Hou, Yonghui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 174-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

INTRODUÇÃO: A prolactina é um hormônio polipeptídico que possui reconhecida ação sistêmica, principalmente no sistema reprodutor. O papel desse hormônio no desenvolvimento e na extensão do câncer da mama ainda é muito debatido. A progressão do câncer de mama em grande parte depende do movimento celular e da capacidade da célula em remodelar seu citoesqueleto de actina. Nesse processo, proteínas envolvidas na migração celular, como moesina, FAK e c-Src, são influenciadas por vários hormônios, incluindo a prolactina. O presente estudo teve por objetivo avaliar os efeitos da PRL na migração de células T47D, MCF-7 e ZR75-1 de câncer de mama, bem como os mecanismos envolvidos. MÉTODOS: As células foram cultivadas em placas de cultura com meio suplementado e divididas em oito grupos diferentes de tratamento: Grupo I (veículo); Grupo II (PRL na concentração de 25 ng/mL); Grupo III (PRL na concentração de 50 ng/mL), Grupo IV (PRL na concentração de 100 ng / mL), Grupo V (RNAi + veículo); Grupo VI (RNAi + PRL na concentração de 25 ng/mL); Grupo VII (RNAi + PRL na concentração de 50 ng/mL) e Grupo VIII (RNAi + PRL na concentração de 100 ng / mL). Nos Grupos de I a IV, a reorganização da actina do citoesqueleto foi analisada por imunofluorescência após 30 minutos do tratamento. Em todos os grupos estudados foram realizadas análise da migração horizontal com auxílio de microscopia de luz e avaliadas as expressões de Moesina, p-Moesina, FAK, p-FAK, c-Src e p-c-Src por Western Blot após 48 horas do tratamento. RESULTADOS: As células de câncer de mama expostas à prolactina apresentaram um aumento da expressão de Moesina, p-Moesina, FAK, p-FAK, c-Src e p-c-Src. Essas alterações moleculares estão associadas à reorganização da actina do citoesqueleto e ao aumento da mobilidade das células. CONCLUSÕES: Nossos dados sugerem que a prolactina aumenta a migração das células T47D, MFC-7 e ZR75-1 de câncer de mama e remodela a actina do citoesqueleto pela via de sinalização intracelular das proteínas c-Src, FAK e moesina / INTRODUCTION: Prolactin is a polypeptide hormone with a recognized systemic action mainly on reproductive physiology. The role of this hormone on breast cancer development and progression has been debated a lot yet. Breast cancer invasion largely depends on cell movement and on the ability to remodel the actin cytoskeleton. In this process, proteins involved in cell migration, such as moesin, FAK and c-Src, are influenced by a large number of hormones, such as prolactin. The present study was aimed for evaluating the effects of PRL on migration of T47D, MCF-7 and ZR75-1 breast cancer cells as well as the molecular mechanisms in this process. METHODS: The cells were cultured in dishes with supplemented medium and were divided in eight different assays: Group I (control); Group II (25ng/ml of prolactin); Group III (50ng/ml of prolactin); Group IV (100ng/ml of prolactin); Group V (RNAi + control); Group VI (RNAi + 25ng/ml of prolactin); Group VII (RNAi + 50ng/ml of prolactin); Group VIII (RNAi + 100ng/ml of prolactin). In Groups I to IV, the actin cytoskeletal reorganization was analyzed by immunofluorescence 30 minutes after the treatment. In all groups, were performed the horizontal migration analysis with light microscopy and evaluated the expression of moesin, p-moesin, FAK, p-FAK, c-Src and p-c-Src by Western blot after 48 hours of treatment. RESULTS: Breast cancer cells exposed to prolactin display an elevated moesin, p-moesin, FAK, p-FAK, c-Src and p-c-Src expression. These molecular changes are associated with the reorganization of actin cytoskeleton and increased mobility of cells. CONCLUSION: Our data suggest that prolactin enhances the migration of T47D, MFC-7 and ZR75-1 breast cancer cells through the actin cytoskeleton remodeling by intracellular signaling pathway of c-Src, FAK and moesin proteins

