published_or_final_version / Pathology / Master / Master of Medical Sciences

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Au cours de la mitose, la ségrégation des chromatides, la partition du matériel cytoplasmique entre cellules filles et leur position relative se fait selon un plan qui est préfiguré par la plaque métaphasique. Ainsi, l'orientation de ce plan est un processus crucial pour le contrôle du destin des cellules, pour la morphogenèse durant l'embryogenèse et pour l'homéostasie tissulaire. Jusqu'à aujourd'hui, les mécanismes intrinsèques impliqués dans le positionnement du plan de division ont reçu beaucoup d'attention. En revanche, peu d'études ont exploré l'implication de signaux extracellulaires dans l'orientation du plan de division. Pourtant, l'axe des divisions cellulaires dont la position est souvent stéréotypée est largement associé aux axes de polarités du tissu. Au cours de ma thèse, je me suis demandé si des signaux extracellulaires capables de délivrer des informations de position spatiale aux cellules dans le cadre de leur migration, de leur différenciation morphologique, ou de leur polarisation, pouvaient influencer l'orientation des divisions cellulaires. En particulier, je me suis intéressée aux facteurs impliqués dans le guidage axonal à travers l'étude des mitoses des progéniteurs neuraux chez l'embryon de souris. Dans la moelle épinière en développement, les progéniteurs neuraux effectuent leur division au contact du canal central, lequel renferme le liquide céphalo-rachidien (LCR), une source de nombreux facteurs extracellulaires comme les morphogènes. Nous avons montré que la présence de molécules du LCR était nécessaire pour une orientation appropriée du plan de divisions des progéniteurs neuraux localisés au contact du canal central. Priver les progéniteurs neuraux de LCR par l'ouverture du tube neural ou provoquer génétiquement l'obstruction du canal central affecte les proportions de divisions planaires et obliques. Nous avons identifié la protéine Sémaphorine 3B, secrétée par les cellules de la plaque du plancher et les plexus choroïdes, comme un signal extrinsèque contrôlant l'orientation des divisions des progéniteurs neuraux dans la moelle épinière. L'invalidation génétique de Sema3B chez la souris phénocopie la perte d'accès au LCR des progéniteurs. Une application exogène de Sema3B sur des embryons dont le tube neural a été ouvert compense la déficience de LCR. Nous avons pu montrer que Sema3B se lie à ses récepteurs Neuropilines à la surface apicale des progéniteurs mitotiques et agit sur l'architecture des microtubules via l'activation de la voie GSK3/CRMP2, voie initialement mise en évidence dans le contexte du guidage axonal. Afin d'identifier de nouveaux facteurs influençant le positionnement du fuseau mitotique en réponse à ce facteur de guidage, une analyse transcriptomique des progéniteurs neuraux des mutants Sema3B-/- a été réalisée et des gènes candidats dérégulés en contexte d'invalidation de Sema3B ont été considérés. Durant la seconde partie de ma thèse, j'ai exploré l'implication du gène Norbin/Neurochondrin. De manière intéressante, le knock- down de Norbin dans les cellules HeLa altère l'orientation du fuseau mitotique. L'ensemble de ces travaux révèle donc la contribution d'une large famille de signaux topographiques jusqu'à présent inexplorée, dans l'orientation des divisions cellulaires et ouvre un large champ d'investigation passionnant concernant leur action moléculaire et cellulaire dans la neurogenèse et la morphogenèse / During development, the orientation of cell division is crucial to correctly organize andshape tissues and organs and also to generate cellular diversity. As cell mitosis proceeds, thesegregation of chromatids and cytoplasmic material occurs along a division axis. Itsorientation largely determines the relative position of daughter cells and the partition ofmother cell subcellular domain between them. The orientation of the cell division isprefigured by the position of a complex microtubule-based scaffold, the mitotic spindle.Until now, the intrinsic molecular machinery positioning the mitotic spindle and its couplingto cell polarities have been study in details. In contrast, the contribution of extracellularsignals to cell