöra sig information, men i vilken utsträckning och på vilket sätt är ännu inte beskrivet. Utifrån fynden i föreliggande studie blev rekommendation att interventioner och stöd till personer med Ushers syndrom utformas specifikt till varje individ, med hänsyn taget både till hens grad av synnedsättning och hörselnedsättning.

Persons with deafblindness experience difficulties in daily life and they experience service to sometimes barrier. The overall aim of this thesis is therefore to discover, evaluate and explain: 1. mechanisms that might have impact on participation restrictions for people who have visual and hearing impairment i.e. deafblindness and 2. mechanisms that might barrier service to these people. Service is used as an umbrella term for health care, education and certain service for persons with disabilities. Materials from multiple sources have been used: literature (Study I No 96 papers). Interviews (Study I and V) with 32 and 3 adults with deafblindness respectively. Questionnaires (Study II and III): answered by 33 and 34 adults and youth with deafblindness. Patient records (Study IV and V): records from 9 and 3 adult females with USH I respectively. Materials mostly retrospectively cover the period from 2005 and about 40–50 years. Both quantitative and qualitative methods were used. International Classification of Functioning, Disability and Health (ICF) were consequently used as a framework to describe as well as a tool to analyze mechanisms. Further, the Ecological approach, Disability as a laminated system and Life course approach were used in order to evaluate and explain mechanisms. The conclusions that can be drawn from an ecological, laminated and life course approach are: Participation restrictions for people with deafblindness are far-reaching and are embedded in a complex process of interaction between the person with deafblindness and the environment. Services entail systematical barriers. In order to improve service it is extremely important to understand the role of participation restrictions in deafblindness. Primary activity limitation is to not see and hear enough for comprehension. Hence, not taking part in the visible and audible world is primary participation restriction. Performing activities without basic information includes risk. One important aspect of deafblindness is exposure. Persons with deafblindness require rehabilitation in a life perspective. In order to increase people’s participation and protection requirement of individually adapted support and assistive devices is necessary. ICF and the UN convention support service alterations.

Made available in DSpace on 2016-06-02T19:46:23Z (GMT). No. of bitstreams: 1 5036.pdf: 14531886 bytes, checksum: e76b181fa8c752c0560ff43476c9b79c (MD5) Previous issue date: 2013-02-25 / The objective of this work was to analyze the efficiency of visual adaptations in activities presented in the student with deafblindness with Usher syndrome and its impact on education. This research was developed in the city of Santa Catarina in a Basic Inclusive School where deaf education policy is performed in Elementary School in the first to ninth-grade and in High school. This is a case study for 12 year old preteen with deafblindness with Usher syndrome who studies in an Association of sensorineural deafness and acquired blindness as a result of Retinitis Pigmentosa. In the year 201, the student attended the 5th grade, in bilingual mode (only for deaf). In 6th grade (2012) in mixed class (with listeners). In the bilingual class the teacher, teaches in sign language as first language and the Portuguese, as second one. Already in the inclusive class the language used is Portuguese with the presence of an educational interpreter. It was used visual resources in the accessible instructional materials for the contents of the subjects. The bilingual students took advantage by the materials. Data collection consisted of the interview with the family, with the student, and the teacher. Also pre and post student s intervention with the application