Dissertations / Theses on the topic 'Usher'

The present thesis concerns people with Usher syndrome (USH) and their health. People with USH have a congenital hearing loss of various degrees and an eye disease with a progressive course; for some, the balance is also affected. Three clinical groups have been identified 1, 2 and 3, and 13 genes have currently been identified. USH is the most common cause of deafblindness. Clinical knowledge and the limited research that exists have shown that people with deafblindness can experience difficulties in everyday life. Depression, anxiety and social withdrawal have been described. The general aim of the present thesis was to describe the health of people with USH. The empirical material employed was based on an extensive survey in which people with USH answered two questionnaires concerning health, anxiety, depression, social trust, work, health-care, financial situation, and alcohol and drug use. The focus of the present thesis is on general health, physical health and psychological health, social trust and finance. Three studies in the present theses focus on USH1, 2 and 3, respectively; finally, the fourth study provides an in-group comparison of people with USH. The results of studies I and III are compared with a crosssection of the Swedish population. The results revealed poor physical and psychological health, a lack of social trust and a strained financial situation regardless of clinical diagnosis. The discussion stresses the importance of taking a biopsychosocial approach when describing the health of people with USH, in which previous research is lacking. Additional research should focus on the mechanisms at different levels that affect people with USH and their health from a life- course perspective. Furthermore, research should include a salutogenic perspective to explore the resources and strengths of people with USH.

Usher syndrome (USH) is the name given to a group of recessively inherited disorders characterised by hearing loss, progressive visual loss due to a retinal degeneration termed retinitis pigmentosa (RP) and in some cases vestibular dysfunction. It is the most common form of syndromic RP and is clinically and genetically heterogeneous. There are three clinical subtypes termed USH1, USH2 and USH3, which are defined by the severity of hearing loss and vestibular dysfunction with visual loss due to RP being common to each subtype. To date, mutations in nine genes have been associated with the three clinical subtypes of Usher syndrome as well as non-syndromic hearing loss and RP. This wide spectrum of clinical and genetic variability provides challenges to clinicians in making a diagnosis of Usher syndrome and delivering prognostic information to affected individuals, whilst the genetic heterogeneity presents problems to geneticists attempting to achieve a molecular diagnosis. This study aims to address these issues by determining the distribution of clinical and molecular subtypes of USH in the United Kingdom (UK). This study represents an original contribution to the knowledge of Usher syndrome, as it is the first prospective clinical study to sequence the coding regions of each of the nine genes associated with this disorder in 187 affected families regardless of their clinical subtype. Detailed ophthalmic phenotyping was performed in 219 individuals. This comprehensive strategy of molecular analysis afforded the opportunity to interrogate for the possibility of digenic effects for which no evidence was found. This strategy enabled the discovery of an atypical and novel phenotype associated with the USH1C gene. A molecular diagnosis was achieved in 80% of families with Usher syndrome and the ophthalmic phenotype of a large cohort of affected individuals with Usher syndrome has been further delineated. This study has resulted in a large cohort of UK patients with a confirmed molecular diagnosis and detailed ophthalmic phenotyping, which will provide a framework for subsequent longitudinal studies enabling the characterisation of how visual function progresses over time. Understanding the natural history of this disorder in genotyped individuals will help pave the way for subsequent gene-directed therapy studies in the future.

Usher syndrome type 1C is an autosomal recessive condition in which profound, congenital sensorineural deafness is found in association with vestibular hypofunction and childhood onset retinitis pigmentosa. The gene responsible for Usher type 1C, USH1C, codes for a PDZ domain-containing protein, harmonin, of unknown function. In addition, the locus for a form of non- syndromic autosomal recessive deafness, DFNB18, overlaps with the USH1C gene. In this thesis the USH1C gene is studied in more detail, both at the molecular level and at the protein level. Two cohorts of patients, individuals diagnosed with Usher type 1, and a group of sibs with recessive non-syndromic deafness concordant for markers flanking the DFNB18 locus, were screened for mutations in USH1C. One Usher type 1 patient was homozygous for a recurrent mutation, and the possibility of a founder effect was investigated by analysing intragenic SNPs. Another Usher type 1 patient had two novel coding mutations that were studied in more detail to establish whether they were likely to be disease-causing or represent rare polymorphisms. USH1C is an alternatively spliced gene with evidence for tissue-specific isoforms of the protein. The repertoire of alternative isoforms and their tissue distributions were studied in human foetal tissues using non-quantitative RT- PCR. Particular attention was paid to a putative isoform thought to utilize an alternative start site in the centre of the gene. This isoform may have importance in other tissues when a mutation at the 5' end of USH1C results in a non-functional protein from the usual start site. The sub-cellular localization of harmonin was investigated in individual human epithelial cells using fluorescent immunocytochemistry, and fluorescent immunohistochemistry was used to study the localization in mouse inner ear sections. Finally, to understand more about the possible role of harmonin in the ear and the eye, an in vitro GST pull-down assay was set up to investigate the interaction of harmonin with another Usher type 1 protein, protocadherin 15.

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.

Le syndrome d’Usher (USH) cause une surdité-cécité chez l’homme. Au moins neuf gènes responsables ont été identifiés. L’origine de l’atteinte auditive a été dévoilée par l’analyse de souris mutantes, mais les causes de la cécité sont encore obscures. Néanmoins, unes des protéines de Usher, la myosine VIIa, a été impliquée dans le transport intracellulaire dans les photorécepteurs. Pour mieux comprendre le rôle dans la rétine, j’ai étudié l’un de ses partenaires, la spectrine βV. Nous avons conclu que cette spectrine, en collaboration avec les protéines USH1, est impliquée dans le trafic cellulaire: elle couple les moteurs (myosine VIIA, kinesine II et le complexe dynéine/dynactine) à leurs cargos en route vers le segment externe des photorécepteurs. La combinaison d’études comparatives dans les cellules ciliées (CC) de l'oreille interne de grenouille et de souris, de tests biochimiques et d’analyses phylogénétiques indique que le transport vers et depuis la surface apicale des cellules est la fonction ancestrale de cette spectrine. Chez les mammifères, une pression évolutive a engendré le recrutement de la spectrine βV à la paroi latérale des CC externes auditives, probablement pour participer à une nouvelle fonction: l’électromotilité. Enfin, j’ai étudié l’origine de la surdité dans le syndrome d’Usher III. Le seul gène causal connu est CLRN1, qui code pour la clarine-1. Nous avons conclu que la clarine-1 est nécessaire à la maturation et le maintien des touffes ciliaires des CC. De plus, la clarine-1 est essentielle pour le regroupement des canaux calciques voltage-dépendants au voisinage immédiat de la machinerie exocytotique de la synapse à ruban des CC internes
Usher syndrome (USH) causes a combined deafness-blindness in humans. At least nine causative genes are known. While the analysis of USH knockout mice has shed light on the origin of the auditory deficit, the causes of vision loss are still unclear. Nevertheless, USH1B protein, myosin VIIa, appears to contribute to intracellular traffic in photoreceptor cells. To better understand the role of this myosin in the retina, I studied the functions of its interacting partner, spectrin βV. We found that spectrin V, along with USH1 proteins, participates in intracellular transport by coupling motor proteins (myosin VIIa, kinesin II, dynein/dynactin complex) to the cargoes en route towards the outer segment of photoreceptor cells. Evidence from comparative studies in frog and mouse inner ear, biochemical assays and phylogenetic analyses point to cargo trafficking to and from the apical cell region, as the likely ancestral function of this spectrin. Our analyses also suggest that evolutionary pressures in the mammalian lineage drove the recruitment of spectrin βV to the lateral wall of auditory outer hair cells, probably to support a new function: electromotility. Finally, I explored the origin of hearing loss in Usher syndrome of type III (USH3). So far, the only causal gene known is CLRN1, which codes for clarin-1. The comparative characterization of two Clrn1 mouse mutants revealed that clarin-1 is required for the maturation and maintenance of the hair bundle in the hair cells. Moreover, our results indicate that clarin-1 is also essential to cluster the voltage-gated Ca2+ channels in close proximity to the exocytotic machinery of the ribbon synapse of inner hair cells

Le syndrome d’Usher (USH) cause une surdité-cécité chez l’homme. Au moins neuf gènes responsables ont été identifiés. L’origine de l’atteinte auditive a été dévoilée par l’analyse de souris mutantes, mais les causes de la cécité sont encore obscures. Néanmoins, unes des protéines de Usher, la myosine VIIa, a été impliquée dans le transport intracellulaire dans les photorécepteurs. Pour mieux comprendre le rôle dans la rétine, j’ai étudié l’un de ses partenaires, la spectrine βV. Nous avons conclu que cette spectrine, en collaboration avec les protéines USH1, est impliquée dans le trafic cellulaire: elle couple les moteurs (myosine VIIA, kinesine II et le complexe dynéine/dynactine) à leurs cargos en route vers le segment externe des photorécepteurs. La combinaison d’études comparatives dans les cellules ciliées (CC) de l'oreille interne de grenouille et de souris, de tests biochimiques et d’analyses phylogénétiques indique que le transport vers et depuis la surface apicale des cellules est la fonction ancestrale de cette spectrine. Chez les mammifères, une pression évolutive a engendré le recrutement de la spectrine βV à la paroi latérale des CC externes auditives, probablement pour participer à une nouvelle fonction: l’électromotilité. Enfin, j’ai étudié l’origine de la surdité dans le syndrome d’Usher III. Le seul gène causal connu est CLRN1, qui code pour la clarine-1. Nous avons conclu que la clarine-1 est nécessaire à la maturation et le maintien des touffes ciliaires des CC. De plus, la clarine-1 est essentielle pour le regroupement des canaux calciques voltage-dépendants au voisinage immédiat de la machinerie exocytotique de la synapse à ruban des CC internes
Usher syndrome (USH) causes a combined deafness-blindness in humans. At least nine causative genes are known. While the analysis of USH knockout mice has shed light on the origin of the auditory deficit, the causes of vision loss are still unclear. Nevertheless, USH1B protein, myosin VIIa, appears to contribute to intracellular traffic in photoreceptor cells. To better understand the role of this myosin in the retina, I studied the functions of its interacting partner, spectrin βV. We found that spectrin V, along with USH1 proteins, participates in intracellular transport by coupling motor proteins (myosin VIIa, kinesin II, dynein/dynactin complex) to the cargoes en route towards the outer segment of photoreceptor cells. Evidence from comparative studies in frog and mouse inner ear, biochemical assays and phylogenetic analyses point to cargo trafficking to and from the apical cell region, as the likely ancestral function of this spectrin. Our analyses also suggest that evolutionary pressures in the mammalian lineage drove the recruitment of spectrin βV to the lateral wall of auditory outer hair cells, probably to support a new function: electromotility. Finally, I explored the origin of hearing loss in Usher syndrome of type III (USH3). So far, the only causal gene known is CLRN1, which codes for clarin-1. The comparative characterization of two Clrn1 mouse mutants revealed that clarin-1 is required for the maturation and maintenance of the hair bundle in the hair cells. Moreover, our results indicate that clarin-1 is also essential to cluster the voltage-gated Ca2+ channels in close proximity to the exocytotic machinery of the ribbon synapse of inner hair cells