division orientation is less characterised. My research shows that these signalsin the CSF contribute to the orientation of cell division in neural progenitors. Removal theCSF cues by opening the neural tube or by genetic engineering affects the proportion ofplanar and oblique divisions. We identified Semaphorin 3B (Sema3B), released from thefloor plate and the nascent choroid plexus, as an important actor in this extrinsic control ofprogenitor division. Knockout of Sema3B phenocopies the loss of progenitor access to CSF.Delivery of exogenous Sema3B to progenitors in living embryos compensates this deficiency.We showed that Sema3B binds to Neuropilin receptors at the apical surface of mitoticprogenitors and exerts its effect through GSK3b activation and subsequent inhibition of themicrotubule stabilizer CRMP2. Thus extrinsic signaling mediated by Semaphorins directs theorientation of progenitor division in neurogenic zones.In order to identify new factors implicated in Sema3B-dependant mitotic spindleposition, we performed a transcriptomic analysis of Sema3B -/- neural progenitors. Severalderegulated candidate genes were considered. In the second part of my thesis, I focus onone of this, Norbin/Neurochondrin. Interestingly, the invalidation of Norbin/Neurochondrinalters the orientation of the mitotic spindle in HeLa cells.My PhD work reveals the contribution of a large family of topographic cues known tofunction in axon guidance has a novel role in the orientation of cell division

Orientador: Pablo Agustin Vargas / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T11:33:31Z (GMT). No. of bitstreams: 1 Fonseca_FelipePaiva_D.pdf: 1634857 bytes, checksum: 43da6be9627bec5197103495826aa6d6 (MD5) Previous issue date: 2015 / Resumo: Tumores de glândulas salivares correspondem a aproximadamente 3% de todas as neoplasias de cabeça e pescoço e as neoplasias malignas derivadas destas estruturas anatômicas ainda representam um grande desafio para a oncologia de cabeça e pescoço devido a sua difícil abordagem cirúrgica e pobre resposta às outras abordagens terapêuticas. Um melhor entendimento do seu perfil molecular contribuiria significativamente para um melhor manejo terapêutico futuro e o estudo do potencial angiogênico dos tumores de glândulas salivares representa um interessante alvo de investigação. Tem sido demonstrado que as semaforinas induzem a apoptose de células tumorais, modulam a migração celular neoplásica e inibem a angiogênese em diferentes neoplasias humanas, competindo com o fator de crescimento endotelial vascular (VEGF) pela ligação aos seus principais receptores, as neuropilinas-1 e -2, desta forma inibindo os efeitos mitogênicos e pró-angiogênicos de VEGF. Assim, o objetivo deste estudo é investigar a expressão das semaforinas de classe 3 A e B (Sema3A e Sema3B), e dos seus receptores neuropilinas-1 e -2 (Np-1 e Np-2) em tumores de glândulas salivares, determinando seus significados clínicos. Duzentos e quarenta e oito tumores benignos e malignos de glândulas salivares selecionados de quatro instituições brasileiras foram organizados em blocos de parafina em microarranjo tecidual em matriz e submetidos a reações de imunoistoquímica contra CD34, Sema3A, Sema3B, Np-1 e Np-2. As imunoreações foram quantificadas utilizando algoritmos digitais e os resultados foram correlacionados com parâmetros clinicopatológicos e índices de sobrevida. Tumores malignos apresentaram uma maior densidade vascular, porém uma menor área vascular do que sua contraparte benigna. Em glândulas salivares normais a expressão de Np-1 e -2 esteve restrita às células ductais, enquanto que Sema3A e Sema3B estiveram principalmente no componente acinar. Tumores benignos e malignos revelaram uma expressão similar de todos os marcadores e a co-expressão de Np-1/Np-2 correlacionou-se significativamente com a ocorrência de parestesias e estágios mais avançados dos tumores. Apesar de não ser estatisticamente significativa, a sobre-expressão simultânea de ambos os receptores também indicou uma menor taxa de sobrevida. Desta forma, Sema3A, Sema3B, Np-1 e Np-2 devem estar envolvidas no desenvolvimento das glândulas salivares normais e na patogênese das neoplasias benignas e malignas derivadas destas estruturas; entretanto, a expressão destas proteínas