No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25494号 / 医博第5094号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 羽賀 博典, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The morphological diversity of the external and internal surfaces of the petrosal bone, which contains the structures of the inner ear, across a broad range of therian mammals is documented, and patterns of variation across taxa are identified. One pattern of variation is the result of ontogenetic changes in the ear region, as described for the external surface morphology of a sample of isolated petrosal bones referred to Proboscidea from Pleistocene deposits in central Texas. The morphology of the aquaeductus Fallopii for passage of the greater petrosal branch of the facial nerve supports an ontogenetic explanation for some variation within the proboscidean sample, and a sequence of ossification surrounding the aquaeductus Fallopii is hypothesized. Further ontogenetic patterns are investigated using digital endocasts of the bony labyrinth (preserved on the internal surfaces of the petrosal) constructed from CT data across a growth series of the opossum Monodelphis domestica. Strong correlation between skull length and age is found, but from 27 days after birth onward, there is no correlation with age among most dimensions of the inner ear. Adult dimensions of several of the inner ear structures are achieved before the inner ear is functional in M. domestica. Morphological variation within the inner ear of several eutherian mammals from the Cretaceous of Asia, including zhelestids from the Bissekty Formation of Uzbekistan, is described. The variation within the fossil sample is compared to that observed within extant species of placental mammals, and it is determined that the amount of variation within the Bissekty zhelestid population is within the range of that measured for extant species. Additional evolutionary and physiological patterns preserved within the walls of the bony labyrinth are identified through a high level anatomical comparison of the inner ear cavities across Placentalia as a whole. In particular, features of the inner ear support monophyly of Cetacea, Carnivora, Primatomorpha, and caviomorph Rodentia. The volumetric percentage of the vestibular apparatus (vestibule plus semicircular canals) of aquatic mammals is smaller than that calculated for terrestrial relatives of comparable body size. Thus, aspects of the bony labyrinth are both phylogenetically and physiologically informative. / text

Depuis le début des années 1990, l'imagerie de l'oreille et de la base du crâne s'est considérablement développée. Des pathologies récentes, mal connues ont été explorées en imagerie, en particulier en IRM. L'objectif de mes travaux de recherche, a été de développer des séquences Haute Résolution (HR) sur l'oreille et la base du crâne permettant une analyse sémiologique fine et une caractérisation de ces différentes pathologies dans l’objectif d’une prise en charge plus adaptée. Le début de mes travaux a été orienté vers le conduit auditif interne. Mon but initial a été de mettre point et de développer une séquence 3 HR T2 sur l'oreille interne et le conduit auditif interne ainsi qu’une séquence spin écho T1 HR en coupes fines. Ces séquences ont permis l’analyse précise de l'extension des schwannomes vestibulaires dans le fond du conduit auditif interne. Cette étude a permis de dégager des critères intéressants en imagerie dans le choix thérapeutique (chirurgie versus radio-chirurgie) et dans le choix de la voie d'abord chirurgicale. Mon travail s’est également orienté vers le développement des séquences de diffusion en base du crâne, le but était de développer une séquence de diffusion HR dans le suivi des cholestéatomes opérés de l'oreille moyenne. Les récidives de cholestéatome sont fréquentes et les oreilles opérées sont parfois difficiles à analyser en TDM en cas de comblement de la cavité opératoire. Des séquences IRM spécifiques sont alors nécessaires pour différencier une récidive de cholestéatome, du tissu fibro-inflammatoire fréquemment présent. Plusieurs auteurs avaient montré l'intérêt des séquences de diffusion du fait de coefficients de diffusion très différents entre le tissu fibro-inflammatoire et le cholestéatome. Cependant ces premières séquences de diffusion EPI basse résolution, étaient peu adaptées au rocher et la base du crâne. Notre travail a été de mettre au point et de développer une séquence de diffusion HR Turbo Spin écho adaptée à l'oreille et à la base du crâne permettant le dépistage des récidives de cholestéatome et de valider cette séquence par comparaison au gadolinium tardif et à la chirurgie. Mon travail sur la base du crâne haute résolution m'a également permis de développer des séquences Spin Echo T1 et 3D Echo de gradient T1 en Haute Résolution avec saturation de la graisse permettant une analyse fine des extensions tumorales périnerveuses à la base du