Très répandus dans la nature, les biofilms sont fréquemment des perturbateurs des processus industriels et peuvent également remettre en cause la sécurité sanitaire dans le domaine médical. La formation de biofilms débute par une étape d'adhésion associée à une importante variété de facteurs extracellulaires qui vont permettre la reconnaissance d'un grand nombre de ligands différents. Parmi ces adhésines, la famille des chaperone-usher constitue un groupe hétérogène et abondant de structures extracellulaires qui participent à la colonisation bactérienne de nombreux environnements. Un des axes de recherche de notre laboratoire vise à identifier et caractériser les acteurs moléculaires permettant à la bactérie modèle E. Coli d'adhérer aux surfaces et de former des biofilms. Cette bactérie possède une dizaine de chaperone-usher "cryptiques" en conditions classiques de laboratoire, mais fonctionnelles lorsqu'elles sont exprimées. Nous nous sommes concentrés sur un fimbriae formant des « fagot» lorsqu'il est exprimé chez E. Cou K-12 : le fimbriae Yad. Notre travail a débuté par une caractérisation structurale de Yad permettant d'identifier YadN comme la piline majeure formant la structure principale du fimbriae et YadC comme la lectine reconnaissant un sucre : le xylose. Un second axe d'étude a permis de mieux comprendre la complexité du réseau de régulation de yad mettant en jeux de nombreux régulateurs ainsi que des facteurs environnementaux tels que l'oxygène ou la température. Le xylose étant un des constituants principal de la biomasse végétale et l'expression de yad étant maximale en condition anaérobie et à des températures inférieures à 37°C, une étude dans la rhizosphère des plantes a été réalisée. Des expériences de compétitions de colonisations bactériennes dans la rhizosphère de graines de maïs germées ont ainsi démontré que les bactéries exprimant Yad colonisent mieux que la souche sauvage. Ces résultats suggèrent donc que le fimbriae Yad participe à la survie d' E. Coli à l'extérieur de son hôte, dans son habitat secondaire
Bacterial cell surface proteins and appendages mediate adhesion to surfaces or host tissues. Initial adhesion is thus a key step in colonization and biofilm formation pro cesses leading to infection. The chaperone-usher family includes many fimbriae promoting interaction with host specific epithelial cell surfaces as well as colonisation of secondary habitats such as plants. Our laboratory objective is to characterize E. Coli arsenal of adhesins since they allow certain microorganisms to survive in the host or on surfaces, to understand their role in E. Cou biology. We recently identified seven E. Cou K-12 chaperone-usher fimbriae silenced under classical laboratory conditions but functional when constitutively expressed. One of these fimbriae is produced upon the expression of the yad operon and forms a network of surfaces appendages forming bundles connecting bacteria. This structural organisation is responsible for the capacity of the Yad fimbriae to induce adhesion to abiotic and biotic surfaces. We undertook a functional characterisation of these fimbriae, thus identifying their structural components. We demonstrate that they promote adhesion of E. Coli to xylose through the lectin, YadC, located at die tip of the fimbriae. The study of yad regulation showed that it is strongly repressed by the nucleoid-associated protein H-NS. Additionally, we demonstrated that a complex regulatory network involving multiple regulators and environmental factors such as temperature and oxygenation also participate in the control of yad expression. Indeed, yad expression is highly increased in anaerobic conditions and at 30°C. Those results and Yad affinity for xylose prompt us to assay Yad involvement in rhizosphere colonization. Yad expressing bacteria colonize the rhizosphere more efficiently in competition experiments with the wild¬type strain. Those results show that E. Con possesses a large arsenal of adhesion factors, which expression is controlled by environmental conditions that could modulate its ability to colonize and persist

Le syndrome de Usher (USH) est une maladie transmise selon le mode autosomique récessif caractérisée par l’association d’une surdité congénitale (HL) et d’une rétinite pigmentaire (RP), et dans certains cas, d’une aréflexie vestibulaire. Une hétérogénéité clinique et génétique est reconnue. Environ 10 % des cas USH restent non résolus après analyse moléculaire exhaustive des différents gènes. Ces cas incluent les patients qui ne portent aucune mutation dans un des gènes USH connus ainsi que les patients porteurs d’une seule mutation dans un gène USH. Au cours de cette thèse, nous nous sommes intéressés à l’étude des patients porteurs d’une seule mutation dans les gènes USH2A et PCDH15.Dans la première partie de la thèse, nous avons analysé une cohorte de patients avec un phénotype USH2A bien défini : 5 patients pour lesquels une seule mutation à l’état hétérozygote avait été identifiée dans le gène USH2A et un patient porteur d’un variant silencieux en trans d’une mutation non-sens.Pour les 5 patients, nous avons émis l’hypothèse que la seconde mutation, restant à être identifiée, pourrait se trouver dans des régions introniques profondes. Pour cela, nous avons développé une approche de séquençage à haut débit (NGS) de l’ADN pour identifier les variants introniques profonds dans le gène USH2A et évaluer leurs conséquences sur l’épissage. Comme preuve de concept et pour valider l’approche, y compris le pipeline bio-informatique et l’évaluation des outils de prédiction de l’épissage, nous avons analysé un patient porteur d’un pseudoexon (PE) connu dans le gène USH2A. Ensuite, les 5 patients ont été étudiés en utilisant le pipeline défini, ce qui a conduit à l’identification de 3 nouveaux variants introniques profonds chez 4 d’entre eux. Tous les variants ont été prédits comme pouvant avoir un impact sur l’épissage et aboutir à l’insertion de PE. Ces prédictions ont été validées par les essais minigènes. Grâce à cette étude, nous présentons une stratégie innovante pour identifier les mutations introniques profondes, lorsque l’analyse des transcrits n’est pas possible. Par ailleurs, le pipeline bio-informatique développé fonctionne indépendamment de la taille du gène analysé, ce qui permet l’application possible de cette approche à n’importe quel gène. Par ailleurs, un oligonucléotide antisens de type morpholino (AMO) a été évalué in vitro afin de rétablir l’altération d’épissage induite par une des mutations identifiées. Les résultats ont montré un taux d’exclusion élevé du transcrit aberrant et suggèrent une application possible en thérapie moléculaire. Nous avons ensuite effectué des études sur le variant USH2A c.1377T>A, un variant silencieux afin d’évaluer son effet sur l’épissage. L’analyse de l’ARN issu de cellules nasales du patient a montré que ce variant conduit au saut de l’exon 8 dans les transcrits USH2A. Ceci a été confirmé par un essai minigène. En outre, des études préliminaires ont été réalisées en utilisant des outils de prédictions et des essais minigènes pour évaluer l’implication des éléments cis-régulateurs dans le défaut d’épissage observé chez le patient. Dans la deuxième partie de la thèse, nous avons analysé une patiente USH1, pour laquelle une seule mutation avait été identifiée dans le gène PCDH15. Dans ce cas, nous avons combiné la culture des cellules épithéliales nasales avec l’analyse des transcrits PCDH15. Celle-ci a été réalisée par séquençage de cinq RT-PCR chevauchantes. Grâce à cette analyse, nous avons réussi à délimiter une région d’intérêt dans le transcrit, dont l’amplification a échoué exclusivement pour l’allèle porteur de la mutation non identifiée. D’autres analyses ont été effectuées dans la région génomique correspondante par capture ciblée couplée au séquençage NGS et LongRange PCR suivi de séquençage Sanger. Cependant, aucun variant candidat n’a été identifié à ce jour. Nous suggérons l’implication de mécanismes moléculaires complexes qui restent à être caractérisés
Usher syndrome (USH) is an autosomal recessive disorder characterized by the association of sensorineural hearing loss (HL) and retinitis pigmentosa (RP), and in some cases, vestibular areflexia. Clinical and genetic heterogeneity are recognised. Indeed, three clinical types can be caused by mutations in one of the 10 known genes and USH2A represents the most frequently involved gene.Approximately 10 % of the USH cases remain genetically unsolved after extensive molecular analysis of the different genes, which includes sequencing of the exons and their intronic boundaries, combined to large rearrangements screening by array CGH. These unsolved cases include patients who do not carry any mutation in any of the known USH genes and patients who carry a single USH mutation. During this thesis we focalised on the study of patients carrying a single mutation in USH2A and PCDH15 gene.First, we have analysed a cohort of well-defined USH2A patients: five patients, for whom a single USH2A heterozygous mutation had been identified and one patient carrying a silent variant in trans to a nonsense mutation. For the 5 patients, we supposed that the second mutation remaining to be found could be localised deep in the introns. Indeed, a deep intronic mutation resulting in the inclusion of a pseudoexon (PE 40) in USH2A transcripts had been identified, following RNA analysis from nasal cells. Unfortunately, analysing USH2A transcripts still represent a challenging approach in a diagnostic settings and it is not always possible. To circumvent this issue, we have developed a DNA-Next Generation Sequencing (NGS) approach to identify deep intronic variants in USH2A and evaluate their consequences on splicing. As a proof of concept and to validate this approach, including the bioinformatics pipeline and the assessment of splicing predictor tools, the patient carrying the PE 40 was analysed at first. Then, the 5 patients were studied using the defined pipeline, which led to the identification of 3 distinct novel deep intronic variants in 4 of them. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. Through this study, we present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. In addition, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Moreover, an antisense morpholino oligonucleotide (AMO) tested in vitro for its ability to restore the splicing alterations caused by one of the identified mutation provided high inhibition rates. These results are indicative of a potential application for molecular therapy.In the second case, we have performed studies on the USH2A c.1377T>A silent variant to investigate its effect on splicing. Analysis of RNA from nasal cells of patients showed that this variant led to the skipping of exon 8 in USH2A transcripts. This was confirmed by minigene assay. Moreover, preliminary studies have been performed using prediction tools and minigene assays to assess the involvement of cis-acting elements in causing the aberrant splicing.In the second part of the thesis, we have analysed an USH1 patient, for whom only one mutation had been identified in the PCDH15 gene. In this case, we combined nasal epithelial cells culture with the analysis of the PCDH15 transcripts. This was performed by sequencing five overlapping RT-PCRs. Through this analysis, we were able to delimit a region within the transcript, which failed to be amplified exclusively in the allele carrying the unidentified mutation. Further analyses have been performed in the corresponding genomic region by NGS-target capture and LongRange PCR associated with Sanger sequencing. However, no evident mutation has been identified so far. Therefore, we suggest the involvement of complex molecular mechanisms that remain to be characterised