não apresentou um potencial prognóstico estatisticamente significativo no presente estudo / Abstract: Salivary gland tumors correspond to approximately 3% of all head and neck neoplasms and the malignant neoplasias derived from these anatomic structures still represent a major pitfall in head and neck oncology because of their difficult surgical approach and poor response to other therapies. A better understanding of their molecular basis would significantly aid to an improved future management and the study of salivary gland tumors angiogenic potential represents an interesting target of investigation. It has been shown that semaphorins induce tumor cell apoptosis, modulate tumor cell migration and inhibit angiogenesis in different human neoplasms, competing with vascular endothelial growth factor (VEGF) for biding to their main receptors, the neuropilins-1 and -2, thereby inhibiting mitogenic and pro-angiogenic effects of VEGF. Hence, the objective of this study is to investigate the expression of class 3 Semaphorins A and B, and their receptors neuropilins-1 and -2 in salivary gland tumors, determining their clinical significance. Two hundred and forty eight benign and malignant salivary gland tumors selected from four Brazilian institutions were organized in tissue microarray paraffin blocks and submitted to immunohistochemical reactions against CD34, Sema3A, Sema3B, Np-1 and Np-2. The immunoreactions were quantified using digital algorithms and the results were correlated with clinicopathological parameters and survival rates. Malignant tumors presented an increased vascular density but a lower vascular area than their benign counterparts. In normal salivary glands Np-1 and Np-2 expression was restricted to ductal cells, whereas Sema3A and Sema3B were positive mainly in serous acinar compartment. Benign and malignant tumors revealed a similar expression of all markers and the co-expression of Np-1/Np-2 significantly correlated with the occurrence of paresthesia and higher stages of the tumors. In addition, although not statistically significant, simultaneous overexpression of both receptors also indicated an inferior survival rate. Hence, these results suggest that Sema3A, Sema3B, Np-1 and Np-2 may be involved in the development of normal salivary glands and in the pathogenesis of benign and malignant neoplasms derived from these structures; however, the expression of these proteins did not present a statistically significant prognostic potential in the current study / Doutorado / Patologia / Doutor em Estomatopatologia

Solid tumors can hijack many of the same programs used in neurogenesis to enhance tumor growth and metastasis, thereby generating a plethora of neurogenesis-related molecules including semaphorins Among them, we have identified Semaphorin7A (SEMA7A) in breast cancer We first used to the DA-3 mammary tumor model to determine the effect of tumor-derived SEMA7A on immune cells We found that tumor-derived SEMA7A can modulate the production of proangiogenic chemokines CXCL2/MIP-2 and CXCL 1, and prometastatic MMP-9 in macrophages We next aimed to determine the expression and function of SEMA7A in mammary tumor cells We found that SEMA7A is highly expressed in both metastatic human and murine breast cancer cells We show that both TGF-β and hypoxia elicits the production of SEMA 7 A in mammary cells SEMA7 A shRNA silencing in 4T1 cells resulted in decreased mesenchymal markers MMP-3, MMP-13, Vimentin and TGF-β) SEMA7A silenced cells show increased stiffness with reduced migratory and proliferative potential In vivo, SEMA7A silenced 4T1 tumor bearing mice showed decreased tumor growth and metastasis Genetic ablation of host-derived SEMA7A synergized to further decrease the growth and metastasis of 4T1 cells Our findings suggest novel functional roles for SEMA7A in breast cancer and that SEMA7A could be a novel therapeutic target to limit tumor growth and metastasis / Includes bibliography / Dissertation (PhD)--Florida Atlantic University, 2016 / FAU Electronic Theses and Dissertations Collection