Chan, Ka Ki Alex. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 245-263). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Structure and function of the enteric nervous system --- p.1 / Chapter 1.2 --- Neural crest cells (NCC) --- p.5 / Chapter 1.2.1 --- Vagal neural crest cells --- p.7 / Chapter 1.2.2 --- Sacral neural crest cells --- p.10 / Chapter 1.3 --- Prespecialization of the neural crest cells to form ENS --- p.15 / Chapter 1.4 --- Signaling pathways involved in ENS development --- p.19 / Chapter 1.4.1 --- Endothelin signaling pathway --- p.20 / Chapter 1.4.2 --- Ret signaling pathway: GDNF/Ret/GFRa1 --- p.22 / Chapter 1.4.3 --- Ret signaling pathway: NRTN/Ret/GFRa2 --- p.26 / Chapter 1.4.4 --- Phox2b --- p.28 / Chapter 1.4.5 --- Sox10 --- p.29 / Chapter 1.5 --- Hirschsprung's Disease (HSCR) --- p.31 / Chapter 1.6 --- Objective of studies --- p.32 / Figures and legends --- p.35 / Chapter Chapter Two --- The early migratory pathways of mouse sacral neural crest cells --- p.39 / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and Methods --- p.46 / Chapter 2.2.1 --- Animals --- p.46 / Chapter 2.2.2 --- Isolation of the mouse embryos at E95 --- p.46 / Chapter 2.2.3 --- Preparation ofWGA-Au --- p.47 / Chapter 2.2.4 --- Preparation of Dil --- p.48 / Chapter 2.2.5 --- Microinjection ofWGA-Au or Dil --- p.48 / Chapter 2.2.6 --- Preparation of rat serum --- p.49 / Chapter 2.2.7 --- Preparation of culture medium --- p.50 / Chapter 2.2.8 --- in vitro whole embryo culture system --- p.50 / Chapter 2.2.9 --- Examination of embryo after culture --- p.51 / Chapter 2.2.10 --- Histological preparation of WGA-Au labelled embryos --- p.51 / Chapter 2.2.11 --- Silver enhancement staining on sections of WGA-Au labelled embryo --- p.52 / Chapter 2.2.12 --- Histological preparation of Dil labelled embryos --- p.53 / Chapter 2.2.13 --- Reconstruction of the mouse embryos --- p.53 / Chapter 2.2.14 --- Cell counting on labelled sacral NCC between the anterior and posterior halves of the somite --- p.54 / Chapter 2.2.15 --- Cell counting on migrating labelled sacral NCC for each somite at different developmental stages --- p.55 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Development of E9.5 mouse embryo in vitro and in vivo --- p.57 / Chapter 2.3.2 --- Labelling of sacral neural crest cells by means of different cell markers --- p.58 / Chapter 2.3.3 --- Migration of sacral neural crest cells at different developmental stages --- p.59 / Chapter 2.3.3.1 --- Distribution of sacral NCC at the 26th somite stage --- p.60 / Chapter 2.3.3.2 --- Distribution of sacral NCC at the 28th somite stage --- p.61 / Chapter 2.3.3.3 --- Distribution of sacral NCC at the 30th somite stage --- p.61 / Chapter 2.3.3.4 --- Distribution of sacral NCC at the 32nd somite stage --- p.63 / Chapter 2.3.3.5 --- Distribution of sacral NCC at the 34th somite stage --- p.64 / Chapter 2.3.4 --- Defined migration pathways of the sacral neural crest cells --- p.65 / Chapter 2.3.5 --- Quantification of migrating sacral NCC at different somite axial levels at different developmental stages --- p.66 / Chapter 2.4 --- Discussion --- p.68 / Chapter 2.4.1 --- E9.5 mouse embryo grew normally in vitro using whole embryo culture --- p.69 / Chapter 2.4.2 --- Migration of sacral neural crest cells at 26th somite stage --- p.70 / Chapter 2.4.3 --- Migration of sacral neural crest cells at 28th somite stage --- p.72 / Chapter 2.4.4 --- Migration or sacral neural crest cells at 30th somite stage --- p.73 / Chapter 2.4.5 --- Migration of sacral neural crest cells at 32nd somite --- p.75 / Chapter 2.4.6 --- Migration of sacral neural crest cells at 34th somite stage --- p.77 / Chapter 2.4.7 --- Majority of sacral neural crest cells migrate along the dorsomedial pathway --- p.80 / Figures and Legends --- p.82 / Tables --- p.136 / Chapter Chapter Three --- The early migratory pathways of Dom mouse sacral neural crest cells --- p.139 / Chapter 3.1 --- Introduction --- p.139 / Chapter 3.2 --- Materials and Methods --- p.145 / Chapter 3.2.1 --- Animals --- p.145 / Chapter 3.2.2 --- In vitro culture of Dom mouse embryos --- p.145 / Chapter 3.2.3 --- Genotyping by polymerase chain reaction (PCR) --- p.146 / Chapter 3.2.4 --- Treatment of the harvested Dom mouse embryos --- p.147 / Chapter 3.2.5 --- Reconstruction of images and cell counting --- p.148 / Chapter 3.2.6 --- Percentage of migrating sacral neural crest cells reduction in Dom mouse embryo --- p.148 / Chapter 3.3 --- Results --- p.150 / Chapter 3.3.1 --- Migration of sacral neural crest cells in Dom mouse embryos at different developmental stages --- p.150 / Chapter 3.3.1.1 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 26th somite stage --- p.150 / Chapter 3.3.1.2 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 28th somite stage --- p.151 / Chapter 3.3.1.3 