crâne principalement dans les cancers du nasopharynx. Ces travaux ont permis de définir les voies d'extension précises de ces tumeurs afin de ne pas sous-évaluer le stade initial de ces tumeurs dans la classification TNM de l'UICC. Ces travaux associés à ceux d'autres chercheurs ont permis de mettre à jour récemment cette classification UICC des tumeurs du nasopharynx. Une de mes principales voies de recherche a été le dépistage, l'analyse et la description des pathologies de l’oreille interne. Une étude longitudinale menée de 1998 à 2008 incluant toutes les pathologies intra-labyrinthiques découvertes au CHU de Lille, a permis de définir et de caractériser trois grands types de pathologies distinctes: les schwannomes intra-labyrinthiques, les labyrinthites, les hémorragies intra-labyrinthiques. Ces pathologies nécessitent des prises en charge différentes. Nous avons pu également définir des critères pronostics en particulier des critères de gravité nécessitant une prise en charge rapide adaptée. Dans le cadre ce travail de recherche sur les pathologies de l’oreille interne, nous nous sommes également intéressés au Syndrome de déhiscence du canal semi-circulaire supérieur, syndrome clinique récemment décrit par Minor en 1998. [...] / Since the beginning of the 1990s, the imaging of the ear and skull base has grown considerably. Recently, poorly known pathologies have been investigated by imaging, in particular with MRI. The objective of my research works was to develop high resolution (HR) sequences dedicated to the ear and the skull base, allowing a fine semiological analysis and a characterization of these various pathologies. The purpose is to contribute to a more appropriate medical management. The beginning of my work was directed towards the internal auditory canal. My initial purpose was to determine and develop a three-dimensional HR T2 sequence for the inner ear and the internal auditory canal as well as a spin echo T1 HR with thin slices. These sequences allowed the precise analysis of the extension of vestibular schwannomas in the fundus of the internal auditory canal. This study has indentified a relevant criteria for the therapeutic choice (surgery versus radiosurgery) and for the choice of the surgical approach. My work has also aimed to the development of diffusion sequences dedicated to the skull base. The purpose was to develop a HR diffusion sequences in the follow-up of operated cholesteatoma of the middle ear. Recurrence of cholesteatoma is frequent and the operated ears are sometimes difficult to analyze on CT, particularly when the postoperative cavity is completely filled. Specific MRI sequences are then necessary to differentiate a recurrent cholesteatoma from fibro-inflammatory tissue. The fact that the diffusion coefficient between fibro-inflammatory tissue and cholesteatoma is different has been shown by several authors. However these early sequences of echoplanar imaging (EPI) diffusion with low resolution were poorly suited to the temporal bone and the skull base. Our work was to finalize and develop a sequence of turbo spin echo HR diffusion, adapted to the ear and skull base for the screening of recurrent cholesteatoma and validate this sequence by comparison with delayed post gadolinium spin echo T1 sequence and surgery. My work on the HR imaging of the skull base also enabled me to develop some other sequences: spin echo T1 and 3D HR gradient echo T1 with fat saturation allowing for the detailed analysis of perineural spread to the skull base, mainly in nasopharyngeal cancers. This work helped to define the precise pathways of extension of these tumours in order not to underestimate the initial stage of these tumours in the TNM UICC classification. This work together with those of other researchers allowed the recent update of the UICC classification in nasopharyngeal tumours. One of my main research topics has been the screening, analysis and description of the pathologies of the inner ear. A longitudinal study from 1998 to 2008, including all the intralabyrinthine pathologies discovered to the University Center of Lille, allowed to define and characterize three major different pathologies: intralabyrinthine schwannoma, labyrinthitis and intralabyrinthine hemorrhage. These pathologies require different treatments. We were also able to define prognostic criteria in particular criteria of severity requiring a rapid and adapted medical management. Within the framework of my research on the pathologies of the inner ear, we were also interested in the syndrome of dehiscence of the superior semicircular canal. This clinical syndrome was recently described by Minor in 1998. A retrospective study of the cases discovered between 2007 and 2009, included a HR TDM of the temporal bone and a HR MRI (in particular imaging fusion between the 3D HR time-of-flight (TOF) and the 3D HR T2 sequences), enabled to identify a variant of the classic form of dehiscence. [...]