Etude de l'implication de CRMP4, un partenaire de MAP6, dans la voie de signalisation sémaphorine 3E.Pendant le développement embryonnaire, les neurones établissent des milliards de connexions. Ces connexions ne sont pas aléatoires, mais précisément orientées, dirigées par des molécules de guidage situées dans l’environnement cellulaire. Le branchement inapproprié de ces neurones a de graves conséquences sur les fonctions sensorielles, motrices et cognitives du système nerveux, aboutissant à des pathologies neurologiques et psychiatriques telle que la schizophrénie. Ainsi la mutation de certaines protéines impliquées dans le guidage de ces connexions, comme MAP6 ou CRMP4, peut entraîner des perturbations conduisant à des prédispositions pour le développement de telles pathologies. En effet l'absence de MAP6 (souris KO MAP6) conduit à l'altération de nombreuse connections neuronales associé a différents troubles comportementaux réminiscent avec des symptômes schizoïdes. Parmi les faisceaux d'axones affectés on remarque la disparition du fornix, un faisceau neuronal connu pour son implication dans la schizophrénie. Cette disparition est en partie causée, en l'absence de MAP6, par l'abolition de la signalisation induite par la molécule de guidage sémaphorine 3E (Sema3E). Dans ce projet de thèse, le lien entre MAP6 et CRMP4 dans cette voie de signalisation Sema3E à pu être établi. De plus, l'impact de l'absence de la protéine CRMP4 sur la formation du fornix a pu être caractérisé par l'étude neuroanatomique des souris KO CRMP4. Nous avons par ailleurs pu mettre en évidence de nouvelles altérations causée par l'absence de MAP6. Dans son ensemble ce travail approfondit les connaissances des défauts des connectivités des souris KO MAP6 et identifie CRMP4 comme un nouvel acteur de la signalisation Sema3E et de la formation du fornix. / Study of the involvement of the MAP6 partners, CRMP4, in the semaphorin 3E signaling pathway.During embryonic development, neurons establish billion of connections between them. Those connections are not random. On the contrary, they are precisely targeted thanks to the driving by cellular environment guidance cues. A wrong branching of those neurons can lead to dramatic impairment of sensory, motor and cognitive function of the central nervous system resulting in neurologic or psychiatric disorders such as Schizophrenia. Thus, mutation of proteins implicated on neurons guidance like MAP6 or CRMP4 can lead to susceptibility for those kind of pathology occurrence. In fact, MAP6 deletion ( MAP6 KO mice) leads to diverse neuronal connectivity alterations associated to schizophrenia-like behavior disorders. Among axonal tracts affected we notice the absence of the fornix known for its implication on Schizophrenia. In MAP6 KO mice, this fornix disruption is partly due to the loss of semaphorin 3E (Sema3E) dependant signaling pathway. This project shows the involvement of CRMP4, a partner of MAP6, in the Sema3E signaling pathway. Furthermore, it characterized the impact of the CRMP4 deletion (CRMP4 KO) on fornix formation. Finally, neuroanatomical studies allowed us to identify unknown alteration of MAP6 KO mice connectivity alteration.

<p>Normal hearing and balance relies on the detection of sound, orientation and acceleration by sensory hair cells (HCs) located in the inner ear. Once sound is detected, that information must be transmitted to the brain by sensory neurons. Damage to the HCs and/or neurons in the auditory or vestibular organs of the inner ear can result in hearing loss or balance disorders. In mammals, these disorders can be permanent, as HCs do not regenerate after damage. While hearing aids and cochlear implants can restore some ability to hear, there are currently no molecular therapies for hearing loss. By examining genes involved in HC development and innervation, basic science can identify candidate genes for potential molecular therapies. This dissertation focuses on molecular regulators involved in establishing and/or maintaining innervation in the chicken inner ear during embryonic development.</p><p>The basilar papilla (BP) is the auditory sensory organ in the chicken and is homologous to the mammalian organ of Corti (oC). The BP houses two types of sensory HCs – tall HCs and short HCs. On the neural side of the BP, tall HC receive primarily afferent innervation (neural-side identity). On the abneural side, short HC receive primarily efferent innervation (abneural-side identity). The patterning of these two identities along the radial axis is dependent upon the precise spatiotemporal expression of certain genes during embryonic development. One such gene is <i>Wingless/integrated (Wnt)9a</i>.