Axons of retinal ganglion cells (RGCs) send visual information to a number of retinorecipient regions in the brain. In rodents, visual thalamus receives dense innervations from RGC axons and is important for both image-forming and nonimage-forming visual functions. Retinal inputs invade visual thalamus during embryonic development, before the arrival of non-retinal inputs (such as local interneurons and axonal inputs from other brain regions). In this dissertation, I explore how early innervation of RGC axons affects circuitry in visual thalamus and the role of visual experience, neural activity, and molecular cues in the development. While the development of astrocytes in cortex has been well-described, they have been largely overlooked in visual thalamus. Using immunohistochemical, functional, and ultrastructural analysis, I show that astrocytes in visual thalamus reach adult-like morphological properties and functionality at retinogeniculate synapses early in development, by eye-opening and before visual experience. These studies reveal that while experience-dependent visual activity from RGC axons is critical for many aspects of visual thalamus development, astrocytic maturation occurs independent of that information about our visual environment. As with astrocytes, little progress has been made in understanding the development of interneurons in the visual thalamus. Here, I show that retinal inputs interact with thalamic astrocytes to influence the recruitment of GABAergic interneurons into visual thalamus. I found that this interaction between RGC axons and astrocytes is not dependent on neural activity of RGCs. Using transcriptomic analysis, in situ hybridization, and reporter lines, I observed thalamus-projecting RGCs express SHH and astrocytes in visual thalamus express SHH signaling molecules. My results reveal that SHH signaling between RGC axons and astrocytes is critical for astrocytic fibroblast growth factor 15 (FGF15) expression in developing visual thalamus. Ultimately, FGF15 serves as a potent motogen that is essential for thalamic interneuron migration. These data identify a novel morphogen-dependent and activity-independent mechanism that mediates crosstalk between RGCs and astrocytes to facilitate the recruitment of interneurons into the developing visual thalamus. / Doctor of Philosophy / The most dominant sense in human is the sight, which we need to interact with our environment efficiently. The retina takes up the information about our visual world and sends it to the brain, which ultimately puts everything together, for us to see properly. The visual information from the retina goes to the brain via nerves (which are essentially cables/wires of brain cells). These nerves from the retina go to many places in the brain, including a region called visual thalamus, which is the focus of my PhD work. For the past five years, I have been trying to understanding if nerves from the retina play a role in the brain formation during early development. To study this, I have used mice as a model system, as their brain regions that process visual information have very similar structural architecture to those in humans. My research shows that retinal nerves are indeed important for the development of visual thalamus. Here, I show that information from the eye is critical for migration (a process during development where brain cells move from their place of origin to their final location) of cells in visual thalamus. Discoveries made in this dissertation are important because they highlight how different cells in the central nervous system communicate with each other at the level of molecules and how these interactions are important for building circuits that are important for vision.

Titre de l'écran-titre (visionné le 5 septembre 2023) / La voie de signalisation Hedgehog est impliquée dans de nombreux processus nécessaires au bon développement et maintien des organes. Elle a lieu par fixation de ses ligands sur un récepteur qui se trouve soit à la surface membranaire des cellules, soit à la surface des cils. Les cils sont des extensions cellulaires, motiles ou non, se trouvant à la surface des cellules. Chez les mammifères, la voie de signalisation Hedgehog a principalement été identifiée à la surface des cils non motiles, appelés cils primaires. En leur sein, de nombreuses protéines, dont des récepteurs, se concentrent, permettant une amplification des différents signaux reçus. Cela permet aux cils primaires d'avoir une fonction d'antenne de signalisation, captant les signaux et les transmettant à la cellule. Bien que les cils primaires soient le lieu privilégié de la voie de signalisation Hedgehog, il a été montré que d'autres types de cils, des cils motiles, étaient également capables de la transduire. En cas de dysfonctionnement de cette voie de signalisation, ou bien des cils en eux-mêmes, plusieurs pathologies peuvent en découler, regroupées sous le nom de ciliopathies. Parmi la multitude de