La radiation adaptative des Equoidea est encore mal comprise en raison notamment de la méconnaissance de la phylogénie de cette super-famille. La principale irrésolution de ces relations de parenté porte sur les pachynolophes, Equoidea européens rapprochés des Equidae ou des Palaeotheriidae. Pendant une grande part de l'Éocène, l'Europe est isolée et subit à la fin de cette période de profonds changements climatiques. Lors de la Grande Coupure son isolement s'achève, tandis que l'aridité du climat s'installe, et des faunes migrantes provoquent l'extinction de nombreux groupes endémiques. Un Equoidea européen basal, richement représenté par un matériel bien préservé permet d'appuyer une des hypothèses phylogénétiques les plus récentes. Cependant, les caractères couramment utilisés pour débattre de cette question n'apportent pas de réponse claire et définitive. Aussi, cette étude se propose de mener des investigations sur des régions encore peu explorées de ces animaux comme le neurocrâne, grâce à la microtomographie (CT scan), qui permet un accès non destructif aux structures (encéphale, pétreux, labyrinthe osseux, sinus). Outre l'intérêt phylogénétique, ces organes peuvent, de par leurs fonctions, receler un intérêt paléoécologique. Jusqu'à présent, peu d'études à large échelle ont porté sur ces structures chez les Perissodactyla, s'agissant pour la plupart de mentions anecdotiques. Comme préalable indispensable, une étude modèle a été réalisée sur un Equidae sauvage actuel afin de mieux appréhender la variabilité de ces structures méconnues. Pour la première fois, un large échantillon d'Equoidea européens a été scanné et leurs structures neurocrâniennes reconstruites en trois dimensions virtuelles. Ce sont au total 20 espèces qui ont été échantillonnées, couvrant l'évolution de ces animaux de leur origine à leur extinction, pendant plus de 20 millions d'années. Leurs crânes ont été scannés, leurs structures internes reconstruites, comparées et analysées au moyen de la cladistique. Une nouvelle hypothèse phylogénétique propose des relations de parenté intra-Equoidea et montre la pertinence des caractères neurocrâniens, tout en conduisant à envisager une étude plus vaste. Les Palaeotheriidae apparaissent comme un groupe très diversifié, notamment au regard des Equidae éocènes d'Amérique du Nord, et caractérisé par une évolution en mosaïque. Ils connaissent une évolution cérébrale précoce par rapport aux faunes contemporaines (Equidae, Cetartiodactyla, Carnivora), ce qui, via le développement de nouvelles stratégies adaptatives, pourrait expliquer partiellement cette grande diversification familiale. Un parallèle est envisagé avec l'évolution endémique des Notoungulata, qui semblent eux aussi montrer une complexification cérébrale précoce. Cependant, face à un environnement biotique et abiotique bouleversé (fin de l'Éocène et lors de la Grande Coupure), ces structures complexes impliquant un coût métabolique important et une trop grande spécialisation, avec en conséquence, moins de potentiel adaptatif, auraient pu les désavantager et les conduire à l'extinction. / The Equoidea adaptive radiation still remains badly known, especially due to the ignorance of their phylogeny. The main indecision of these relationships concerns the pachynolophs, European Equoidea either approached to the Equidae or to the Palaeotheriidae. During a great part of the Eocene times, Europe was isolated, and, at the end of this period, has undergone strong climatic changes. That isolation ended at the « Grande Coupure » event, whereas an arid climate moved, and migrant faunas caused the extinction of many endemic groups. A basal European Equoidea, richly represented by well-preserved material, can support one of the latest phylogenetic hypotheses. However, commonly used characters to discuss this issue do not provide a clear and definitive answer.Therefore, this study aims to investigate on unexplored regions of these animals as the neurocranium through microtomography (CT), which allows access to non-destructive structures (brain, petrosal, bony labyrinth, and sinus).Furthermore phylogenetic interest these bodies may, through their functions, harbor paleoecological interest. Until now, few large-scale studies have focused on those structures in the Perissodactyla, with regard to most were anecdotal reports. As a prerequisite, a model study was performed on a wild current Equidae to better understand the variability of these unknown structures. For the first time, a large sample of European Equoidea has been scanned and their neurocranium structures virtually reconstructed in three-dimensions. A total of 20 species were sampled, covering the evolution of these animals from their origin to their extinction, for over 20 million years. Their skulls were scanned; their internal structures reconstructed compared and analyzed using cladistics. A new phylogenetic hypothesis provides intra Equoidea relationships and shows the relevance of neurocranium characters, while driving to consider a larger study. The Palaeotheriidae appears as a highly diverse group, particularly with regard to Eocene Equidae in North America, and characterized by a mosaic evolution. Their brain evolved earlier than that of contemporary faunas (Equidae, Cetartiodactyla, Carnivora); which may partially explain the strong diversification of that family, through the development of new adaptive strategies.