</p><p>Previous work has shown that <i>Wnt9a</i>is expressed on the neural edge of the BP and is likely secreted in a gradient across the prosensory domain during crucial time points when proliferation, differentiation, and innervation are occurring. When <i>Wnt9a </i>was overexpressed, we observed an increase in the width of the BP as well as an expansion of the neural-side identity, likely at the expense of the abneural-side identity. RNA sequencing of <i>Wnt9a</i>-overexpressing and control BPs identified genes involved in the Wnt signaling pathway, cytoskeletal remodeling, and axon guidance signaling that were differentially expressed. This dissertation focuses on axon guidance genes, specifically those involved in Slit/Robo (Roundabout), Contactin (Cntn), and Semaphorin (Sema) signaling, that were differentially expressed in this RNA sequencing data set.</p><p>Slits typically act as repulsive cues for neurites expressing Robo receptors. RNA sequencing data indicates that <i>Slit2</i>transcripts increased by 1.2 fold when <i>Wnt9a </i>was overexpressed. When examining Slit2 spatial expression pattern in <i>Wnt9a-</i>overexpressing BPs, we did not observe an upregulation of <i>Slit2 </i>but rather an expansion of the <i>Slit2</i>-expression domain that is likely due to increased proliferation in response to <i>Wnt9a</i>. To better understand the role of Slit/Robo signaling in the developing BP, we examined the radial expression patterns of <i>Slit2</i>, <i>Robo1</i>, and <i>Robo2</i>. <i>Slit2 </i>is expressed on the anterior and posterior walls of the cochlear duct (CD). <i>Robo1</i>and <i>Robo2 </i>had graded expression in the prosensory domain of the BP, highest on the abneural side. <i>Robo1</i>is also present in the auditory ganglion. While only a small population of cochleovestibular ganglion neurites have been previously shown to respond to Slits, Slit-Robo has also been shown to activate TCF transcription factor by non-canonically activating β-catenin through Abl kinase. We examined Abl kinase-activated b-catenin in <i>Slit2-</i>and <i>Wnt9a-</i>overexpressing BPs but did not observe a change in phosphorylated b-catenin. We also overexpressed a dominant-negative Robo1. In some dominant-negative Robo1 overexpressing ears, we observed a reduction in ganglion size; however, this affect did not reliably replicate. These data suggests that Slit-Robo signaling could be involved in neuroblast delamination and/or migration.</p><p>RNA sequencing results indicate that <i>Contactin 6</i><i>Cntn6 </i>transcripts increased by 1.5 fold when <i>Wnt9a </i>was overexpressed. Contactins are cell adhesion molecules that have been previously shown to impact neurite outgrowth and innervation. In the auditory field, clinical studies have also shown that patients diagnosed with autism who also have mutations in <i>Cntn5 </i>and <i>Cntn6 </i>are more likely to exhibit increased sensitivity to sound. Based on RNA sequencing in the embryonic day (E)6 chicken ear, <i>Cntn6 </i>has low levels of expression in controls. We attempted to examine the spatial expression of <i>Cntn6 </i>but found that <i>in situ </i>hybridization is not sensitive enough to detect low levels of <i>Cntn6 </i>in control or <i>Wnt9a-</i>overexpressing BPs.</p><p>Class III Semaphorinsecreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. <i>Sema3D </i>and <i>Nrp2 </i>were downregulated in the presence of exogenous <i>Wnt9a</i>; however, the spatial expression of these transcripts did<i></i>not support their role in establishing or maintaining radial innervation patterns. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used <i>in situ </i>hybridization or immunohistochemistry to map the expression of <i>Sema3D</i>, <i>Sema3F</i>, Nrp1<i>, Nrp2</i>, and <i>PlxnA1 </i>in the chicken inner ear from E5 to E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. <i>Sema3D</i>expression flanking the sensory tissue in vestibular organs suggests that it may repel <i>Nrp2</i>- and <i>PlxnA1</i>-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8–E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, <i>Sema3D</i>in the future tegmentum vasculosum opposes Nrp1 and <i>PlxnA1 </i>in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. <i>Sema3D </i>immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.