La vue et l'ouïe font intervenir des cellules capables de rapidement traduire une onde, lumineuse ou sonore, en un message électrochimique transmissible au cerveau. La fonction de ces cellules sensitives repose sur leurs morphologies uniques. Les mutations de onze gènes sont la cause des syndromes Usher, associant cécité et surdité. Les protéines Usher sont indispensables à l'architecture de ces deux types cellulaires ; elles forment des complexes dont les interactions clés sont maintenues principalement par des domaines PDZ. L'une des protéines centrales de ce réseau est la Whirline, une protéine multi-domaine contenant trois domaines PDZ. Pour comprendre les bases moléculaires des syndromes Usher, nous nous sommes concentrés sur la caractérisation biochimique et biophysique de la Whirline. Nous avons identifié un nouveau domaine HHD2 de la Whirline dont nous avons obtenue la structure à haute résolution et déterminé le comportement en solution, isolé et avec les domaines adjacents. Nous avons ensuite caractérisé un supramodule transitoire entre deux domaines PDZ, maintenu par des extensions structurées de chacun des domaines. Nous avons résolu la structure de la conformation compacte unique de ce complexe et étudié son équilibre avec un ensemble de conformations étendues. Nous avons enfin caractérisé in vitro le réseau d'interaction des domaines PDZ de la Whirline avec les protéines Usher. L'ensemble de nos résultats sur la structure modulaire et l'interactome de la Whirline permet de mieux comprendre le rôle de la Whirline dans les différents complexes Usher et d'expliquer les conséquences de ses mutations sur les mécanismes moléculaires de l'audition et de la vision
Vision and hearing rely on the capacity of cells to rapidly transduce electromagnetic waves or sound waves into chemical messages that are transmissible to the brain. The function of these sensory cells requires unique morphologies. The mutations of eleven genes are responsible for Usher syndromes, associating blindness and deafness. The Usher proteins are pivotal to the architecture of the photoreceptor and hearing cells. They form complexes in which the critical interactions are mainly maintained by PDZ domains. One of these central proteins is Whirlin, a multi-domain protein encompassing three PDZ domains. To understand the molecular basis of the Usher syndromes, we focused our project on the biochemical and biophysical characterization of Whirlin. We identified a new HHD2 domain on Whirlin, for which we solved the structure at high resolution and determined the behavior in solution, isolated or with adjacent domains. We then identified a transient supramodule between two PDZ domains, maintained by PDZ structured extensions. We determined the structure of the compact and unique conformation of this tandem and we characterized its equilibrium with an ensemble of more extended conformations. Finally, we characterized in vitro the network of interaction of the PDZ domains of Whirlin, with the majority of the Usher proteins. Our results on the modular structure and the interactome of Whirlin get insight into the role of Whirlin in the numerous complexes formed by the Usher proteins and allow to better explain the consequences of its mutation on the molecular mechanisms of hearing and vision

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

Les photorécepteurs sont des neurones très spécifiques dédiés à la phototransduction et reposant sur une machinerie cellulaire très complexe. La dépolarisation permanente dans le noir des photorécepteurs déclenche une transmission synaptique constante et extrêmement spécifique qui requièrent une quantité d'énergie considérable. Les photorécepteurs peuvent dégénérer lorsque la phototransduction ou l'apport énergétique sont altérés. Le syndrome d'Usher conduit à une surdité et une cécité. La recherche du rôle des protéines usher dans les photorécepteurs a été freinée par l'absence de phénotype rétinien dans les modèles. De la même façon, la compréhension des mécanismes moléculaires conduisant à l'atteinte des cônes dans la rétinopathie diabétique a été entravée par l'absence de symptômes vasculaires et neuronaux dans les modèles. Durant ma thèse, j'ai caractérisé deux modèles animaux des syndromes Usher et HANAC. Des atteintes neuronales ont été démontrées par électrorétinogramme et par l'observation de changements morphologiques des cellules. Dans les modèles Usher, j'ai également montré une neuroprotection des photorécepteurs par plusieurs stratégies. Dans le modèle HANAC, les atteintes neuronales étaient associées à une tortuosité vasculaire anormale une augmentation de la perméabilité vasculaire et l'expression accrue de VEGF. Les évaluations phénotypiques de ces trois modèles fournissent un nouvel aperçu de la physiopathologie des dégénérescences des cônes dans le syndrome d'Usher et dans les maladies vasculaires complexes. Ce travail ouvre surtout la voie au développement et à l'évaluation de nouvelles stratégies thérapeutiques pour ces maladies menant à la cécité
Photoreceptors are very specific neurons dedicated to phototransduction, which relies on very complex machinery. The maintained depolarization in darkness triggers a constant and thus very specific type of synaptic transmission. These require high energy need. As a consequence, photoreceptors can degenerate in various hereditary retinal diseases when phototransduction or energy consumption are altered. The Usher syndrome is such a hereditary disease leading to both deafness and blindness. If Usher proteins are involved in the mechanotransduction in hair cells, investigating their role in photoreceptors has been hamperedby the lack of a retinal phenotype in murine models. Similarly, understanding themolecular mechanisms of cone dysfunction in diabetic retinopathy has beenhampered by the lack of vascular and neuronal symptoms and neuronal models. During my PhD, I have developed animal models of Usher and HANAC syndromes both leading to cone photoreceptor dysfunction and damage. Cone dysfunction was demonstrated by electroretinogram recording and by morphological changes, retinal gliosis and microglial activation. In the Usher models, I also demonstrated photoreceptor neuroprotection by different strategies. In the HANAC model, neuronal dysfunction was associated as in diabetic retinopathy to blood vessel tortuosity, blood vessel permeability and incresead VEGF expression levels. These phenotypic evaluations of mouse models provide new insights into the physiopathology of cone photoreceptor degeneration in Usher syndrome and in complex vascular diseases. It also open the way for the development and assessment of new therapeutic strategies for these diseases leading to blindness

La vue et l'ouïe font intervenir des cellules capables de rapidement traduire une onde, lumineuse ou sonore, en un message électrochimique transmissible au cerveau. La fonction de ces cellules sensitives repose sur leurs morphologies uniques. Les mutations de onze gènes sont la cause des syndromes Usher, associant cécité et surdité. Les protéines Usher sont indispensables à l'architecture de ces deux types cellulaires ; elles forment des complexes dont les interactions clés sont maintenues principalement par des domaines PDZ. L'une des protéines centrales de ce réseau est la Whirline, une protéine multi-domaine contenant trois domaines PDZ. Pour comprendre les bases moléculaires des syndromes Usher, nous nous sommes concentrés sur la caractérisation biochimique et biophysique de la Whirline. Nous avons identifié un nouveau domaine HHD2 de la Whirline dont nous avons obtenue la structure à haute résolution et déterminé le comportement en solution, isolé et avec les domaines adjacents. Nous avons ensuite caractérisé un supramodule transitoire entre deux domaines PDZ, maintenu par des extensions structurées de chacun des domaines. Nous avons résolu la structure de la conformation compacte unique de ce complexe et étudié son équilibre avec un ensemble de conformations étendues. Nous avons enfin caractérisé in vitro le réseau d'interaction des domaines PDZ de la Whirline avec les protéines Usher. L'ensemble de nos résultats sur la structure modulaire et l'interactome de la Whirline permet de mieux comprendre le rôle de la Whirline dans les différents complexes Usher et d'expliquer les conséquences de ses mutations sur les mécanismes moléculaires de l'audition et de la vision
Vision and hearing rely on the capacity of cells to rapidly transduce electromagnetic waves or sound waves into chemical messages that are transmissible to the brain. The function of these sensory cells requires unique morphologies. The mutations of eleven genes are responsible for Usher syndromes, associating blindness and deafness. The Usher proteins are pivotal to the architecture of the photoreceptor and hearing cells. They form complexes in which the critical interactions are mainly maintained by PDZ domains. One of these central proteins is Whirlin, a multi-domain protein encompassing three PDZ domains. To understand the molecular basis of the Usher syndromes, we focused our project on the biochemical and biophysical characterization of Whirlin. We identified a new HHD2 domain on Whirlin, for which we solved the structure at high resolution and determined the behavior in solution, isolated or with adjacent domains. We then identified a transient supramodule between two PDZ domains, maintained by PDZ structured extensions. We determined the structure of the compact and unique conformation of this tandem and we characterized its equilibrium with an ensemble of more extended conformations. Finally, we characterized in vitro the network of interaction of the PDZ domains of Whirlin, with the majority of the Usher proteins. Our results on the modular structure and the interactome of Whirlin get insight into the role of Whirlin in the numerous complexes formed by the Usher proteins and allow to better explain the consequences of its mutation on the molecular mechanisms of hearing and vision