of functional vision instrument. Observations were conducted in different situations and in different spaces. The obtained results were analyzed qualitatively taking as a criterion the student s own performance analysis. The student makes use of sign language easily. He presented the first symptoms of Usher syndrome: dazzing in some situations which implied difficulty to adapt to bright; decreased peripheral vision and night blindness. In the process of perception of the difficulties he doesn t see as deafblind but like a deaf. During the collection of data it was observed signs of decrease in the peripheral vision, in the locomotion, and in the sign language when the colleague uses it in his side. The results suggest that the materials used in the classroom were mainly appropriate for the amplified material with 12 points to 20 or 20 points letters as gradual exposure in different sizes. The Times New Roman font was modified to the Arial font one and after to Verdana because the changes provided more suitable traits. The results indicate, therefore, that the accessible visual resources were used properly. However, there is no support of optical and non-optical resources by the institution on this new reality: the presence of the student with Usher syndrome, in the classroom, and his constitutional right is guaranteed. / O objetivo desse trabalho foi analisar a eficiência das adaptações visuais nas atividades apresentadas para o aluno com surdocegueira por Síndrome de Usher e seu impacto na escolarização. Essa pesquisa foi desenvolvida em uma cidade de Santa Catarina em uma Escola Básica Inclusiva onde a Política de Educação de Surdos, é executada no Ensino Fundamental da primeira a nona série e no Ensino Médio. Trata-se de um estudo de caso de um pré-adolescente 12 anos, com surdocegueira por Síndrome de Usher, que é uma associação de surdez neurossensorial e cegueira adquirida como consequência da Retinose Pigmentar. No ano de 2011 frequentou a 5ª série, na modalidade bilingue (somente surdos) e na 6ª série (2012) em turma mista (surdos e ouvintes). Na classe bilingue a professora, ministra as disciplinas em Língua de Sinais como primeira língua e o Português, como segunda. Já na classe mista a língua de instrução é o Português com a presença de um intérprete educacional. Utilizou-se de recursos visuais acessíveis nos materiais instrucionais nos conteúdos das disciplinas e aproveitados por todos os alunos da turma bilíngue. A coleta de dados constou de entrevista com a família, com o aluno e a professora, avaliação pré e pós - intervenção do participante com a aplicação do instrumento de Avaliação Funcional da Visão. Realizaram-se observações em diversas situações e em diferentes espaços. Os dados obtidos foram analisados qualitativamente tomando como critério para a análise o desempenho do próprio participante. Faz uso da Língua de Sinais com desenvoltura. Apresenta os primeiros sintomas da Síndrome de Usher: deslumbramento em algumas situações o que implica na dificuldade para adaptar-se à luz brilhante; diminuição da visão periférica e cegueira noturna. No processo de percepção das suas dificuldades não se vê como surdocego e sim como surdo. Durante a coleta de dados foi observado indícios de diminuição na visão periférica na locomoção e na leitura de Libras quando o colega usa língua de sinais ao seu lado. Os resultados sugerem que os materiais utilizados em sala de aula foram adequados principalmente na ampliação do material impresso com letras de 12 pontos para 20 ou 22 pontos, conforme exposição gradual a diferentes tamanhos. A fonte Times New Roman foi modificada para a fonte Arial e, posteriormente, para Verdana porque as mudanças proporcionaram traços mais adequados. Os resultados indicam, portanto, que os recursos visuais acessíveis foram utilizados adequadamente. Entretanto, não existe um apoio de recursos ópticos e não ópticos pela Instituição diante dessa nova realidade: a presença do aluno com Síndrome de Usher, na sala de aula, para que o seu direito constitucional seja exercido.