The FDA-approved platinum compound, cisplatin, is commonly used as a chemotherapy drug to treat many forms of cancer. However, this compound also has several associated side-effects, including ototoxicity. This has made the development of novel platinum compounds that reduce cancer cell viability, while causing fewer and milder side-effects, an area of significant research interest. In the present study, we examined the apoptotic effects that four monofunctional platinum compounds, pyriplatin, phenanthriplatin, Pt(diethylenetriamine)Cl, and Pt(N,Ndiethyldiethylenetriamine) Cl, had on zebrafish inner ear auditory epithelial cells. We then compared the apoptotic effects of these compounds to those of cisplatin, which is a bifunctional platinum compound. Our hypothesis was that the four monofunctional platinum compounds would cause reduced inner ear apoptosis compared to cisplatin. Zebrafish were injected with either vehicle solution, cisplatin or with one of the monofunctional compounds. Later, at 24-hour and 48-hour time points, the zebrafish were euthanized, and two of their auditory inner ear endorgans, the utricle and saccule, were dissected out. A terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was then used to label apoptotic cells, and the inner ear organs were viewed under a microscope. The number of apoptotic cells on each sample was quantified and the data were analyzed for significant differences between treatments. We found that with the exception of pyriplatin in the saccules, and with the exception of pyriplatin, Pt(N,N-diethyldiethylenetriamine)Cl, and phenanthriplatin in the utricles, the monofunctional compounds and cisplatin did not induce apoptosis in the inner ear of zebrafish at either time point. Based on these results we conclude that the monofunctional platinum compounds largely do not induce zebrafish inner ear apoptosis and if they were to produce ototoxicity, it would not be through an apoptotic mechanism.

The experiments described in this thesis report were aimed at studying the functions of Sox2 and Serrate1 during the development of the neurosensory elements of the inner ear. First, we have described the expression pattern of Sox2 during inner ear development and compared to that of Sox3 and Serate1. Secondly, we have shown the results of plasmid based in ovo electroporation experiments, designed to manipulate gene expression exogenously, and to study the gain of function of Sox2 and Serrate1. Effects on cell fate and downstream targets were assessed by in situ hybridization immunohistochemistry and quantitative real-time PCR (qRT-PCR).The results show that Sox2 is expressed in the neurosensory domain of the otic epithelium during the neurogenic period of otic development and, later on, during the development of the prosensory patches and sensory organs. As differentiation proceeds, Sox2 is excluded from differentiated neurones and hair cells, but remains expressed in the supporting cells of the sensory organs. Sox3 is co-expressed with Sox2 in the neurogenic domain of the otic cup. But Sox3 is then down-regulated and only Sox2 expression persists in the sensory precursors, where it is co-expressed with the Notch ligand Serrate1. The expression domain of Serrate1 is initially nested within Sox2, however, later in development Sox2 becomes restricted within the boundaries of Serrate1 expression, a process that is concomitant to the formation of the sensory patches. These expression patterns suggest: 1) that Sox2 correlates with neurosensory fate in the otic placode, 2) that neurogenesis is associated with Sox2 and Sox3 and 3) that sensory development is associated with Sox2 and Serrate1.Gain of function studies show that Serrate1 regulates prosensory fate and sensory organ development by maintaining Sox2 expression in restricted domains of the otocyst, without affecting neurogenesis. Serrate1 operates in a Notch-dependent