symptômes, des cas d'infertilité masculine sont décrits. Plusieurs études ont montré que cette voie de signalisation existe dans le système reproducteur mâle et femelle, tel dans l'épididyme ou l'endomètre. Dans cette étude, nous avons tenté d'élucider le rôle de la voie de signalisation Hedgehog dans le contrôle de la fertilité mâle de la maturation des spermatozoïdes. Nous avons tout d'abord étudié la voie Hedgehog au sein de l'épididyme. C'est l'organe en charge de la maturation des spermatozoïdes, ils y acquièrent une motilité primaire et d'autres modifications, nécessaires à la fertilité. Dans cet organe, les cellules susceptibles de répondre à la voie Hedgehog sont les cellules basales, car elles sont les seules cellules de l'épithélium à posséder un cil. La première partie de ce projet a été d'étudier le rôle in vitro de la voie de signalisation Hedgehog dans des cellules épididymaires immortalisées. Nous avons pu montrer grâce à ce modèle que la voie de signalisation Hedgehog dépendait bien de la présence du cil primaire. Après traitement pharmacologique, les cellules épithéliales répondent à la voie Hedgehog contrôlant ainsi l'expression de nombreux gènes, régulant la réponse immunitaire et la prolifération cellulaire. Nous avons dans un deuxième temps voulu comprendre in vivo le rôle de la voie Hedgehog, dépendante des cils primaires, dans les cellules basales de l'épididyme. Pour cela, nous avons créé un modèle murin présentant une délétion d'un facteur nécessaire à la voie Hedgehog spécifiquement dans les cellules basales, et comparé son phénotype à celui des souris contrôles. Nous avons pu montrer que la voie Hedgehog permettait de réguler l'homéostasie du tissu et plus particulièrement les propriétés des cellules basales. Ces dernières auraient de multiples fonctions dont le rôle de cellule souche épithéliale. Dans notre modèle, l'ablation d'une protéine ciliaire a empêché la régénération du tissu après dommages (ligation des canaux efférents). La voie Hedgehog pourrait donc être primordiale dans la réponse immunitaire après blessure ou inflammation, en régulant la prolifération cellulaire et la régénération tissulaire. Ces phénomènes pourraient donc avoir un lien indirect avec la maturation des spermatozoïdes dans l'épididyme, et donc dans le contrôle de la fertilité. Dans un troisième temps, nous avons voulu explorer pour la première fois le rôle direct de la voie Hedgehog sur les spermatozoïdes. En effet, ces cellules possèdent pareillement un cil : le flagelle. Nous avons donc posé l'hypothèse que les spermatozoïdes pourraient également être capable d'y répondre. En travaillant avec des spermatozoïdes de trois espèces mammifères différentes, nous avons pu montrer la présence des facteurs de la voie Hedgehog dans les spermatozoïdes. Les spermatozoïdes étaient également capables de répondre à cette voie, induisant un changement dans leur capacitation (motilité ; réaction acrosomique) et dans leur capacité à féconder un ovocyte. La voie Hedgehog pourrait donc avoir également un effet direct sur la maturation des spermatozoïdes. En conclusion, l'ensemble de ces travaux de thèse contribue à une meilleure compréhension du contrôle de la fertilité mâle par la voie Hedgehog et ouvre la voie à l'identification de nouvelles cibles thérapeutiques. / The Hedgehog signaling pathway is involved in many processes necessary for the proper development and maintenance of organs. Signalling takes place by binding of ligands to a receptor, which is either on the membrane surface of the cells, or on the surface of a cilium. Cilia are cellular extensions, motile or non-motile, found on the surface of cells. In mammals, the Hedgehog signaling pathway has primarily been identified on the surface of nonmotile cilia, called primary cilia. Within them, many proteins, including receptors, are concentrated, allowing amplification of the various signals received. This allows the primary cilia to act as a signalling antenna, picking up extracellular signals and transmitting them to the cell. Although the primary cilia are the main site of the Hedgehog signaling pathway, it has been shown that other types of cilia, motile cilia, are also able to transduce Hedgehog signalling. In case of dysfunctions of this signaling pathway, or of cilia themselves, several pathologies can result, grouped under the name of ciliopathies. Among the multitude of symptoms, cases of male infertility are described. Several studies have shown that this signaling pathway exists in the male and female reproductive system, such as in the epididymis or the endometrium. In this study, we attempted to elucidate the role of Hedgehog signaling pathway in male fertility and more specifically in the control of sperm maturation. We first studied the Hedgehog pathway within the epididymis. It is the organ in charge of the maturation of spermatozoa, there they acquire primary motility and other changes, necessary for fertility. In this organ, the cells likely to respond to