Le syndrome d’Usher (USH) associe une surdité neurosensorielle congénitale et une perte progressive de la vision par rétinite pigmentaire. Pendant ma thèse, l’essentiel de mon travail a porté sur un gène responsable du syndrome d’Usher de type 2, USH2A. Ce gène code pour l’usherine, une protéine associée aux liens fibreux interstéréociliaires situés à la base de la touffe ciliaire des cellules ciliées de la cochlée. Ces liens transitoires disparaissent autour du 9e jour post-natal (P9), chez la souris et le complexe moléculaire associé à ces liens inclut l’usherine, adgrv1 (un récepteur membranaire couplé aux protéines G), la whirline, et pdzd7 (deux protéines sous-membranaires d’échafaudage contenant des domaines PDZ). Des travaux précédents ont montré que l’interaction de ces quatre protéines était nécessaire à un développement correct de la touffe ciliaire, qui lui-même conditionne la transduction mécano-électrique opérée par les cellules ciliées. Pendant ma thèse, j’ai étudié les effets, à court terme et à long terme, de l’absence de la plus longue des 2 isoformes connues de l’usherine, l’isoforme-b transmembranaire, sur des souris mutantes pour le gène Ush2a (Ush2aΔTM/ΔTM). Chez ces souris, j’ai effectué des mesures de courants de transduction mécano-électrique, des enregistrements des potentiels évoqués auditifs (PEA), des tests de masquage auditif, et une analyse morphologique des cellules ciliées par imagerie au microscope électronique à balayage. Ainsi, j’ai pu montrer que les liens interstéréociliaires basaux étaient présents à P4 et que les courants de transduction mécano-électrique étaient normaux à P7. L’absence de l’isoforme-b de l’usherine n’a, en fait, que très peu de conséquences morphologiques et fonctionnelles sur la touffe ciliaire des cellules ciliées de la cochlée durant les 3 ou 4 premiers mois de vie chez la souris. A partir de l’âge de 4 mois cependant, les souris Ush2aΔTM/ΔTM souffrent d’une perte progressive de l’audition et d’anomalies de la sélectivité dans l’analyse des fréquences du son, dues surtout à un dysfonctionnement des cellules ciliées externes. Ces résultats viennent alimenter le débat sur le caractère progressif de la surdité du syndrome d’Usher de type 2A. La surdité des patients USH2A est considérée comme étant le plus souvent non progressive, mais plusieurs études ont révélé que certains patients souffrent en fait d’une surdité progressive. Mon travail a permis de montrer que chez la souris, la surdité en rapport avec des mutations d’Ush2a peut également être progressive. L’existence potentielle d’une fenêtre temporelle chez les patients USH2A dont la surdité moins sévère à la naissance, va ensuite s’aggraver, pourrait permettre d’envisager dans le futur un traitement curatif précoce du déficit auditif de ces patients, par thérapie génique. / Usher syndrome (USH) is characterised by a sensorineural congenital deafness and a progressive loss of vision by retinitis pigmentosa. During my PhD, my main focus of study was a gene responsible for Usher syndrome type 2, USH2A. This gene codes for usherin, a protein associated with the fibrous links located at the base of the hair bundle of cochlear, and vestibular hair cells. In mice, these transitory links start to disappear as of postnatal day 9 (P9), and the molecular complex with which they are associated is composed of usherin, adgrv1 (an adhesion G protein coupled receptor), whirlin, and pdzd7 (two submembranous PDZ domain-containing scaffold proteins). Previous work has shown that the interaction in between these 4 proteins is essential for the development of the hair bundle, the structure responsible for the initiation of the mechano-electrical transduction (MET) process in the hair cells. During my thesis, I studied the short term and long term effects of the absence of the longest of the 2 usherin isoforms, the transmembrane b-isoform, in mice carrying a mutation in the Ush2a gene (Ush2aΔTM/ΔTM). In these mice, I measured mechano-electrical currents, auditory brainstem responses, undertook auditory masking tests, and analysed scanning electron micrographs of cochlear hair bundles. Through this work, I showed that basal lateral links similar to ankle links could be observed on P4, and that MET currents were normal on P7. The absence of the long b-isoform of usherin actually has very little effect on the morphology or the function of the cochlear hair bundle in mice, until 3 or 4 months of age. As of 4 months old however, Ush2aΔTM/ΔTM mice suffer from a progressive hearing loss, and frequency selectivity defects, mainly cause by a dysfunction of outer hair cells. These results will further add to the debate on whether the hearing loss in Usher syndrome type 2A is progressive or not. Hearing loss in USH2A patients is generally considered non progressive, but several studies have given indication to the contrary. My work has shown that in mice, deafness caused by mutations to the Ush2a gene can also follow a progressive pattern. The potential existence of this temporal window in USH2A patients whose hearing impairment is less severe at birth, but gets worse over time, could allow clinicians to use gene therapy as curative treatment for patients who fall into this category.