[ES] El síndrome de Usher (USH) es un trastorno raro autosómico recesivo definido principalmente por sordera neurosensorial (SNHL), y una distrofia retiniana conocida como retinosis pigmentaria (RP). La patología muestra heterogeneidad genética, puesto que se conocen al menos 10 genes responsables. No obstante, las mutaciones en USH2A son la causa más frecuente de la enfermedad, en gran medida por la recurrencia de la variante patogénica c.2299delG. En esta tesis se ha desarrollado un ensayo de edición génica para revertir dicha anomalía genética por medio del sistema CRISPR/Cas9. Se diseñaron y probaron varios complejos CRISPR específicos de locus, y el más eficiente fue usado para la corrección de la mutación c.2299delG en células derivadas de pacientes. La tasa de corrección de la mutación obtenida fue del 2.5%. Otro objetivo de esta tesis ha sido la caracterización genética de pacientes USH aún sin diagnóstico molecular. Una primera fase implicó la secuenciación masiva dirigida de las regiones codificantes de todos los genes asociados a la enfermedad. Este estudio, cuya cohorte incluyó 58 pacientes no escrutados previamente, permitió la identificación de 42 nuevas mutaciones presuntamente patológicas, y una tasa general de detección de alelos responsables de la enfermedad de prácticamente el 83%. Sorprendentemente, uno de los sujetos presentaba mutaciones en CEP250, uno de los últimos genes correlacionados con la enfermedad. Una exhaustiva revisión clínica reveló que la degeneración retiniana se trataba en realidad de una distrofia de conos y bastones en lugar de RP clásica, lo cual permitió consolidación del gen CEP250 como responsable de un fenotipo similar al USH. El resto de casos sin resolver induce a sospechar de la existencia de otros genes vinculados con USH. Así pues, se analizó el exoma íntegro de dichos casos negativos del panel por medio de secuenciación de exoma completo, lo cual proporcionó resultados relevantes en seis de las muestras estudiadas. Uno de tales sujetos resultó ser un claro caso de fenocopia de USH, al albergar mutaciones patogénicas en dos genes independientes, TECTA y REEP6, siendo el primero responsable de la SNHL y el segundo de la RP. De forma parecida, en otro paciente se detectaron variantes patológicas para RP en el gen EYS, pero no se identificó paralelamente ningún cambio genético que explicara la SNHL. Tres individuos adicionales resultaron haber sido erróneamente diagnosticados como USH, dada la conclusiva inexistencia o ambigüedad de la sordera. Uno de ellos fue definido como homocigoto de una mutación en CNGB1, ya reconocido como responsable de RP. En el segundo de dichos sujetos se identificó una mutación en homocigosis en el gen GRN, cuyos defectos en estado heterocigoto están asociados a demencia frontotemporal y más raramente combinada con RP si ambos alelos se encuentran alterados. Por otro lado, el tercer paciente fue resuelto como heterocigoto compuesto de variantes en WDR19, un gen asociado en mayor medida a una distrofia retiniana acompañada de trastornos renales y, más raramente, a la forma aislada del síntoma. En el último de los seis casos resaltados de este objetivo se detectó una mutación homocigota sin sentido en el gen ASIC5, cuyo papel en el organismo todavía se desconoce. Sin embargo, se han correlacionado funciones visuales y auditivas para miembros de la misma familia proteica. En conjunto, los hallazgos obtenidos en este trabajo avalan la importancia del uso de las más novedosas tecnologías en la búsqueda de soluciones para enfermedades raras, las cuales presentan por ahora un pronóstico terapéutico bastante desamparado. Asimismo, otras consecuencias positivas en cuanto a la caracterización genética de los pacientes son la corroboración (o rectificación) del diagnóstico inicial, así como la contribución a la estimación demográfica y correlaciones de genotipo-fenotipo, que en definit
[CAT] La síndrome d'Usher (USH) és una malaltia rara autosòmic recessiu definit principalment per sordera neurosensorial (SNHL) i una distròfia retiniana coneguda com a retinosi pigmentària (RP). La patologia mostra heterogeneïtat genètica, ja que es coneixen almenys 10 gens responsables. No obstant això, les mutacions en USH2A són la causa més freqüent de la malaltia, a causa de la recurrència de la variant patogènica c.2299delG. En aquesta tesi s'ha desenvolupat un assaig d'edició gènica per a revertir la dita anomalia genètica per mitjà del sistema CRISPR/Cas9. Es van dissenyar i van probar diversos complexos CRISPR específics de locus, i el més eficient va ser usat per a la correcció de la mutació en cèl·lules derivades de pacients. La taxa de correcció de la mutació obtinguda va ser del 2.5%. Un altre objectiu d'aquesta tesi ha sigut la caracterització genètica de pacients USH encara sense diagnòstic molecular. Una primera fase va implicar la seqüenciació massiva dirigida de les regions codificants de tots els gens associats a la malaltia. Aquest estudi, la cohort de la qual va incloure 58 pacients no escrutats prèviament, va permetre la identificació de 42 noves mutacions presumptament patològiques, i una taxa general de detecció d'al·lels responsables de la malaltia de pràcticament el 83%. Sorprenentment, un dels subjectes presentava mutacions en CEP250, un dels últims gens correlacionats amb la malaltia. Una exhaustiva revisió clínica va revelar que la degeneració retiniana es tractava en realitat d'una distròfia de cons i bastons en lloc de RP clàssica. Aquestes troballes han permés la consolidació del gen CEP250 com a responsable d'un fenotip similar al USH. La resta de casos sense resoldre induïx a sospitar de l'existència d'altres gens vinculats amb USH. Així, doncs, es va analitzar l'exoma íntegre dels casos negatius del panell a través de seqüenciació d'exoma complet, cosa que va proporcionar resultats rellevants en sis de les mostres estudiades. Un de tals subjectes va resultar ser un clar cas de fenocopia d'USH, a l'albergar mutacions patogèniques en dos gens independents, TECTA i REEP6, sent el primer responsable de la SNHL i el segon de la RP. De forma semblant, en un altre pacient es van detectar variants patològiques per a RP al gen EYS, però no es va identificar paral·lelament cap canvi genètic que explicara la SNHL. Tres individus addicionals van resultar haver sigut erròniament diagnosticats com USH, donada la final inexistència o ambigüitat de la sordera. Un d'ells va ser definit com a homozigot d'una mutació en CNGB1, ja reconegut com a responsable de RP. En el segon d'aquestes subjectes es va identificar una mutació en homozigosi en el gen GRN, els defectes del qual estan associats a demència frontotemporal en estat heterozigot, i més rarament en combinació amb RP si ambdós al·lels es troben alterats. D'altra banda, el tercer pacient va ser resolt com a heterozigot compost de variants en WDR19, un gen associat en major grau a una distròfia retiniana acompanyada de trastorns renals i, més rarament, a la forma aïllada del símptoma. En l'últim dels sis casos ressaltats d'aquest objectiu es va detectar una mutació homozigota sense sentit en el gen ASIC5, el paper en l'organisme del qual encara es desconeix. Amb tot, s'han correlacionat funcions visuals i auditives per a membres de la mateixa família proteica. En conjunt, les troballes obtingudes en aquest treball avalen la importància de l'ús de les més noves tecnologies en la recerca de solucions per a malalties rares, les quals presenten per ara un pronòstic terapèutic prou desemparat. Així mateix, altres conseqüències positives quant a la caracterització genètica dels pacients són la corroboració (o rectificació) del diagnòstic inicial, així com la contribució a l'estimació demogràfica i correlacions de genotip-fenotip, que en definitiva ajuden en la compressió d'US
[EN] Usher syndrome (USH) is a rare autosomal recessive disorder defined essentially by sensorineural hearing loss (SNHL) and a retinal dystrophy known as retinitis pigmentosa (RP). The condition shows a genetic heterogeneity, since there are at least 10 genes known to be causative of the syndrome. However, mutations in USH2A are the most frequent cause of the disease, due in a large measure to the recurrence of the c.2299delG pathogenic variant. A gene editing assay to reverse this specific genetic anomaly was developed in this thesis by means of the groundbreaking CRISPR/Cas9 system. Several locus-specific CRISPR complexes were designed and tested, and the most efficient was used to proceed with the c.2299delG mutation correction on patient-derived cells. The trial resulted in a mutation correction rate of 2.5%. Another goal of this thesis was the genetic characterization of molecularly undiagnosed USH patients. Given the genetic diversity of the disease, the procedure required the implementation of high-throughput sequencing, a technology that enables in bulk sequencing of any number of selected loci (or the indiscriminate totality) of the genome. The first phase implied the targeted sequencing of the coding-relevant regions of all known causative or disease-associated genes at the moment. The study, comprising a cohort of 58 previously unscreened patients, enabled the identification of 42 novel putative pathogenic mutations, and an etiologic-allele detection ratio shy of 83%. Remarkably, one of the subjects harbored nonsense mutations in CEP250, which is one of the latest USH-associated genes. However, an exhaustive review of the clinical features unmasked the retinal degeneration as a cone-rod dystrophy rather than RP, which reinforced the linkage of the gene to an USH-like phenotype. The remaining portion of unresolved cases lead to suspicion of the existence of other genes accountable for USH. Hence, the complete exome of such panel-negative cases was screened through whole exome sequencing. This venture provided relevant findings in six of the surveyed samples. One subject was plainly exposed as an USH phenocopy by harboring pathogenic splice-site mutations in two independent genes, TECTA and REEP6, the former responsible for the SNHL and the latter for the RP. Similarly, RP-causative variants in EYS were detected in another patient, yet no pathogenic changes explaining the HL were discovered. Three additional individuals were ultimately unveiled as USH misdiagnosed cases, being the HL actually absent or ambiguous. One of the patients in this set was homozygous for a mutation in CNGB1, already known to be accountable for RP. The other two cases showed a more peculiar outcome being compound heterozygous for putatively pathogenic variants in genes generally associated to other disorders. One presented a homozygous mutation in GRN, a gene associated to frontotemporal dementia under heterozygous condition and less commonly to combined RP for homozygous alterations. The third subject was found to be a carrier of mutations in WDR19, a gene best associated with retinal disorders accompanied by renal signs and rarely with the isolated visual symptom. The last case presented a homozygous nonsense variant in the ASIC5 gene, whose role has yet to be learned. However, some correlations to visual and hearing functions have been reported for members of the same protein family. Altogether, the results obtained from this work attest to the importance of applying the most up-to-date technologies in the search of solutions for rare diseases that realistically pose a despairing therapeutic prognosis. In addition, the positive consequences of the genetic characterization of the patients are the corroboration (or else correction) of the initial diagnosis, and the contribution to the appraisal of demographic and genotype-phenotype correlations, which ultimately aid in the understanding USH and other related diseases.
This work was financially supported by the Institute of Health Carlos III and FEDER funds (ISCIII; grants PI13/00638, PI16/00425, PI16/00539, and PIE13/00046), Fundación ONCE (grant 2015/0398), XVIII Fundaluce-FARPE, and “Telemaratón: Todos Somos Raros, Todos Somos Únicos” (grant IP58). C.F.-G. is a recipient of a fellowship (grant IFI14/00021) from the ISCIII. R.P.V.-M. is a Miguel Servet researcher (grant CP11/00090 funded by ISCIII, Madrid, Spain). The funds from the ISCIII are partially supported by the European Regional Development Fund. R.-P.V.M. is also a Marie Curie fellow (grant CIG322034 from the European Commission).
Fuster García, C. (2020). Therapeutic approaches and development of genomic diagnostic tools for Usher syndrome [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137034
TESIS

Thesis: S.M. in Science Writing, Massachusetts Institute of Technology, Department of Comparative Media Studies/Writing, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 21-27).
Usher Syndrome is an inherited disease that leads to the progressive loss of hearing and vision (retinitis pigmentosa). Increasingly, genetic testing, either through panels or whole exome sequencing, lets people know which of the twelve genes identified to date is responsible for the loss of their senses. Researchers are using these genetic ascertainment data to identify patients for clinical trials: There is no approved treatment for retinitis pigmentosa. A philanthropically-funded translational research program led by Dr. Edwin Stone at the University of Iowa seeks to provide an at-cost personalized gene therapy for everybody with Ushers, regardless how rare. His efforts focus transfecting patient-derived induced pluripotent stem cells with a viral gene vector to replace the broken Ushers gene. Meanwhile, a phase 1/11 clinical trial led by Dr. Eric Pierce and ReNeuron takes a different approach-injecting participants' subretinal space with healthy donor stem cells. Critically, both of these methods risk remaining vision. This is the story of two people with Ushers -- an infant with MYO7A -- associated Ushers who was genetically diagnosed in her first year of life, and a retired man who likely suffers from USH2A-associated Ushers, whose life experience exemplifies the condition, but whose specific genetic mutation has never been identified. Both have opted for cochlear implants to improve their hearing, and both work to adapt each day to their changing senses.
by Kate Telma.
S.M. in Science Writing

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este trabalho tem como objetivo analisar as estruturas narrativa e fílmica do conto The Fall of the House of Usher (1839), de Edgar Allan Poe (1809-1849), e do filme La Chute de la Maison Usher (1928), dirigido por Jean Epstein (1897-1953), considerando o modo como se constrói o espaço e os sentidos resultantes dessa composição. As obras que tomamos como base teórica para a análise são: Lima Barreto e o Espaço Romanesco (1976), de Osman Lins, A linguagem cinematográfica (2003), de Marcel Martin, A narrativa cinematográfica (2009), de André Gaudreault e François Jost. Assim, analisaremos os procedimentos narrativos utilizados por Poe na composição do espaço e as técnicas cinematográficas que aproximam ou afastam as características do espaço fílmico da narrativa original, o conto
This study aims to analyze the narrative and filmic structures of the tale The Fall of the House of Usher (1839), by Edgar Allan Poe (1809-1849), and the film La Chute de la Maison Usher (1928), directed by Jean Epstein (1897-1953), considering how space is constructed and the meanings resulting from this construction. The works we considered as theoretical basis for the analysis are: Lima Barreto e o Espaço Romanesco (1976), by Osman Lins, A Linguagem Cinematográfica (2003), by Marcel Martin, A narrativa cinematográfica (2009), by André Gaudreault and François Jost. Thus, we analyze the narrative techniques used by Poe in the composition of the space, and the cinematic techniques which bring together the characteristics of the filmic space of the original narrative, namely the tale, or which set them apart

Orientador: Fabiane Renata Borsato
Banca: Maria de Lourdes Ortiz Gandini Baldan
Banca: Alvaro Luiz Hattnher
Resumo: Este trabalho tem como objetivo analisar as estruturas narrativa e fílmica do conto The Fall of the House of Usher (1839), de Edgar Allan Poe (1809-1849), e do filme La Chute de la Maison Usher (1928), dirigido por Jean Epstein (1897-1953), considerando o modo como se constrói o espaço e os sentidos resultantes dessa composição. As obras que tomamos como base teórica para a análise são: Lima Barreto e o Espaço Romanesco (1976), de Osman Lins, A linguagem cinematográfica (2003), de Marcel Martin, A narrativa cinematográfica (2009), de André Gaudreault e François Jost. Assim, analisaremos os procedimentos narrativos utilizados por Poe na composição do espaço e as técnicas cinematográficas que aproximam ou afastam as características do espaço fílmico da narrativa original, o conto
Abstract: This study aims to analyze the narrative and filmic structures of the tale The Fall of the House of Usher (1839), by Edgar Allan Poe (1809-1849), and the film La Chute de la Maison Usher (1928), directed by Jean Epstein (1897-1953), considering how space is constructed and the meanings resulting from this construction. The works we considered as theoretical basis for the analysis are: Lima Barreto e o Espaço Romanesco (1976), by Osman Lins, A Linguagem Cinematográfica (2003), by Marcel Martin, A narrativa cinematográfica (2009), by André Gaudreault and François Jost. Thus, we analyze the narrative techniques used by Poe in the composition of the space, and the cinematic techniques which bring together the characteristics of the filmic space of the original narrative, namely the tale, or which set them apart
Mestre