</p><p>To better understand innervation patterns in the avian BP, we also examined the developing efferent innervation patterns from E11 to E17 using NeuroVue lipophilic tracer dye. Our data suggest that efferents have already begun to penetrate the sensory epithelium at E11 and that efferents arrive to the ipsilateral BP earlier than the contralateral BP. By E12, many efferents appear to send back branches out to short HCs. At E15, many efferents appear to have reached the abneural edge of the BP, are innervating the hyaline cells, and are projecting apically.</p><p>In summary, this work suggests that Slit and Sema signaling are not involved in establishing radial innervation patterns but may have alternative roles in inner ear development. Additionally, while efferents appear to arrive to the ipsilateral BP sooner than the contralateral BP, both ears send projections across the radial axis and back branch around the same time.</p>

Yra žinoma, jog nervinės ląstelės gali regeneruoti savo aksonus po periferinės, o tam tikrais atvejais ir po centrinės nervų sistemos pažeidimų. Tačiau aksonų augimas yra sudėtingas, o jo reguliacija turi kritinę įtaką tiek neuronų vystymęsi, tiek regeneracijoje. Vekiami aplinkinių ląstelių, išskiriamų pritraukiančiųjų ir atstumiančiųjų molekulių, aksonai augdami nuolat keičia augimo kryptis iki kol pasiekia galutinius taikinius. Dorsalinių ragų ganglijų (DRG) aksonų augimas priklauso nuo semaforinų klasės molekulių. Sekretuojantys, ar su membrana surišti semaforinai dalyvauja įvairiuose biologiniuose procesuose, tokiuose, kaip centrinės ir periferinės nervų sistemos (CNS ir PNS) vystymęsi ir regeneracijoje, širdies ir kraujagyslių vystymęsi ir imuninės sistemos funkcijose. DRG aksonų vystymasis ir išlikimas smarkiai priklauso nuo nervų augimo faktoriaus (NGF). Darbo tikslas buvo įvertinti NGF koncentracijos įtaką DRG aksonų augimo atsakams į semaforiną 3A. 15 parų pelių embrionų DRG buvo preparuojami iš C57/Bl linijos pelių embrionų. DRG neuronų auginimui naudoti sterilūs dengiamieji stikleliai buvo padengiami poli-L-lizino 0,01 mg/ml ir laminino 0,01 mg/ml tirpalu, pagamintu GBSS terpėje. Aksonų augimo kūgelių vertinimas buvo atliekamas praėjus 60 minučių, o aksonų ilgio vertinimas - praėjus 16 valandų po DRG pasodinimo. Tam, kad nustayti DRG apoptozės lygį, DRG neuronuose priklausomai nuo NGF koncentracijos buvo įvertinta Bcl-2, Bax, c-jun genų raiška, naudojant RT - PGR... [toliau žr. visą tekstą] / It is known that nerve cells can regenerate their axons after damage to peripheral and in some cases central nervous system (PNS and CNS). However, axon growth over longer distances, especially in central nervous system, is complicated. Regulation of axon growth is a critical event both in neuronal development and regeneration. To reach their proper targets, axons rely upon the actions of attractive and repulsive guidance molecules. It is known that growth of dorsal root ganglion (DRG) axons depend on guidance molecules of semaphorin class. Secreted and membrane bound semaphorins participate in diverse biological processes, including development and regeneration of central and peripheral nervous system, cardiovascular development, and immune system functioning. In addition to regulation of DRG axon growth by semaphorin class molecules, DRG axon growth and survival is strongly dependent on nerve growth factor (NGF). The aim of this study was to evaluate responses of DRG axons to semaphorin 3A in dependence of NGF concentration. DRG were dissected from C57/Bl mice E15 embryos in dissection HBSS/glucose medium. DRG were plated on cover slips coated with laminin and poly-L-lysine and grown in Neurobasal medium supplemented with 2% of B27 supplement. To evaluate collapse rate the morphology of axons growth cones were evaluated after 60 minutes and axons length were evaluated 16 hours after DRG plating. To evaluate DRG survival and level of apoptosis in dependence of NGF... [to full text]

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.