the Hedgehog pathway are the basal cells, because they are the only cells of the epithelium to possess a cilia. The first part of this project was to study the in vitro role of the Hedgehog signaling pathway in immortalized epididymal cells. We were able to show, thanks to this in vitro model, that the Hedgehog signaling pathway did indeed depend on the presence of the primary cilium. After pharmacological treatment, the epithelial cells respond to the Hedgehog pathway thus controlling the expression of many genes, regulating the immune response and cell proliferation. Secondly, we wanted to better understand in vivo the role of the Hedgehog pathway, dependent on primary cilia, in the basal cells of the epididymis. For this, we created a mouse model presenting a deletion of a factor necessary for the Hedgehog pathway specifically in the basal cells and compared it with control mice. We have been able to show that the Hedgehog pathway regulates tissue homeostasis and more particularly the properties of basal cells in the epididymis. Basal cells have multiple functions including the role of epithelial stem cell. In our model, the ablation of a ciliary protein prevented tissue regeneration after efferent duct ligation. The Hedgehog pathway could therefore be essential in the immune response after injury or inflammation, by regulating cell proliferation and tissue regeneration. These phenomena could consequently have an indirect link with the maturation of spermatozoa in the epididymis, and thus in the control of male fertility. Thirdly, we wanted to explore for the first time the direct role of the Hedgehog pathway on spermatozoa. Indeed, these cells also have a cilium, the flagellum, and we therefore hypothesized that they could also be able to respond to it. By working with spermatozoa from three different mammalian species, we were able to show the presence of Hedgehog pathway factors in spermatozoa. Sperm cells were also able to respond to this pathway inducing a change in vitro in their capacitation (motility; acrosomal reaction) and in their ability to fertilize an oocyte. The Hedgehog pathway could therefore also have a direct impact on sperm maturation. In conclusion, this thesis research contributes to a better understanding of the control of male fertility by Hedgehog signalling and could open the way to the identification of new therapeutic targets.

Le corps calleux (CC) est la principale commissure cérébrale connectant les aires corticales homologues des deux hémisphères chez les vertébrés placentaires. Les malformations du corps calleux (MCC) représentent la malformation cérébrale la plus fréquente à la naissance et sont présentes chez 5% des individus avec anomalie neuro-développementale. Une meilleure connaissance de l’ontogenèse du corps calleux et de ses causes génétiques devrait permettre d’ouvrir la voie à des corrélations cliniques pour un meilleur conseil génétique. Cet aspect constitue probablement l’enjeu de la prochaine décennie concernant les foetus avec MCC. Le travail de thèse a porté sur 138 foetus avec MCC, pour lesquels nous avons fait un examen foeto-neuropathologique et une classification en plusieurs catégories. Au total, ce travail a permis : 1/le démantèlement des causes génétiques des MCC par une triple approche de CGH array, d’exome en trio et de panels ciblés, avec une augmentation considérable des causes identifiables de MCC au sein de cette cohorte, 2/ l’identification et la caractérisation fonctionnelle d’un nouveau gène de ciliopathie dans un phénotype extrême ; 3/ l’identification de 3 nouvelles mutations de ZBTB20, récemment identifié comme responsable du syndrome de Primrose, démontrant que ce syndrome est une cause fréquente de MCC et permettant une description clinico-radiologique plus précise. 4/ L’identification de plusieurs gènes candidats en cours de validation. / Corpus callosum is the main cerebral commissure connecting homologous cortical areas in placental mammals. Malformations of corpus callosum (MCC) are the most frequent brain malformation at birth and are present in 5% of patients with neurodevelopmental delay. A good knowledge of genetics of corpus callosum development should pave the way to better clinical correlations for a more accurate genetic counselling. This is the challenge of the next decade. This thesis concerns a cohort of 138 fetuses with MCCs, well classified on neuropathological examination. It allowed 1/ to unravel the genetic causes of MCC through a triple approach combining CGH array, whole exome and NGS panels sequencing, with a considerable increase in the number of causes of MCC identified ; 2/ identification of a new gene in an extreme ciliopathy phenotype; and 3/ identification of novel ZBTB20 mutations , a gene recently identified as responsible for Primrose syndrome, showing that this syndrome is frequent among MCCs and allowing a precise clinico-radiological description of the syndrome. 4/ Several new candidate genes are under study.