Usher syndrome is a rare inherited genetic condition which is one of the main causes of acquired deafblindness in the United Kingdom (UK). Although the condition is not life threatening, it is life altering and will have a significant impact on the lives of not only the person diagnosed with the condition, but also their families, friendship groups and new and existing relationships. The aim of the study was to develop an understanding of the experiences of diagnosis of and living with Usher syndrome, from the perspective of adults living in England. Specific objectives of the study were to explore the experience of being diagnosed with Usher syndrome; explore the transition from adolescence to adulthood for people who have Usher syndrome; to develop an understanding of the experience of living with Usher syndrome, including support, developmental opportunities and the role of the Deaf community; to disseminate original findings; inform future practice, service development, policy and education and make recommendations for further research relating to the experience of living with Usher syndrome. To address these aims and objectives, this qualitative, descriptive phenomenological study, conducted interviews with 20 males and females aged between 18-82 years from a variety of demographic locations. To contribute to the trustworthiness of the study, I developed a methodological innovation called ‘Multiple Sensory Communication and Interview Methods’ (MSCIM) which ensured that as far as possible communication and interview methods were participant led. Three overarching messages from findings were revealed: the importance of ensuring communication is timely, supportive and appropriate; Usher support at the right time: providing physical and virtual support networks and essentiality of Usher awareness: raising the profile. This study is unique because it is the first qualitative, descriptive phenomenological study to demonstrate new knowledge to better understand and support people living with Usher in England.

As shallow reefs continue to decline, scientists are searching for the key to their persistence; as it turns out, they may just need to look deeper. Below many shallow tropical reefs, there exist healthy and more stable mesophotic coral reef communities. The ability of these reefs to act as a refuge for declining shallow populations has garnered significant interest among the scientific community; however, the reproductive and larval aspects necessary for this to occur are unknown. This study assesses the ability of deep reefs to act as a reproductive refuge for shallow counterparts by examining gametic compatibility, viability and larval settlement preferences. Gametes from Orbicella franksi inhabiting the shallow (14-20m) and the upper mesophotic (27-32m) were introduced in a series of inter- and intra-depth crosses and found to be compatible. Larval settlement experiments found no natal depth preference, with deep larvae significantly preferring to settle on shallow conditioned substrate. Our findings support the plausibility of healthy mesophotic reefs acting as a refuge for depauperate shallow populations by (1) providing gametes to mix with limited shallow gametes resulting in increased fertilization and (2) providing larvae that recruit and repopulate shallow reefs. This is the first study to comprehensively evaluate the Deep Reef Refugia Hypothesis from a reproductive and larval settlement standpoint. Our results suggest a close coupling between shallow and mesophotic reefs through gamete and larval export and illustrate the current and future importance of these mesophotic reefs.

In my thesis I will discuss Edgar Allan Poe’s “The Fall of the House of Usher” in relation to the expectations that scholars have of the gothic genre. I will break this project into four chapters, along with an introduction: (Ch.1) a critical review of scholarship on Poe’s “Usher” that will demonstrate the difficulty in coming to a critical consensus on the tale, (Ch.2) a discussion of Brown’s outline of Gothic conventions, (Ch.3) a look at Poe’s “The Philosophy of Composition” juxtaposed with Aristotle’s Poetics to illumine aspects of Poe’s approach to writing and how it has been informed, and (Ch.4) a close reading of Poe’s “Usher.”

Le syndrome de Usher (USH) est la cause la plus fréquente de surdité-cécité héréditaire chez l’Homme. USH1B est causé par une mutation dans le gène codant la myosine VIIa. Pour comprendre le rôle de cette myosine dans la dystrophie rétinienne, nous avons identifié et caractérisé son interaction avec une spectrine dite non-conventionnelle, la spectrine βV, dans les cellules photoréceptrices de la rétine. Nos avons montré que la spectrine βV s’associe également à d’autres protéines USH1, l’opsine et d’autres protéines de la phototransduction, et à des moteurs moléculaires associés aux microtubules. Ainsi, cette spectrine contribuerait au trafic protéique vers le segment externe des photorécepteurs, lieu de la transduction du signal lumineux. Nous avons également montré que la spectrine βV a subi une sélection positive dans la branche des mammifères ce qui pourrait expliquer pourquoi la localisation et le rôle de la protéine varient en fonction du degré de spécialisation cellulaire et de l’espèce étudiée
Usher syndrome is the most frequent cause of deaf-blindness in Humans. Defects in myosin VIIa causes the USH1B syndrome. To understand the role of this actin-based motor in the retinal pathology, we identified and characterized its interaction with a non-conventional spectrin, spectrin βV, in the retinal photoreceptor cells. We found that spectrin βV associates also with other USH1 proteins, opsin and some other phototransduction proteins, as well as to the microtubule-based motors. Together our data led us suggest that spectrin βV contribute to protein transport towards the photoreceptor outer disks, site of phototransduction. Moreover, we found that βV spectrin has been submitted to a positive selection in mammalian lineage, which could explain the differences we observed in the localization and the function of the protein in different cell types and species

This qualitative study investigated the remembered lived experiences of six individuals who were diagnosed with Usher syndrome, the effect that the progressive condition had upon their lives, and their experiences with vocational rehabilitation. Usher syndrome is an autosomal recessive condition that presents as deafness or hearing loss with comorbid retinitis pigmentosa sometimes with vestibular areflexia. The participants recalled details of their own reaction to the diagnoses as well as the reactions of their parents. Themes were identified in their responses that included independent dependence, Usher support, parental reaction, lowered expectations, hope, and ongoing change. The participants, three men and three women, reported periods of adjustment and sadness as well as hopes for their future, career accomplishments, and social interactions.

Dans l’environnement, les bactéries vivent le plus souvent attachées à une surface, en un mode de vie communautaire pluricellulaire, le biofilm. Pseudomonas aeruginosa est un pathogène humain opportuniste à Gram négatif, responsable d’infections nosocomiales et d’infections respiratoires chez les patients atteints de mucoviscidose. La vie communautaire chez cette bactérie est un moyen de persistance chez l'hôte qu'il colonise et constitue un mécanisme passif ou actif de résistance aux molécules antimicrobiennes. L’établissement de P. Aeruginosa sous forme communautaire est le fait de machineries macromoléculaires, dont la voie « chaperone-usher » (CU). Mon travail de thèse a consisté à caractériser les systèmes CU, CupB et CupC de P. Aeruginosa. Ces deux systèmes sont fonctionnels et assemblent des fimbriae de façon spécifique au travers de leur protéine de membrane externe, le « usher ». Le cluster cupB code pour deux chaperones périplasmiques CupB2 et CupB4 dont nous montrons qu’elles adressent respectivement la piline majeure CupB1 et l’adhésine CupB6 à la protéine usher CupB3. Le cluster cupB code également pour un substrat des systèmes TPS, CupB5 dont la sécrétion est assurée par le « P-usher », résultant de la fusion d’un domaine POTRA et d’un domaine usher. Le « P-usher » est une protéine bi-fonctionnelle capable d’assembler un fimbriae CU et de sécréter un substrat TPS. Le cluster cupB de P. Aeruginosa aurait subi au cours de l’évolution un réarrangement génétique aboutissant à la disparition du gène tpsB et à la création du gène hybride cupB3 codant pour le « P-usher », hypothèse confirmée par la sécrétion de la protéine TPS de P. Aeruginosa par la protéine de membrane externe TpsB de P. Fluorescens présente dans le cluster cupB de cette bactérie. L’étude de ces fimbriae CupB et CupC a montré leur implication dans la formation du biofilm et la réponse de la cellule hôte, plus particulièrement la protéine CupB5 qui joue un rôle dans la phagocytose
In the environment, bacteria live mostly attached to a surface in a sedentary and multicellular community called biofilm. Pseudomonas aeruginosa is a human opportunistic Gram negative pathogen, responsible for nosocomial infections and respiratory infections of cystic fibrosis patients. This bacterial lifestyle represents a strategy to persist in colonized hosts and constitutes a passive or active mechanism of resistance towards antimicrobial molecules. The establishment of P. Aeruginosa in biofilm is due to macromolecular machines of the “chaperone-usher” (CU) family. My thesis work consisted in the characterisation of P. Aeruginosa CupB and CupC systems of the CU family. These two systems are functional and specifically assemble fimbriae through their own outer membrane protein, the usher. The cupB cluster encodes two periplasmic chaperones CupB2 and CupB4, which are respectively responsible for the capture in the périplasme and targeting of the major pilin CupB1 and the adhesin CupB6, respectively, to the CupB3 usher protein. The cupB cluster encodes a TpsA substrate of the two partner secretion system (TPS), CupB5, which secretion occurs through the “P-usher”. The “P-usher” is therefore a bi-functional protein capable of both the assembly of CupB fimbriae and the secretion of CupB5 protein. The “P-usher” results from the fusion of a POTRA domain and of an “usher” domain, highlighting a genetic rearrangement in the P. Aeruginosa cupB gene cluster. This is strongly supported by the restoration of the P. Aeruginosa TPS substrate (CupB5) secretion, in the absence of the “P-usher”, by the introduction of the heterologous TpsB outer membrane protein from the P. Fluorescens cupB cluster. The study of the fimbriae CupB and CupC fimbriae showed their implication in biofilm formation and host cell response, particularly of the CupB5 protein which plays a role in phagocytosis

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 & 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.

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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.