Human Usher syndrome is a severe and congenital form of syndromic deafness that affects 1 person in 25,000 people in the world population. Normally the stereocilia, microvillar protrusions of the apical membrane of inner ear hair cells, are organized into coherent bundles. This precise organization is critical for mechanosensing, i.e. for hearing. Mutation in any of the five known Usher syndrome genes is sufficient to alter the precise organization of stereocilia, a condition that results in deafness. To date, however, the molecular mechanisms responsible for the splaying of stereocilia and genesis of the disease are not well understood. Here, I identified Drosophila melanogaster genes related to human Usher syndrome and characterized some of them (Cad99C, DSANS and crinkled) during Drosophila development, in the processes of microvilli morphogenesis in the follicular and wing imaginal disc epithelia. Cadherin Cad99C is a transmembrane protein with putative cell adhesion properties. Similar to its human ortholog Protocadherin 15, Drosophila Cad99C localizes to microvillar protrusions in the follicular epithelium. In this epithelium, Cad99C is required for the proper morphogenesis and organization of microvilli into bundles, similar to human Protocadherin 15. Further, overexpression of the full-length Cad99C or of a deleted version, devoid of the cytoplasmic region, promotes microvilli bundling. This finding suggests that Cad99C establishes adhesive interactions between microvilli via its extracellular region. Interestingly, morphological alteration of follicle cell microvilli associates with defective deposition of the vitelline membrane, an extracellular matrix that protects the embryo from osmotic stresses. These findings suggest that microvilli are normally required for the even deposition of the extracellular matrix. In order to test whether Cad99C is involved in microvilli morphogenesis and bundling in other tissues, I analyzed the function of Cad99C in a larval tissue, the wing imaginal disc. Cad99C overexpression, but not Cad99C removal, is sufficient to alter microvilli morphology and organization in the columnar epithelium of the wing imaginal disc. Likely, other molecules can compensate for Cad99C loss of function in this tissue. To possibly get some insights on the molecular function of other Usher syndrome proteins, I analyzed the function of Drosophila SANS and crinkled in the follicular epithelium, where both these genes are expressed. crinkled is the ortholog of myosinVIIa, that encodes a motor protein of the actin cytoskeleton. DSANS is related to human SANS and encodes a cytoplasmic protein of unknown function. It has been puzzling how removal of SANS, a cytoplasmic protein, could impair adhesion and bundling of stereocilia. To study the function of DSANS, I generated null mutant flies and observed that, in the absence of DSANS, delivery of Cad99C to microvilli is impaired. Cad99C localization is however unperturbed in crinkled mutant follicle cells. By immunostaining, DSANS immunoreactivity was detected diffusively in the cytoplasm and in dot-like structures, possibly corresponding to vesicles. In conclusion, DSANS is a cytoplasmic protein that is required for the efficient delivery of Cad99C to microvilli protrusions. Taken together, the analysis that I here performed of Drosophila Usher syndrome related genes indicates two novel molecular mechanisms of function for the corresponding human Usher syndrome proteins. First, human Protocadherin 15, like Drosophila Cad99C, could be involved in establishing adhesive interactions between microvilli protrusions of the inner ear (stereocilia). Removal of Protocadherin 15 would then cause splaying of stereocilia due to lack of inter-stereocilia adhesive links. Second, the analysis here performed suggests that SANS is involved in the efficient delivery of Protocadherin 15 to stereocilia. Mutations in SANS would then lead to splaying of stereocilia and deafness due to poor localization of Protocadherin 15 to stereocilia.