Le corps calleux (CC) est la principale commissure cérébrale connectant les aires corticales homologues des deux hémisphères chez les vertébrés placentaires. Les malformations du corps calleux (MCC) représentent la malformation cérébrale la plus fréquente à la naissance et sont présentes chez 5% des individus avec anomalie neuro-développementale. Une meilleure connaissance de l’ontogenèse du corps calleux et de ses causes génétiques devrait permettre d’ouvrir la voie à des corrélations cliniques pour un meilleur conseil génétique. Cet aspect constitue probablement l’enjeu de la prochaine décennie concernant les foetus avec MCC. Le travail de thèse a porté sur 138 foetus avec MCC, pour lesquels nous avons fait un examen foeto-neuropathologique et une classification en plusieurs catégories. Au total, ce travail a permis : 1/le démantèlement des causes génétiques des MCC par une triple approche de CGH array, d’exome en trio et de panels ciblés, avec une augmentation considérable des causes identifiables de MCC au sein de cette cohorte, 2/ l’identification et la caractérisation fonctionnelle d’un nouveau gène de ciliopathie dans un phénotype extrême ; 3/ l’identification de 3 nouvelles mutations de ZBTB20, récemment identifié comme responsable du syndrome de Primrose, démontrant que ce syndrome est une cause fréquente de MCC et permettant une description clinico-radiologique plus précise. 4/ L’identification de plusieurs gènes candidats en cours de validation. / Corpus callosum is the main cerebral commissure connecting homologous cortical areas in placental mammals. Malformations of corpus callosum (MCC) are the most frequent brain malformation at birth and are present in 5% of patients with neurodevelopmental delay. A good knowledge of genetics of corpus callosum development should pave the way to better clinical correlations for a more accurate genetic counselling. This is the challenge of the next decade. This thesis concerns a cohort of 138 fetuses with MCCs, well classified on neuropathological examination. It allowed 1/ to unravel the genetic causes of MCC through a triple approach combining CGH array, whole exome and NGS panels sequencing, with a considerable increase in the number of causes of MCC identified ; 2/ identification of a new gene in an extreme ciliopathy phenotype; and 3/ identification of novel ZBTB20 mutations , a gene recently identified as responsible for Primrose syndrome, showing that this syndrome is frequent among MCCs and allowing a precise clinico-radiological description of the syndrome. 4/ Several new candidate genes are under study.