Plusieurs études ont souligné le rôle important de différents fimbriae dans la formation des biofilms. Ce projet de thèse a visé à l'identification de nouvelles adhésines chezE. Coli K-12, dans la fraction de son génome (34%), qui n'a pas été caractérisée expérimentalement. Les résultats obtenus nous ont permis de caractériser sept opérons,ycbtybg,yfc,yadtyra, sfm etyeh, qui présentent des homologies avec les fimbriae de type 1, membre prototypique de la famille des fimbriae «Chaperone/Usher». A l'aide d'une fusion lacZzeo et des souches exprimant constitutivement chacun de ces opérons, nous avons démontré que, malgré leur, très faible expression en conditions de laboratoire, six sur sept opérons sont fonctionnels et promeuvent l'adhésion sur des surfaces abiotiques et biotiques. L'extraction de ces fimbriae a révélé les unités majeures pour certains de ces appendices, tandis que la microscopie électronique a permis la visualisation des filaments pour ycb etyad. Une analyse détaillée a montré également que d'une part, ces fimbriae potentiels ont des profils d'adhésion et d'affinité pour des sucres distincts, et d'autre part que des interactions physiques avec d'autres adhésines peuvent avoir lieu. De plus, nous avons prouvé que leur expression est sous le contrôle de H-NS et, à l'exception deyad, sous celui de CRP. En conclusion cette étude a mis en évidence que les opérons ycb, yfc, yad, yra, sfm et yeh codent pour des fimbriae cryptiques mais cependant fonctionnels, dont l'expression pourrait, en j conditions environnementales qui restent à déterminer, contribuer à la capacité d' E. Coli à adhérer une grande variété de surfaces et de niches ont d'explorer aussi les propriétés d'autres protéines d'enveloppe de différents types de virus pour l'application virologique fondamentale et clinique
The study of the bacteria/surface interactions has revealed the important role played by different fimbriae in biofilm formation. Although extensively studied as a model bacterium, E. Coli K-12 genome still contains 34% of genes of unknown function and we hypothesized that some of them could correspond to functional adhesins. In this project we characterized E. Coli K-12 ycb, ybg, yfc, yad, yra, sfm and yeh operons, which display sequence and organization homologies to type 1 fimbriae exported by the chaperone usher pathway. By using a reporter lacZzeo cassette as well as overexpressing strains we showed that, although they are not or are poorly expressed in laboratory conditions, six of them are j functional when expressed and promote adhesion to various abiotic and/or epithelial cell surfaces. Fimbriae extraction revealed the major subunits for certain appendages, whereas electron microscopy analysis allowed visualization of fimbrial filaments for ycb and yad. While the studied fimbriae display different binding specificities and potentially distinct sugar affinities, we demonstrated the possibility for synergy or interference witth other adhesins such as Ag43 or type 1 fimbriae. We furthermore showed that their régulation is under the negative control of H-NS and, except for yad, subjected to cAMP receptor protein-mediated activation and carbon catabolite repression. These results therefore demonstrate that ycb, yfc, yad, yra, sfm and yeh operons encode cryptic but functional fimbrial adhesins, whose expression, upon still undertermined environmental conditions, could contribute to E. Coli's ability to adhere to a large diversity of surfaces in its various ecological niches

Den här uppsatsen handlar om elever med Ushers syndrom och inkludering i klassrummet. Ushers syndrom består av en kombinerad syn- och hörselnedsättning och synen försämras med tiden framförallt i synfältet. Med Ushers syndrom innebär det även svårigheter att se i mörkret. Syftet med uppsatsen är att undersöka hur inkludering kan skapas i klassrummet utifrån elever på aktörsnivå och skolan och lärare på institutionell nivå. Anpassningarna är viktiga faktorer för att eleverna ska bli inkluderade och jag undersöker även vilka anpassningar som uppstår och hur går de tillväga för att lösa problem i klassrummet. Denna uppsats utgår från en etnografisk ansats och studiens empiri består av intervjuer med elever, läraren, dövblindsamordnaren, en klassrumsobservation och styrdokument. Dessa empirier presenteras i resultatredovisningen med fokus på elever med Ushers syndrom och inkludering på olika nivåer som skolan, läraren och klassrummet. Studien har visat att skolan och lärarens bemötande är en betydande faktor för inkluderingsprocessen. Forskningen har betonat att det är hela verksamheten som ska förändras i ett inkluderingsarbete. Ett bra bemötande innebär att dessa aktörer med anpassningar ser till att eleverna med Ushers syndrom får uppleva inkludering. Anpassningarna som visats i studien är lärarens klädsel, belysning, turtagning i samtal och tekniska hjälpmedel. Det har konstaterats att elever med Ushers syndrom också själva har en del i inkluderingsarbetet genom att framföra sina behov eftersom behovet varierar från elev till elev. Det har framträtt i studien att det inte är lätt att framföra sina behov direkt eftersom elever inte vill avvika från normen i klassrummet som består av döva elever och teckenspråk.

Le modèle de Usher est un modèle matriciel qui décrit l'évolution en temps discret d'une population
structurée par taille et qui restreint les transitions entre les classes d'état. Il est particulièrement
adapté pour décrire la dynamique d'un peuplement forestier et sert de guide dans la gestion des
forêts. Cette étude porte sur les prédictions dans l'état stationnaire du modèle. L'objectif principal
est la construction d'intervalles de confiance de ces prédictions. Dans un premier temps, des
intervalles de confiance asymptotiques sont construits en utilisant les estimateurs du maximum de
vraisemblance des prédictions. La distribution asymptotique de ces estimateurs est obtenue grâce
à la delta-méthode. Les résultats sont étendus, dans un autre chapitre, au cas du modèle densitédépendant,
dans lequel les paramètres sont fonctions des caractéristiques courantes de la population.
Dans un deuxième temps, les intervalles de confiance asymptotiques sont affinés en cherchant des
estimateurs robustes des paramètres de transition du modèle. Cette recherche est guidée par deux
types de contraintes du modèle portant sur sa structure discrète et sur la dynamique de la population.
Les estimateurs des paramètres ainsi construits sont des L-estimateurs exprimés dans un
modèle statistique multidimensionnel. Le critère de robustesse utilisé est la sensibilité des estimateurs,
basé sur la notion de fonction d'influence. Les résultats théoriques sont appliqués un jeu de
données réelles d'un peuplement forestier en Guyane Française et les implications pratiques sont
discutées.

The fraction 1 antigenic capsule of Yersinia pestis is a homopolymer assembled by the two-component chaperone-usher system; a terminal branch of the general secretory pathway. A periplasmic chaperone (CaflM) is responsible for folding and capping of monomeric Cafl, whilst preserving it in an energy competent conformation for fiber formation. Translocation of Cafl polymer to the cell surface occurs via the outer membrane usher CaflA, and circumstantial evidence suggests that polymerisation is catalysed by the usher. Recovery of OlT-sensitive CaflT10CT139C polymer at the surface of recombinant E. coli demonstrated that donor strand alignment corresponding to the register T10/T139 (donor strand/F strand) was used throughout the fiber. Sucrose density gradient centrifugation was used to show that periplasmic CaflM :Cafl fiber preassembly complexes specifically target CaflA within the outer membrane. CaflM bound to polymerisation-deficient subunit was shown to be degraded in the presence of usher, consistent with a secondary binding event upon interaction with CaflA, resulting sensitivity to periplasmic OegP. Deletion mutagenesis within the N-terminus of CaflA (CaflAN) showed that this domain is essential for capsular Fl biogenesis and that loss of even the 27 N-terminal residues of CaflA led to drastic reduction in assembly. Fl biogenesis was completely abolished in this mutant when periplasmic CaflAN was coexpressed, suggesting that the domain was able to compete for periplasmic Fl preassembly complexes. The usher N-terminus was overproduced as a soluble periplasmic domain and purified. This has recently been used for collaborative crystallographic studies of both the Nterminus alone and in complex with chaperone and subunit. Histagged CaflAN was purified and used for in vitro Biacore binding studies with purified CaflM:Cafl~3 binary complex and Cafl:CaflA9R2 ternary complex. Qualitative analysis was obtained for an interaction between CaflAN and the preassembly complexes, consistent with similar observations made in related chaperone/usher systems for the usher N-terminus.

Le syndrome d'Usher (USH) est la première cause de surdité et de cécité chez l'homme. 3 types cliniques USH (USH1-3) sont définis. La perte auditive des patients de forme clinique de type III n'est pas congénitale, mais progressive, survenant généralement pendant ou après l'adolescence, et la présence de défauts vestibulaires et l'âge d'apparition de la rétinite pigmentaire sont variables. J'ai étudié le rôle de la clarine-1, à l'origine de l'USH3A. Le gène CLRN1 code pour la clarine-1. La caractérisation des souris mutantes Clrn1 a révélé que la clarine-1 est essentielle pour l'organisation structurelle et la fonction des canaux présynaptiques Cav1.3 Ca2+ au niveau de la synapse du ruban des cellules ciliées internes et pour la distribution des récepteurs AMPA postsynaptiques. Le transfert à médiation virale de la Clrn1 intacte dans les souris mutantes clarin-1 dans la cochlée a empêché durablement les défauts synaptiques et l'apparition de la perte auditive. J'ai également exploré le rôle de clarine-2, dont l'absence entraîne une perte auditive. Les souris mutantes clarin-2 présentent une perte auditive progressive et précoce. Nos résultats démontrent un rôle clé pour la clarin-2 dans l'audition des mammifères, fournissant des informations sur l'interaction entre la transduction mécano-électrique et le maintien des stéréocils. Enfin, j'ai étudié les mécanismes compensatoires impliquant les deux clarines qui pourraient cacher des fonctions importantes dans l'oreille interne. L'inactivation de Clrn1 et Clrn2 altère prématurément la fonction vestibulaire, la perte totale de transduction mécano-électrique et les perturbations extrêmes des stéréocils du faisceau de cheveux. Une élucidation supplémentaire des mécanismes par lesquels les deux clarines interagissent, et l'importance de ces interactions dans les systèmes vestibulaire et cochléaire est en cours
Usher Syndrome (USH) is the first cause of deafness blindness in humans. 3 USH clinical types (USH1-3) are defined. Type III clinical form patients hearing loss is not congenital, but progressive, usually occurring during or after adolescence, and the presence of vestibular defects and age of onset of retinitis pigmentosa is variable. I studied the role of clarin-1, causing USH3A. CLRN1 gene encodes clarin-1. The characterization of Clrn1 mutant mice revealed that clarin-1 is essential for the structural organization and function of the presynaptic channels Cav1.3 Ca2+ at the inner hair cell ribbon synapse and for the distribution of postsynaptic AMPA receptors. The viral-mediated transfer of the intact Clrn1 into the clarin-1 mutant mice in cochlea durably prevented synaptic defects and occurrence of the hearing loss. I also explored the role of clarin-2 another member of the clarin family, the absence of which leads to hearing loss. The clarin-2 mutant mice have a progressive, early-onset hearing loss. Our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance. Finally, I studied the compensatory mechanisms involving the two clarins which might conceal important functions in the inner ear. The inactivation of both Clrn1 and Clrn2 impairs prematurely the vestibular function, total loss of mechano-electrical transduction & extreme disruptions of the hair bundle stereocilia. Further elucidation of the mechanisms through which the two clarins interact, and the importance of such interactions in the vestibular and cochlear systems is underway