manner, consistently with a mechanism of lateral induction that includes the induction of its own expression and downstream targets of the Notch signalling pathway Hes1, Hey1 and Hey2. Similar studies on Sox2 indicate that it specifies neurosensory fate in the otic epithelium. However, high concentrations of Sox2 suppress sensory fate and promote neuronal fate. Besides, Sox2 prevents cell differentiation though the cooperation with Notch and BMP signalling pathways.We like to propose a model in which an extended neural competence is early established in the otic placode with the early expression of Sox2 and Sox3 genes. The cooperation between Sox2 and Sox3 then provides a high concentration of SoxB1 protein and promote neuronal fate. In parallel, Serrate1 maintains Sox2 expression in restricted domains, after Sox3 down-regulation. These domains retain the neurosensory competence and thereby develop as sensory patches. / Los experimentos descritos en esta tesis tuvieron por objetivo estudiar la función de Sox2 y Serrate1 en el desarrollo de los elementos neurosensoriales del oído. En primer lugar describimos el patrón de expresión de Sox2 durante el desarrollo del oído y lo comparamos con el de Sox3 y Serrate1. En segundo lugar, mostramos los resultados de experimentos de electroporación in ovo, diseñados para manipular exógenamente la expresión génica y estudiar la ganancia de función de Sox2 y Serrate1. Los efectos sobre el destino celular y las dianas moleculares se analizaron mediante hibridación in situ, inmunocitoquímica y real-time PCR (qRT-PCR).Los resultados muestran que Sox2 se expresa en el dominio neurosensoerial del epitelio ótico durante la fase de neurogénesis y, más adelante, durante el desarrollo de los parches prosensoriales y los órganos sensoriales. Con la diferenciación, Sox2 es excluido de las neuronas diferenciadas y las células ciliadas, pero permanece expresado en las células de soporte. Sox3 se coexpresa con Sox2 en el dominio neurogénico de la copa ótica. Pero entonces, la expresión de Sox3 se reduce y sólo Sox2 persiste en los precursores sensoriales, en donde se co-expresa con el ligando de Notch Serrate1. El dominio de expresión de Serrate1 está inicialmente contenido en el de Sox2, sin embargo, más adelante, Sox2 se restringe dentro de los límites de Serrate1, un proceso que es concomitante con la formación de los parches sensoriales. Estos experimentos sugieren que : 1) Sox2 se correlaciona con el destino neurosensorial de la placoda ótica, 2) la neurogénesis está asociada con Sox2 y Sox3, y 3) el desarrollo sensorial está asociado a la expresión de Sox2 y Serrate1Los estudios de ganancia de función muestran que Serrate1 regula el destino prosensorial y el desarrollo de los órganos sensoriales mediante el mantenimiento de la expresión de Sox2 en dominios restringidos del otocisto, sin afectar a la neurogénesis. Serrate1 opera en un modo dependiente de Notch, consistente con un mecanismo de inducción lateral que comprende la inducción de su propia expresión y la de las dianas de Notch Hes1, Hey1 and Hey2. Estudios similares sobre Sox2 indican que Sox2 especifica el destino neurosensorial en el epitelio ótico. Sin embargo, las concentraciones altas de Sox2 suprimen el destino sensorial y promueven el destino neuronal. Además, Sox2 previene la diferencoiación celular mediante la cooperación con Notch y Bmp. Se propone un modelo en el cual la competencia neural se establece tempranamente en la placoda ótica mediante la expresión temprana de Sox2 y Sox3. La cooperación entre Sox2 y Sox3 provee una alta concentración de factores SoxB1 que promueven el destino neuronal de los progenitores. En paralelo, Serrate1 mantiene la expresión de Sox2 en dominios restringidos tras la supresión de Sox3. Estos dominios, retienen el potencial neurosensorial y, más adelante, se desarrollan como parches sensoriales.

No description available.