Le corps calleux (CC) est la principale commissure cérébrale connectant les aires corticales homologues des deux hémisphères chez les vertébrés placentaires. Les malformations du corps calleux (MCC) représentent la malformation cérébrale la plus fréquente à la naissance et sont présentes chez 5% des individus avec anomalie neuro-développementale. Une meilleure connaissance de l’ontogenèse du corps calleux et de ses causes génétiques devrait permettre d’ouvrir la voie à des corrélations cliniques pour un meilleur conseil génétique. Cet aspect constitue probablement l’enjeu de la prochaine décennie concernant les foetus avec MCC. Le travail de thèse a porté sur 138 foetus avec MCC, pour lesquels nous avons fait un examen foeto-neuropathologique et une classification en plusieurs catégories. Au total, ce travail a permis : 1/le démantèlement des causes génétiques des MCC par une triple approche de CGH array, d’exome en trio et de panels ciblés, avec une augmentation considérable des causes identifiables de MCC au sein de cette cohorte, 2/ l’identification et la caractérisation fonctionnelle d’un nouveau gène de ciliopathie dans un phénotype extrême ; 3/ l’identification de 3 nouvelles mutations de ZBTB20, récemment identifié comme responsable du syndrome de Primrose, démontrant que ce syndrome est une cause fréquente de MCC et permettant une description clinico-radiologique plus précise. 4/ L’identification de plusieurs gènes candidats en cours de validation. / Corpus callosum is the main cerebral commissure connecting homologous cortical areas in placental mammals. Malformations of corpus callosum (MCC) are the most frequent brain malformation at birth and are present in 5% of patients with neurodevelopmental delay. A good knowledge of genetics of corpus callosum development should pave the way to better clinical correlations for a more accurate genetic counselling. This is the challenge of the next decade. This thesis concerns a cohort of 138 fetuses with MCCs, well classified on neuropathological examination. It allowed 1/ to unravel the genetic causes of MCC through a triple approach combining CGH array, whole exome and NGS panels sequencing, with a considerable increase in the number of causes of MCC identified ; 2/ identification of a new gene in an extreme ciliopathy phenotype; and 3/ identification of novel ZBTB20 mutations , a gene recently identified as responsible for Primrose syndrome, showing that this syndrome is frequent among MCCs and allowing a precise clinico-radiological description of the syndrome. 4/ Several new candidate genes are under study.

Les genodermatoses sont des maladies génétiques rares à expression cutanée. Parmi elles, les dysplasies ectodermiques (DE) caractérisées par des anomalies du développement d’au moins deux structures ectodermiques (dents, ongles, glandes sudorales et poils), constituent un groupe hétérogène de genodermatoses de plus de 200 syndromes rares. Si la plupart de ces syndromes associent des anomalies des seuls dérivés ectodermiques, d'autres plus complexes, tels que le syndrome de Bazex-Dupré-Christol et l’Incontinentia Pigmenti, rassemblent en plus des manifestations disparates. Le syndrome de Bazex-Dupré-Christol (SBDC) associe une DE à la prédisposition aux carcinomes basocellulaires (CBCs) de survenue précoce. L’étude de 6 familles nous a permis d’identifier, chez 2 d’entre elles, une mutation tronquante dans le gène ACTRT1, codant la protéine Arp-T1. Dans l’épiderme, la protéine Arp-T1 est diminuée chez tous les patients atteints de SBDC, porteurs ou non de mutations dans le gène ACTRT1. Le séquençage à haut débit de la région candidate a permis d’identifier des mutations dans des régions transcrites, régulatrices du gène ACTRT1 chez les patients des 4 autres familles. Notant que la voie Hedgehog est dérégulée dans 70 % des CBCs, nous avons démontré qu’ACTRT1 est un nouvel inhibiteur de cette voie, en particulier par sa liaison au promoteur de GLI1 dont il inhibe l’expression. Enfin, ACTRT1 est un nouveau gène suppresseur de tumeur capable de réduire in vivo la progression tumorale de certaines lignées cancéreuses par la régulation de gènes impliqués dans la prolifération, la mort et la survie cellulaire, ou encore la migration. L’Incontinentia Pigmenti (IP) est une affection multisystémique caractérisée par une atteinte de la peau, des dents, des yeux et parfois du système nerveux central. Elle résulte de mutation dans le gène NEMO et l’abolition de l’activation de la voie NF-KB. L’étude d’une famille concernée par l’IP à permis d’identifier