La bactérie à Gram négatif Pseudomonas aeruginosa est un pathogène opportuniste possédant de nombreux systèmes moléculaires contribuant à sa pathogénicité et à son développement selon un mode de vie en biofilm, qui permet une meilleure résistance aux défenses de l’hôte et aux antibiotiques. Parmi ces systèmes, on retrouve les fimbriae assemblés par la voie chaperonne – « usher » (CU). La voie CU implique une protéine formant un pore dans la membrane externe, la protéine « usher », une chaperonne périplasmique et au moins une sous unité piline qui va être assemblée en fimbriae à la surface de la bactérie.Mon travail de thèse a principalement porté sur l’étude du cluster de gènes cupE, qui code pour un système CU du clade σ-fimbriae. Ce système est différent des quatre autres systèmes CU cupA – cupD déjà caractérisés chez P. aeruginosa et appartenant au clade γ4-fimbriae. Indépendamment des gènes codant la protéine « usher » et la chaperonne, ce cluster comprend quatre autres gènes qui codent pour des sous unités pilines atypiques (une piline majeure, deux pilines mineures et une adhésine). Nous avons montré que le système CupE est fonctionnel et permet l’assemblage de fimbriae à la surface de la cellule, assemblage (oligomérisation de la sous unité majeure en fimbirae) pour lequel nous avons montré que l’adhésine est essentielle, au contraire des deux sous unités mineures. Ces fimbriae jouent un rôle important dans la formation et la structuration du biofilm, tant à des étapes précoces que lors des étapes tardives. Excepté une piline mineure, tous les éléments de la fibre sont importants pour la formation du biofilm. Ce cluster de gènes est spécifiquement exprimé en conditions de formation du biofilm et par une approche de mutagenèse aléatoire par transposition, le système à deux composants (TCS) PprA – PprB a été identifié comme un régulateur positif potentiel de ce cluster. L’implication de ce TCS dans la régulation de cupE a été vérifiée et nous avons pu démontrer par des techniques de retard sur gel que ce contrôle est direct et que PprB se lie sur des boites putatives en amont du +1 de transcription de cupE défini par 5’-RACE PCR.Ce TCS ayant été identifié au préalable comme un régulateur positif de pili de type IVB Flp, eux-mêmes acteurs du biofilm chez P. aeruginosa, nous avons caractérisé le régulon du régulateur de réponse PprB. Parmi les nouvelles cibles régulées positivement par PprB, nous avons identifié deux cibles nouvelles dont nous avons débuté la caractérisation. La première est un opéron de quatre gènes codant pour un transporteur ABC impliqué dans la résistance aux antibiotiques spécifiquement en conditions de biofilm et pour une protéine de haut poids moléculaire, substrat potentiel de ce transporteur ABC. Cette protéine, que nous avons renommé AdhA, est effectivement sécrétée par le transporteur ABC et est impliquée dans la cohésion des cellules au cours de la formation du biofilm. Il s’agit d’une nouvelle adhésine participant à la structuration du biofilm chez P. aeruginosa. La seconde cible est un gène codant pour une protéine que nous avons renommée Hvn, homologue aux halovibrines HvnA et HvnB de Vibrio fischeri. Le sécrétome d’une souche délétée du gène hvn est considérablement modifié et son absence d’effet sur la morphologie des cellules eucaryotes par rapport au sécrétome de la souche PAO1 suggère que la protéine Hvn pourrait jouer un rôle dans la virulence de P. aeruginosa.Au travers de ce travail, nous avons caractérisé le système CupE de la voie CU chez P. aeruginosa et montré qu’il assemblait des fimbriae atypiques pouvant avoir un rôle dans les différentes phases de la formation du biofilm et qu’il était sous la régulation directe et positive du TCS PprA – PprB. [...]
The Gram negative opportunistic pathogen Pseudomonas aeruginosa is equipped with molecular systems that contribute to bacterial pathogenesis and biofilm development, this latter being associated with increased resistance to host defenses and antibiotics. Among them, are the fimbriae assembled by the chaperone usher (CU) pathway. The CU pathway involves a protein called the usher that forms a pore in the outer membrane, a periplasmic chaperone and at least one fimbrial subunit assembled into fimbriae at the cell surface.My PhD study mainly focuses on the cupE gene cluster, encoding a CU system from the σ-fimbrial clade. This system is different from all the CU systems cupA – cupD already characterized in P. aeruginosa, all belonging to the γ4-fimbrial clade. Independently from the genes encoding the usher and the chaperone, this cluster comprises four other genes encoding atypical pilins (one major pilin, two minor pilins and one adhesin). We showed that this CupE system is functional and allows the assembly of fimbriae at the cell surface. Unlike the two minor pilins, the adhesin is necessary for the fimbriae assembly (oligomerisation of the major subunit into the fiber). These fimbriae play an important role in biofilm formation and structuration, at early and late steps. Except one minor pilin, all subunits are important for the CupE-dependent biofilm formation. This gene cluster is specifically expressed in biofilm conditions and a random transposon mutagenesis allowed us to identify the two component system (TCS) PprA-PprB as an activator of cupE genes. We verified the implication of this TCS in cupE regulation and, using EMSA, we showed that the PprB control on cupE is direct, with PprB binding onto putative boxes upstream the transcription start of cupE, defined by 5’-RACE PCR.As this TCS was identified before as a positive regulator for the type IVB Flp pilus, another actor in the biofilm formation, we defined the PprB regulon. Among the new targets positively controlled by PprB, we found two new targets that we started to characterize. The first one is a four gene operon encoding an ABC transporter involved in antibiotic resistance specifically in biofilm conditions and a high molecular weight protein, a potential substrate for this ABC transporter. This protein that we renamed AdhA is indeed secreted by this ABC transporter and is implicated in the cohesion between cells during the biofilm formation. It is a new adhesin participating into the biofilm structuration of P. aeruginosa. The second target is a gene encoding a protein that we renamed Hvn, and homologous to HvnA and HvnB halovibrins from Vibrio fischeri. Secretome from an hvn mutant is highly modified and the lack of effect on eukaryotic cell’s morphology in comparison to the PAO1 secretome suggests the Hvn protein can play a role in P. aeruginosa virulence.Through this work, we characterized the cupE system from the CU pathway and showed that this system can assemble atypical fimbriae having a role in the different phases of biofilm formation. This system is under the positive and direct regulation of the TCS PprA – PprB.[...]

La (Pcdh15) est localisée dans les touffes ciliaires des cellules ciliées internes et externes de la cochlée. Elle forme des liens interstéréociliaires entre les différents stéréocils, des protrusions riches en actine qui forment ensemble la touffe ciliaire. L’absence de Pcdh15 entraîne la désorganisation des touffes ciliaires et la perte de la mécanotransduction auditive. Pcdh15 forme en effet la partie basse du lien de bout de cil (en anglais tip-link), le lien contrôlant l’ouverture du canal de transduction, qui se situe au point d’insertion inferieur de ce lien. Il existe trois isoformes de Pcdh15 (CD1, CD2 et CD3). J’ai étudié la distribution de ces isoformes dans la touffe ciliaire à différents stades de sa maturation. Différents modèles murins « KO conditionnel » ont été générés, ils m’ont permis d’analyser les conséquences de l’absence de chacune des isoformes individuellement, ainsi que celles de l’absence de Pcdh15-CD2 et Pcdh15-CD3, et celles de l’absence simultanée des trois isoformes. J’ai ainsi pu conclure que Pcdh15-CD2 est la seule isoforme essentielle pour la formation des tip-links dans les cellules ciliées matures. Dans les touffes ciliaires matures, Pcdh15 joue également un rôle dans l’interaction entre les touffes ciliaires et la membrane tectoriale, dans le contrôle de la taille des stéréocils, et dans la formation des liens interstéréociliaires apicaux. Pour ces fonctions, mais pas pour la formation du tip-link, Pcdh15-CD1 et Pcdh15-CD2 (les isoformes présentes dans la touffe ciliaire mature) sont redondantes. Cependant, Pcdh15-CD1 ne peut compenser que partiellement l’absence de Pcdh15-CD2 et Pcdh15-CD3. Pour étudier comment Pcdh15 interagit avec les autres protéines impliquées dans le syndrome de Usher, les interactions avec l’harmonine et la whirline (deux protéines d’échafaudage qui sont colocalisées avec Pcdh15 à l’apex des stéréocils) ont été analysées in vitro
Protocadherin-15 (Pcdh15 is located in the stereociliary hair bundles of inner and outer hair cells (IHCs and OHCs) of the cochlea, where it forms fibrous links between different stereocilia. Absence of Pcdh15 leads to deafness due to the disorganization of hair bundles and absence of mechano-electrical transduction. The latter is explained as Pcdh15 forms the lower component of the tip-link, that gate hair cell mechano-electrical transduction channels. There are three different splice isoforms of Pcdh15 (CD1, CD2 and CD3), I studied their distribution in the developing and mature auditory hair cells. Different conditional Pcdh15 knockout mouse models were generated, permitting analysis of the absence of each of the different Pcdh15 isoforms individually, of the combined absence of Pcdh15-CD2 and Pcdh15-CD3, and of the absence of all isoforms. I was able to conclude that Pcdh15-CD2 is essential for the formation of tip-links in mature hair cells. In mature hair bundles Pcdh15 also plays a role in the coupling of the hair bundles to the tectorial membrane, in the control of the size of the stereocilia, and in the formation of apical links between stereocilia. The different Pcdh15 isoforms present in mature hair bundles (Pcdh15-CD1 and Pcdh15-CD2) are functionally redundant for these functions, but not for tip-link formation. In immature hair bundles, the different Pcdh15 isoforms are functionally redundant, although Pcdh15-CD1 can only partially compensate the absence of Pcdh15-CD2 and Pcdh15-CD3. To discover how Pcdh15 interacts with other proteins implicated in Usher syndrome, interactions with harmonin and whirlin were analyzed by biophysical techniques

In this thesis, I try to situate the effects of the text, specifically on the reader, by looking at ideas of transformation. My primary investigation is to determine the extent of the effect on the reader and the reader's reality, and if it is possible to alter the reader by inducing a transformation. I argue that transformation is possible as a “becoming”. Transformation depends on the text's reflection and verisimilitude to reality, which aids introspection and the consequent transitioning toward a new identity. I confront these concerns via close analysis of Edgar Allan Poe's “The Fall of the House of Usher”. Whereas critics have read Poe while considering authorial intent and biography, and while limiting effect to emotion, I argue that the reader determines meaning and effect which can impose on identity. This inquiry deals directly with the interaction between the text and the reader, while acknowledging language as the common ground and means of communicating meaning and effect between them. Arnold van Gennep's theory of liminality provides a framework for transition, which I apply to character and reader becoming. And, it explains the interstitial space between the textual realm and the reader's reality. My close analysis of Poe's characters elucidates these tasks as I engage the text as a reflection of the reader's development, and as the narrator's interactions with the Usher siblings mimics the reader's relation to the text. Mikhail Bakhtin's polyphonic theory depicts the text as life-like and appropriate for this exchange. I consider metafiction for its ability to dissemble illusory distinctions between the text and reality, and as it induces consciousness in the reader. I have also placed Poe in conversation with Julia Kristeva for her insights into the psychoanalytic process of abjection, and as she illustrates the revision of identity. Much of this project deals with finding unity and reconciling the inherently contradictory elements of human existence. Ultimately, I consider how the process of textual interaction contributes to potential reader “becoming”. And, I argue that becoming and identity are intimately dependent on selfconsciousness of the vastness of human potential, as well as the dissolution of the very borders designed to limit and make sense of that vastness.