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-02T13:12:16Z No. of bitstreams: 2 Dissertação - Ricardo Campos Cruvinel Filho - 2014.pdf: 8513057 bytes, checksum: e42a2106d7e7abda6b1ee9d65068229d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-02T13:28:23Z (GMT) No. of bitstreams: 2 Dissertação - Ricardo Campos Cruvinel Filho - 2014.pdf: 8513057 bytes, checksum: e42a2106d7e7abda6b1ee9d65068229d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-02T13:28:23Z (GMT). No. of bitstreams: 2 Dissertação - Ricardo Campos Cruvinel Filho - 2014.pdf: 8513057 bytes, checksum: e42a2106d7e7abda6b1ee9d65068229d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-16 / Cross-sectional study conducted at the Center of Reference in Ophthalmology UFG in conjunction with Oregon Health and Science University and the Brazilian Center for Eye Surgery (CBCO). To evaluate the genotype of patients with Usher syndrome of Reference Center for Ophthalmology, Federal University of Goias (UFG-CEROF). Patients clinically diagnosed with SU underwent complete ophthalmic examination, Goldmann manual kinetic perimetry, audiometry and subsequent collection of peripheral blood chromosomal microarray for sequencing. We examined 19 patients with clinical suspicion of SU with a mean age at first visit was 42.5 years (± 12.2) and a slight predominance of males (52.63%). The most prevalent subtype in clinical diagnosis of type I disease (68.4%). The visual acuity measured on the day of the exam for eye examination was 20/92 on the Snellen chart. Examinations audiometry showed hearing loss in all patients ranging from moderate in 12.5% of patients, deep (56.25%) and severe (31.25%). In 36.8% of patients analyzed, we found at least two mutations in the same gene, and of these, 21% were heterozygous mutations, and 15.8% homozygous. The homozygous mutations, which were of the type no sense, occurred in the gene CLRN1 whose patients had a previous diagnosis of USH 2. Met 26.31% of the sample analyzed in heterozygous. Of these, two patients showed mutations in the MYO7A gene (40%), both with clinical suspicion of USH 1. For the proposed methodology, we found no disease-causing mutations in 79% of the sample analyzed. Following the proposed methodology, the authors were able to determine the mutation in seven patients of nineteen patients inclued in this study. Of these, three patients were diagnosed with homozygous mutations in gene CLRN1, and had previous clinical diagnosis of type 2. Two patients had heterozygous mutations in gene MYO7A, both with previous clinical diagnosis of type 1. / Estudo transversal, desenvolvido no Centro de Referência em Oftalmologia da UFG em conjunto com a Oregon Health and Science University e Centro Brasileiro de Cirurgia de Olhos (CBCO), que teve como objetivo avaliar o genótipo de pacientes com síndrome de Usher do Centro de Referência em Oftalmologia da Universidade Federal de Goias (CEROF-UFG). Pacientes clinicamente diagnosticados com SU foram submetidos a exame oftalmológico completo, perimetria cinética manual de Goldmann, audiometria e posterior coleta de sangue periférico para sequenciamento cromossômico por microarray. Foram examinados 19 pacientes com diagnostico clínico de SU com média de idade na primeira consulta de 42,5 anos (± 12,2) e pequena predominância do sexo masculino (52,63%). O subtipo mais prevalente no diagnóstico clínico foi do tipo I da doença (68,4%). A acuidade visual média medida no dia do exame por olho examinado foi de 20/92 na escala de Snellen. Os exames audiométricos mostraram perda de audição em todos pacientes variando de moderada em 12,5% dos pacientes, profunda (56,25%) e severa (31,25%). Em 36,8% dos pacientes analisados, encontraram-se ao menos duas mutações em um mesmo gene, sendo que destes, 21% eram mutações heterozigotas e, 15,8% homozigotas. As mutações homozigotas, as quais eram do tipo sem senso, ocorreram no gene CLRN1, cujos pacientes tinham o diagnóstico clínico prévio de USH 2. Encontrou-se 26,31% da amostra analisada em heterozigose. Desses, dois pacientes mostraram mutações para o gene MYO7A (40%), ambos com suspeita clínica de USH 1. Pela metodologia proposta, não foram encontradas mutações causadoras de doença em 79% da amostra analisada. Dos 19 pacientes incluídos no presente estudo os autores conseguiram determinar a mutação de sete deles segundo a metodologia proposta. Desses, três pacientes foram diagnosticados com mutações homozigoticas todas no gene CLRN1 e possuíam diagnostico clinico prévio de SU tipo 2. Dois pacientes apresentaram mutações heterozigóticas para o gene MYO7A, ambos com diagnostico clinico prévio de SU tipo 1 e um paciente apresentou mutação heterozigótica para o gene ALMS1 que apresentava diagnostico clinico de SU tipo 1.