une nouvelle mutation d’épissage du gène NEMO aboutissant à l’expression d’une protéine tronquée. Cette protéine conserve l’intégralité des domaines fonctionnels de NEMO connus à ce jour. Sa caractérisation a révélé une perte d’interaction avec SHARPIN, composants du complexe LUBAC permettant l’ubiquitination linéaire. Il s’agit de la première mutation humaine de NEMO montrant l’importance de son ubiquitination linéaire dans l’activation de la voie NF-KB. Mes travaux de thèse ont ainsi mis en évidence de nouveaux mécanismes physiopathlogiques responsables de deux formes de dysplasie ectodermique. Ces mécanismes reflètent la complexité des voies moléculaires impliquées dans le développement de la peau et le maintien de son homéostasie durant la vie adulte. / Genodermatoses are rare genetic diseases with cutaneous expression. Among them, ectodermal dysplasia (ED) characterized by abnormal development of at least two ectodermal structures (teeth, nails, sweat glands and hair) constitute a heterogeneous group of genodermatoses of more than 200 rare syndromes. While most of these syndromes associate only abnormalities of the ectodermal derivatives, others more complex, such as Bazex-Dupré-Christol syndrome and Incontinentia Pigmenti, bring together disparate manifestations. Bazex-Dupré-Christol syndrome (BDCS) associates ED with predisposition to early basal cell carcinoma (BCCs). The study of 6 families allowed us to identify, in 2 of them, a truncated mutation in the ACTRT1 gene, encoding the Arp-T1 protein. In the epidermis, Arp-T1 protein is decreased in all patients with BDCS, carrying or not of mutations in ACTRT1 gene. High-throughput sequencing of the candidate region allowed to identify mutations in transcribed enhancer regions, regulating the ACTRT1 gene in patients of the remaining 4 families. Noting that the Hedgehog pathway is deregulated in more than 70% of BCCs, we have demonstrated that ACTRT1 is a novel inhibitor of this pathway, via its binding to GLI1 promoter and inhibiting its expression. Finally, ACTRT1 is a new tumor suppressor gene capable of reducing in vivo the tumor progression of certain cancer lines by the regulation of genes involved in proliferation, death and cell survival, or migration. Incontinentia Pigmenti (IP) is a multisystemic disorder characterized by involvement of skin, teeth, eyes and sometimes the central nervous system. It results from mutation in the NEMO gene and the abolition of activation of NF-KB pathway. The study of a family concerned with IP allowed to identify a new splicing mutation of NEMO gene leading to a truncated protein expression. This protein retains all the functional domains of NEMO known. Its characterization revealed a loss of interaction with SHARPIN, components of LUBAC complex allowing linear ubiquitination. This is the first human mutation of NEMO showing the importance of its linear ubiquitination in the activation of the NF-KB pathway. Thus, my thesis work revealed novel physiopathological mechanisms responsible for two forms of ectodermal dysplasia. These mechanisms reflect the complexity of the molecular pathways involved in the development of the skin and the maintenance of its homeostasis during adult life.

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.

No description available.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:22:54Z No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:23:08Z (GMT) No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Made available in DSpace on 2015-05-12T13:23:08Z (GMT). No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O adenoma pleomórfico (AP), o carcinoma mucoepidermóide (CME) e o carcinoma adenóide cístico (CAC) representam tumores frequentes em glândula salivar. A via de sinalização Sonic Hedgehog (Hh) e o Transdutor de sinal e ativador da transcrição 3 (STAT3) desempenham funções importantes na proliferação celular, favorecendo o desenvolvimento tumoral e a proteína MCM3 tem sido considerada uma nova classe de marcadores de proliferação celular. Portanto, o presente trabalho propõe-se a estudar componentes da via Hh, bem como o STAT3 e o MCM3 em neoplasias de glândula salivar, na tentativa de adicionar informações sobre as características biológicas dessas neoplasias. Foram utilizados 9 casos de AP, 17 casos de CAC e 20 casos de CME e, por meio da técnica imunoistoquímica, realizou-se a detecção das seguintes proteínas: SHH, GLI1, SUFU, HHIP, STAT3 e MCM3. No AP, observou-se alta expressão citoplasmática de SHH e SUFU, e baixa expressão de STAT3 e MCM3. No