La surdité et les troubles vestibulaires sont des pathologies fréquentes, source de handicap et d’altération de la qualité de vie. Actuellement, il n’existe pas de traitement curatif pour ces pathologies. La thérapie génique utilisant les AAVr semble une alternative prometteuse notamment dans le traitement des surdités et troubles vestibulaires d’origine génétique. Cependant, de nombreux défis restent à relever avant d’envisager une application chez l’Homme. Dans ce travail, nous avons cherché à identifier les étapes clés à franchir pour une application clinique de la thérapie génique pour 2 surdités génétiques humaines, le syndrome USH1G et la surdité DFNB9, à l'aide des modèles murins correspondants, d'études chez le primate non-humain et d'un modèle d’explant d’organes vestibulaires humains. Nous avons pu montrer que la fenêtre thérapeutique était un facteur majeur à prendre en compte dans un objectif translationnel. Le stade de maturation de l’oreille interne influe grandement sur l’efficacité de la thérapie, d’autant plus lorsque la pathologie implique des anomalies de développement comme dans le syndrome USH1. Pourtant, nous avons pu apporter la preuve d’une extension de la fenêtre thérapeutique chez la souris Ush1g-/-, et montrer que la thérapie génique permettait une restauration à un niveau proche de la normale de la fonction vestibulaire et dans une moindre mesure de la fonction auditive après injection à un stade mature. Dans la surdité DFNB9 pour laquelle il n’existe pas d’anomalie de développement, nous avons pu montrer que la thérapie génique permettait une restauration complète de l’audition, et posé les fondements d’une future thérapie chez l’Homme
Deafness and vestibular disorders are frequent pathologies, and sources of disability and impaired quality of life. Deafness is the most common sensory disorder in humans, and 1 child is born deaf for every 700 births. Currently, there is no cure for these disorders. A promising therapeutic alternative is gene therapy using rAAV, and numerous preclinical studies have provided proof of its efficacy in the treatment of deafness and vestibular disorders of genetic origin. However, many challenges remain to be overcome before considering application in humans. In this work, we sought to identify the key steps to be taken for a clinical application of gene therapy for 2 human genetic causes of deafness, USH1G syndrome and DFNB9 deafness. We used the corresponding mouse models for this, as well as studies in non-human primates and an in vitro human vestibular organ explant model. We were able to show that the therapeutic window was a major factor to take into account in a translational objective. The stage of maturation of the inner ear greatly influences the effectiveness of therapy, especially when the pathology involves developmental abnormalities such as in USH1 syndrome. However, we were able to provide evidence of an extension of the therapeutic window in Ush1g-/- mice, and to show that viral gene therapy performed at a mature stage allowed vestibular function to be restored to a level close to normal, and to a lesser extent a restauration of hearing function. In DFNB9 deafness for which there is no developmental abnormality, we were able to show that gene therapy allowed a complete restoration of hearing, and laid the foundations for a future therapy in humans

The Fraction I antigen of Yersinia pestis forms an immunogenic capsule around the cell when invading a mammalian host. This capsule is assembled by the chaperone-usher pathway, one of many strategies Enterobacteriacea have evolved for protein translocation across the outer membrane. Chaperone-usher systems are involved in assembly of polymeric surface fibres such as fibrillae and fimbriae, many of which are virulence factors. These fibres comprise repeating subunits; FI being a polymer of Caf1 subunits. Subunits lack the information required to form a stable fold therefore each system has a specific chaperone, providing the missing information by donating a β-strand which promotes subunits to form an immunoglobulin fold. Chaperone- subunit complexes bind the N-terminal periplasmic domain of the usher and are translocated to the cell surface through the pore of the usher, a process coupled with polymerisation of the subunits and catalysed by the usher. Each subunit has an N-terminal extension which displaces the donor strand of the chaperone forming a highly stable polymer with a condensed immunoglobulin fold. This thesis investigates the role of the Caf1A usher using deletion and point mutagenesis together with functional studies to investigate the domain structure of Caf1A and functions of periplasmic domains. A soluble periplasmic domain was identified within the transmembrane β-barrel and was shown to be dispensable for assembly of the β-barrel but required for function. The role of this domain as a plug was investigated and the region in and around the predicted A-strand was shown to be critical for FI' assembly (particularly residues M237, P239 and Y241). The functional requirement for the predicted C-terminal domain of CaflA was confirmed and its periplasmic location deduced. A protease-sensitive 10 kDa region and a stable 10 kDa periplasmic domain were identified. Hydrophobic patches on the surface of the stable domain were shown to be critical for F1 assembly. The identity of the initial residue of Caf1A and its signal sequence cleavage site was investigated, but was unable to be ascertained. The successful purification of the components of the Caf system allowed attempts to create an in vitro polymerisation system, which would allow individual components to be controlled and investigated, but in vitro polymerisation was unable to be observed under conditions used.

Strain-specific Klebsiella pneumoniae virulence determinants have been described but almost exclusively for hypervirulent liver abscess-associated strains. Island-tagging and fosmid-based marker rescue were used to capture and sequence KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116. This 14.0 kb island exhibited a genome-anomalous G+C content, possessed near-perfect 46 bp direct repeats, and encoded a putative γ1-chaperone/usher fimbrial cluster (fim2). This island was shown to belong to a previously unknown KpGI-5-like island family and was hypothesized to have undergone substantial reductive evolution. Transcriptional analysis demonstrated expression of three fim2 genes and suggested that the eight-gene fim2A-fim2K cluster comprised an operon. In vivo models of urinary tract and lung infection, and large intestinal colonization, in addition to in vitro assays were used to examine the role of fim2 in pathogenesis by comparing KR2107, a streptomycin-resistant derivative of KR116, to three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) constructed using optimized allelic exchange techniques. Although no statistically significant in vivo role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence as fimbrial clusters were lost: KR2107 (most virulent), KR2107Δfim2, KR2107Δfim and KR2107ΔfimΔfim2 (least virulent). Despite using several methods the putative Fim2 fimbriae could not be visualised. Furthermore, the fim2-encoded putative phosphodiesterase Fim2K was determined to alter several c-di-GMP-dependent phenotypes, including biofilm formation and exopolysaccharide production. Additionally, fim2 was present in 14 % of Klebsiella strains studied and appeared intact in the majority of fim2-positive strains. Given the above findings, fim2 may confer a niche-specific evolutionary advantage, potentially related to its ability to encourage dissemination. This thesis is the first study of fim2 and KpGI-5-like islands and will aid further investigations into the full impact of these loci on the lifestyle of Klebsiella species.

The opportunistic human pathogen Pseudomonas aeruginosa is a threat for immunocompromised individuals, a major cause of nosocomial infections, and is prevalent in patients with cystic fibrosis. The bacterium can form biofilms that help it evade the immune response. It adheres to host cells using molecular adhesins, such as pili assembled by chaperone-usher pathways (Cup). Understanding the adhesion could, therefore, help develop treatments that prevent the establishment of infections. This thesis considers the CupB system, consisting of an usher (CupB3), two chaperones (CupB2 and CupB4) and two pilin subunits (CupB1 and CupB6). The chaperones target the pilin subunits to the usher assembling a CupB1-containing pilus with a putative CupB6 adhesin at its tip. The cupB operon also encodes the TpsA-like protein (two partner secretion) CupB5, previously suggested to be secreted by CupB3. The aim of this work was to understand the CupB1-containing pilus assembly and CupB5 secretion mechanism. Genetic and biochemical techniques were used, such as deletion or point mutation, qRT-PCR, pull-down assays, shearing assays, and protein structure prediction or resolution. They led to the following results. First, each chaperone likely has a cognate substrate: CupB1 interacts with CupB2 and CupB6 with CupB4. Second, the crystal structure solved for CupB6 showed that it has a pilin and a putative adhesin domain, connected by a poly-proline linker. Third, CupB5 secretion was observed to be CupB3-independent and TpsB4-dependent. tpsB4 is encoded with its substrate tpsA4. The expression of the cupB and tpsB4/tpsA4 operons was shown to be controlled by the same regulatory pathway, Roc1, and deletion of the tpsB4 transporter gene abolished CupB5 secretion. Fourth, a structural analysis indicated that TpsB4 has two POTRA domains, and POTRA-1 interacts with the highly homologue TPS motifs of CupB5 and TpsA4. Based on these results, the thesis presents a model of CupB pilus assembly and CupB5 secretion.

Denna uppsats innehåller en komparativ studie kring karaktäriseringen av den fiktiva figuren Madeleine Usher i Edgar Allan Poes 1800-tals novell "The Fall of the House of Usher" och Bethany Griffins re-telling av novellen i sin skräckroman The Fall, publicerad 2014. Syftet är att synliggöra hur Madeleine porträtteras och karaktäriseras ur ett genuskritiskt perspektiv med hjälp av bland annat Yvonne Hirdmans teori om genussystem och genuskontrakt, samt Maria Nikolajevas narratologiska teori om konvenansen kring litterärt kön. Utifrån denna analys ämnar jag tolka effekterna av Madeleine Ushers skilda subjektspositionering i verken, samt vilken kritik Griffins re-telling därigenom framför.

La (Pcdh15) est localisée dans les touffes ciliaires des cellules ciliées internes et externes de la cochlée. Elle forme des liens interstéréociliaires entre les différents stéréocils, des protrusions riches en actine qui forment ensemble la touffe ciliaire. L’absence de Pcdh15 entraîne la désorganisation des touffes ciliaires et la perte de la mécanotransduction auditive. Pcdh15 forme en effet la partie basse du lien de bout de cil (en anglais tip-link), le lien contrôlant l’ouverture du canal de transduction, qui se situe au point d’insertion inferieur de ce lien. Il existe trois isoformes de Pcdh15 (CD1, CD2 et CD3). J’ai étudié la distribution de ces isoformes dans la touffe ciliaire à différents stades de sa maturation. Différents modèles murins « KO conditionnel » ont été générés, ils m’ont permis d’analyser les conséquences de l’absence de chacune des isoformes individuellement, ainsi que celles de l’absence de Pcdh15-CD2 et Pcdh15-CD3, et celles de l’absence simultanée des trois isoformes. J’ai ainsi pu conclure que Pcdh15-CD2 est la seule isoforme essentielle pour la formation des tip-links dans les cellules ciliées matures. Dans les touffes ciliaires matures, Pcdh15 joue également un rôle dans l’interaction entre les touffes ciliaires et la membrane tectoriale, dans le contrôle de la taille des stéréocils, et dans la formation des liens interstéréociliaires apicaux. Pour ces fonctions, mais pas pour la formation du tip-link, Pcdh15-CD1 et Pcdh15-CD2 (les isoformes présentes dans la touffe ciliaire mature) sont redondantes. Cependant, Pcdh15-CD1 ne peut compenser que partiellement l’absence de Pcdh15-CD2 et Pcdh15-CD3. Pour étudier comment Pcdh15 interagit avec les autres protéines impliquées dans le syndrome de Usher, les interactions avec l’harmonine et la whirline (deux protéines d’échafaudage qui sont colocalisées avec Pcdh15 à l’apex des stéréocils) ont été analysées in vitro
Protocadherin-15 (Pcdh15 is located in the stereociliary hair bundles of inner and outer hair cells (IHCs and OHCs) of the cochlea, where it forms fibrous links between different stereocilia. Absence of Pcdh15 leads to deafness due to the disorganization of hair bundles and absence of mechano-electrical transduction. The latter is explained as Pcdh15 forms the lower component of the tip-link, that gate hair cell mechano-electrical transduction channels. There are three different splice isoforms of Pcdh15 (CD1, CD2 and CD3), I studied their distribution in the developing and mature auditory hair cells. Different conditional Pcdh15 knockout mouse models were generated, permitting analysis of the absence of each of the different Pcdh15 isoforms individually, of the combined absence of Pcdh15-CD2 and Pcdh15-CD3, and of the absence of all isoforms. I was able to conclude that Pcdh15-CD2 is essential for the formation of tip-links in mature hair cells. In mature hair bundles Pcdh15 also plays a role in the coupling of the hair bundles to the tectorial membrane, in the control of the size of the stereocilia, and in the formation of apical links between stereocilia. The different Pcdh15 isoforms present in mature hair bundles (Pcdh15-CD1 and Pcdh15-CD2) are functionally redundant for these functions, but not for tip-link formation. In immature hair bundles, the different Pcdh15 isoforms are functionally redundant, although Pcdh15-CD1 can only partially compensate the absence of Pcdh15-CD2 and Pcdh15-CD3. To discover how Pcdh15 interacts with other proteins implicated in Usher syndrome, interactions with harmonin and whirlin were analyzed by biophysical techniques

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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.