The embryonic mouse inner ear was used as a model with which to study ototoxicity and tissue interactions. The inner ear anlage can be explanted and cultured in vitro from about the 12th gestational day (gd), and will differentiate parallel with the inner ear developing in vivo until a time corresponding to birth (21st gd). During this period the ovoid sac develops into the labyrinth. In the present thesis work, otic anlagen from gd 12, 13, 13.5, 15 and 16 were used. As a rule the explants were kept in culture until a time point equivalent to the 21st gd. Analyses using freeze-fracture technique and transmission electron microscopy showed that in cultured 13th gd otocysts the development of junctional complexes followed the same principal pattern as in vivo. Tight junctions develop into many strands lying parallel to the apical surface of all epithelial cells. Uncoupling of the hair cells occurs with loss of gap junctions. Some tight junctions had an aberrant appearence, with in part very thick strands and strands running at right angles to the apical surface. All aminoglycosides are potentially ototoxic. In the inner ear, outer hair cells of the organ of Corti and vestibular type I hair cells are affected by these antibiotics. The access route to the hair cells and the sites and mechanisms of action of aminoglycosides are not precisely defined. The uptake of tritiated tobramycin in 16th gd inner ears was studied. An initial rapid uptake of the drug, within 10 min, was followed by a slower accumulation, reaching a steady state after 60 min. Most of the tobramycin was bound reversibly, at least after a short period of incubation (2 h). The irreversibly bound fraction was of the same magnitude as the uptake within 10 min. Uptake took place against a concentration gradient. The otocyst can differentiate even without the statoacoustic ganglion. The interaction of the sensory epithelium with the ganglion was investigated by explanting the statoacoustic ganglion without target tissue. Twenty-five percent of the ganglions survived and had outgrowth of neurites but there was no differentiation into either the cochlear or vestibular type of neuron cells. Exposure of cultured otocysts (13 or 13.5 gd) to l-azetidine-2-carboxylic acid, a 1-proline analog that disrupts formation of collagen, resulted in retarded morphogenesis of the labyrinth and a dose- dependent derangement of the basal lamina. The expression of intermediate filaments (IFs) was analysed using monoclonal antibodies. The same IF pattem was found in cultured inner ears as in vivo. Explants were taken on 13th, 15th or 16th gd. Exposure to gentamicin, ethacrynic acid or cisplatin did not alter the IF composition. Cytokeratins (CKs) 8 and 18 were identified in all inner ear epithelia. In addition CKs 7 and 19 were visualized in the epithelia involved in maintaining endolymph homeostasis. The ganglion cells showed coexpression of CK, vimentin and neurofilaments. The elemental composition of the endolymph compartment of 16th gd inner ears cultured for 5 days was studied using energy-dispersive X-ray microanalysis. Na to K ratios characteristic of endolymph were found. / <p>S. 1-34: sammanfattning, s. 37-88: Härtill 6 uppsatser</p> / digitalisering@umu

Sensory neurons are key mediators of the transduction of external stimuli from the ear to the brain, essential for the sense of balance and hearing. Understanding when, where and how the sensory nervous system is assembled during development can provide insights on deafness and balance disorders. Here, I show in zebrafish that Her9 transcription factor is a key element in the regulation of the otic neurogenesis. Loss of Her9 function leads to the ectopic expression of neurogenic genes neurod and neurod4. Moreover, I show that Her9 acts downstream of Tbx1, and both genes are activated by retinoic acid signaling emanating from the paraxial mesoderm and negatively regulated by Hedgehog signaling. Altogether, the data demonstrates a role of retinoic acid in axial patterning and the establishment of a neurogenic domain through Tbx1 and Her9. At later stages, retinoic acid has an additional role by regulating neuronal differentiation in the statoacoustic ganglion. / Les neurones sensorials de l’oïda interna són mediadores claus en la transducció dels estímuls externs des de l’oïda interna al cervell. Entendre a on, quan i com el sistema nerviós sensorial s’organitza durant el desenvolupament embrionari pot ajudar en l’estudi de les malalties neurosensorials. En el present treball, mostro en peix zebra que el factor de transcripció Her9 és un element clau en el control de la neurogènesi òtica i que Her9 es troba sota el control directe del factor Tbx1. A més, ambdos factors estan regulats de manera positva per la via de senyalització de l’àcid retinoic i negativament per la vía de hedgehog. En resum, la tesis demostra un paper de l’àcid retinoic en la regionalització axial del primordi òtic en l’eix anteroposterior i l’establiment d’un domini neurogènic a través de Tbx1 i Her9. En estadis tardans, l’àcid retinoic regula la diferenciació neuronal en el gangli estato-acústic.