CAC, observou-se alta expressão de GLI1, HHIP e STAT3 e baixa expressão de SHH, SUFU e MCM3. No CME, observou-se alta expressão de SHH, GLI1, SUFU e HHIP e baixa expressão de STAT3 e MCM3. Quando comparado entre os tipos tumorais, observou-se diferença estatisticamente significante para expressão de SHH (p=0.0064), STAT3 (p=0.0003) e MCM3 (p=0.0257). Ao comparar a marcação parenquimal e estromal, observou-se maior expressão em parênquima para todos os tumores (p<0.05). Em glândula salivar normal, observou-se marcação citoplasmática das células ductais para SHH, GLI1, SUFU e HHIP, uma discreta marcação citoplasmática para STAT3 e ausência de marcação para MCM3. Não foi observada correlação entre a via Hh e STAT3 nos tumores de glândula salivar (p>0.05). Os resultados desse trabalho apontam para uma possível participação da via Hh na morfogênese e no desenvolvimento do AP, CAC e CME, assim como a participação do STAT3 no desenvolvimento do CAC. Em adição, o MCM3 não parece ser um bom marcador de proliferação para tumores de glândula salivar. Sugerimos que novos estudos sejam realizados visando compreender o mecanismo pelo qual a via Hh e o STAT3 promovem o crescimento e progressão desses tumores e identificar inibidores dessas vias. / The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC) and the adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands. The Sonic Hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The MCM3 protein has been considered as a novel class of cell proliferation markers. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 and MCM3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC and 20 cases of MEC. Using immunohistochemistry, were investigated: SHH, GLI1, Sufu, HHIP, STAT3 and MCM3. In PA, there was high expression of cytoplasmic SHH and Sufu, and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP and STAT3 and low expression of SHH, SUFU and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p=0.0064), STAT3 (p=0.0003) and MCM3 (p=0.0257) when all tumors were compared. And a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p<0.05). In normal salivary gland, ductal segment showed imunolabeling for SHH, GLI1, SUFU and HHIP, a discrete cytoplasmic labeling for STAT3 and MCM3 was negative. No correlation was observed between the Hh pathway and STAT3 in salivary gland tumors (p>0.05). The findings suggest a possible role of Hh pathway in the morphogenesis and development of AP, CAC and CME, as well as the participation of STAT3 in the development of ACC. In addition, the MCM3 did not seem to be a good marker of proliferation for salivary gland tumors. It is important that further studies be conducted to understand the mechanism by which the Hh pathway and the STAT3 promote the growth and progression of these tumors and inhibitors of these pathways might be identified.

Made available in DSpace on 2016-12-23T13:56:07Z (GMT). No. of bitstreams: 1 Dissertacao de Thiago de Almeida Pereira.pdf: 7083594 bytes, checksum: b087de1012807ea82b96d6e7ada7b3cc (MD5) Previous issue date: 2010-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally-mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCC) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCC develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarcinogenesis in chronic viral hepatitis. Methods Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hhresponsive cell types in archival liver biopsies from 45 patients with chronic HBV or HCV with different stages of fibrosis (F0-4), some of which also presented HCC. Hh signaling was manipulated with cyclopamine in primary human hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). Angiogenesis assay was used to investigate if the pathway plays a role in tube formation. SECs were incubated with recombinant Shh, conditioned media from HSC, cyclopamine, short hairpin RNA against smoothened or their respective controls and the length of vascular tubes were measured by morphometry. Hedgehog production was investigated in Huh7 cells infected with JFH-1 and in a rat hepatoma cell line that express the HBV X gene by qRT-PCR. Results We found increased hepatic expression of Hh ligands (Shh and Ihh) in all patients with chronic viral hepatitis (p<0.005), and demonstrated that infection with JFH-1 or ectopic expression of the HBV X gene stimulated cultured hepatocytes to produce Shh (p<0.005). The major cell populations that expanded during cirrhosis and HCC (i.e., liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells Krt7, CD133, Epcam and survivin) were Hh-responsive (Patched and Gli-2 positive) and higher levels of Hh pathway activity associated with cirrhosis and HCC (p<0.005). Inhibiting pathway activity in Hh-responsive target cells (HSC and SEC) reduced fibrogenesis (p<0.005 for &#945;SMA and p<0.05 for col1&#945;1 expression) and angiogenesis (p<0.05). Conclusions HBV/HCV infection increases hepatocyte production of Hh ligands in hepatocytes and expands types of Hh-responsive cells, including myofibroblasts and xiv sinusoidal endothelial cells that associate with fibrosis progression to cirrhosis and hepatocellular carcinoma. Moreover, hedgehog-responsive progenitors that are undergoing epithelial-to-mesenchymal transition accumulate during the progression of viral hepatitis and may play a role in the development or progression of hepatocellular carcinoma. In vitro inhibition of Hh-signaling in primary human hepatic stellate cells and sinusoidal endothelial cells suggest the possibility of using Hh inhibitors as potential tools to prevent cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis B and C / Ativação da via do Hedgehog (Hh) promove vários processos que ocorrem durante o reparo fibrogênico hepático. O papel da via do Hh na lesão causada pela hepatite crônica B e C ainda não foi investigado. Estudos de expressão global em carcinomas hepatocelulares (CHC) demostraram a ativação da via do Hh em pacientes com CHC relacionados à infecção crônica com o vírus da hepatite B (VHB) ou vírus da hepatite C (VHC). Como a maioria dos CHCs desenvolve em fígados cirróticos, levantamos a hipótese de que a ativação da via do Hh ocorre durante o reparo fibrogênico relacionado à hepatite viral crônica B e C e que células Hh-reatoras poderiam orquestrar a progressão da doença e a hepatocarcinogênese na hepatite viral crônica. Métodos Immunohistoquímica e análise por PCR quantitativo em tempo real (qRTPCR) foram utilizados para investigar a ativação da via do Hh e identificar os tipos celulares que respondem aos ligantes dessa via em biópsias arquivadas de 45 pacientes com hepatite crônica B ou C, de diferentes graus de fibrose (F0-4), sendo que 7 também apresentavam CHC. A via do Hh foi manipulada com Ciclopamina em células estreladas hepáticas (HSC) e em células endoteliais sinusoidais hepáticas (SEC) primárias humanas. O ensaio da angiogênese foi usado para investigar o papel da via do Hh na formação de tubos. SECs foram incubadas com Shh recombinante, meio condicionado de HSC, ciclopamina, short hairpin RNA (shRNA) contra o Smoothened ou os seus controles respectivos e o comprimento dos tubos vasculares foram quantificados por morfometria. A produção de ligantes Hh foi investigada por qRT-PCR em células Huh7 infectadas com o virus JFH-1 e em uma linhagem de hepatoma de rato que expressa o gene da proteína X do VHB. Resultados Observamos um aumento dos níveis de expressão de ligantes Hh (Shh e Ihh) em todos os pacientes com hepatite crônica viral (p<0,005) e demonstramos que a infecção pelo vírus da hepatite C JFH-1 ou a expressão ectópica do gene X do VHB estimulam culturas de hepatoma a produzirem Shh (p<0,005). Os principais tipos celulares que proliferam durante a cirrose e CHC (miofibroblastos, células endoteliais ativadas e progenitores que expressam marcadores de células tronco/iniciadoras de tumor CD133, Krt7, EpCAM e survivina) são reatores aos ligantes Hh e os níveis mais altos de expressão de componentes da via do Hh (Shh, Ihh, Gli-2 e Ptch) estão associados xii com cirrose e CHC (p<0,005). Inibição da via do Hh em células reatoras (HSC e SEC) reduziu fibrogênese (p<0,005 para expressão de &#945;SMA e p<0,05 para expressão de col1&#945;1) e angiogênese (p<0,05). Conclusões A Infecção pelo VHB/VHC aumenta a produção de ligantes hedgehog em hepatócitos e expande o número de células estreladas hepáticas e células endoteliais sinusoidais primárias humanas reatoras a esses ligantes, em relação direta com a progressão da fibrose para a cirrose e o carcinoma hepatocelular. Também induz o acúmulo progressivo de células progenitoras reatoras a via do Hh e que estão em processo de transição epitélio mesenquimal, o que pode estar relacionado com o desenvolvimento ou a progressão do carcinoma hepatocelular. A inibição in vitro da ativação da via do Hedgehog em células estreladas e em células sinusoidais endoteliais hepáticas primárias humanas sugere a possibilidade de investigar inibidores dessa via como potenciais ferramentas para reduzir a progressão da fibrose hepática para cirrose e carcinoma hepatocelular nas hepatites crônicas produzidas pelos vírus B e C