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 30th somite stage --- p.152 / Chapter 3.3.1.4 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 32nd somite stage --- p.154 / Chapter 3.3.1.5 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 34th somite stage --- p.156 / Chapter 3.3.2 --- Number of migrating sacral NCC of different genotypes of Dom mouse embryos at different developmental stage --- p.158 / Chapter 3.4 --- Discussion --- p.160 / Chapter 3.4.1 --- The use of Dom mouse model to study the etiology of Hirschsprung's disease (HSCR) --- p.161 / Chapter 3.4.2 --- Migration of sacral NCC in Dom mouse embryos --- p.164 / Figures and legends --- p.169 / Tables --- p.230 / Chapter Chapter Four --- General discussion and conclusions --- p.236 / Appendix --- p.241 / References --- p.245

A hallmark feature of Dictyostelium development is the establishment and maintenance of precise cell-type proportions. In the case of D. discoideum, roughly 20% of the cells that aggregate form the stalk while the remaining 80% form the spores. In order to identify genes involved in cell-type proportioning Jaiswal et al. (2006) carried out random insertional mutagenesis (REMI) of the D. discoideum genome. This led to the identification of a novel gene, which was named trishanku (triA). A knock-out of triA did not show any defects during growth and early development but multiple defects later during development. To understand the reasons for the multiple developmental defects in the absence of triA, I looked at the genomic organization and the pattern of expression of the triA gene. In silico analysis points to the presence of more than one consensus D. discoideum promoter sequence upstream to exons1 and 2, raising the possibility that the triA gene could code for more than one transcript. Northern blot analysis confirms this prediction and provides evidence for the presence of two transcripts: triA1-2-3 (~ 2.9 kb, containing exons 1+2+3) and triA2-3 (~ 2 kb, containing exons 2+3). Both transcripts have exons 2 and 3 in common. In triA- cells, the REMI cassette is inserted in exon 2, which is common to both transcripts; thus, the absence of triA results in the lack of both. The transcripts are absent in vegetative cells but expressed during development. triA2-3 is expressed earlier, by 3h, while triA1-2-3 is expressed later, by 9h, and both remain till the end of development. triA2-3 and triA1-2-3 are differentially regulated by different aspects of the extracellular environment which include mode of development of cells (solid substratum versus shaken suspension), the presence of a high level of extracellular cAMP and formation of stable cell-cell contacts. The expression of triA2-3 and triA1-2-3 in triA- cells, one at a time under a constitutive promoter (Actin15 promoter), suggests that the two transcripts have both specific as well as overlapping functions in the cell. The triA2-3 transcript can specifically restore spore forming efficiency and stalk thickness, while the triA1-2-3 transcript can rescue the stream break up defect. Both the transcripts can rescue the sub-terminal position of the sorus, spore shape and spore viability. To address the question of stream break-up during mid to late aggregation in triA- cells, I have looked at the cell adhesion profile of triA- cells and compared it with the wild type (Ax2). triA- cells show transient disaggregation in buffer and a 2h delay in agglutination in presence of buffer with 10mM EDTA. This aberrant cell adhesion profile seen in triA- cells is in accordance with the expression pattern of genes encoding known cell adhesion molecules. triA- cells also overproduce an extracellular factor which significantly decreases the aggregate size of both Ax2 and triA-. The nature of the extracellular factor overproduced by in triA- cells is currently unknown, but it is not the same as cell-counting factor which is overproduced by smlA null cells. To look at the mis-expression of cell type-specific genes, I have monitored the movement of prestalk cells into the prespore region and vice versa in both Ax2 and triA- slugs. My studies show that there is extensive movement of prestalk cells into the prespore region and of prespore cells into the prestalk region in triA- slugs, which is absent in Ax2 slugs. Also, cells that move into the ‘wrong’ region show a change their cell fate (transdifferentiate) appropriate to the new location; whether transdifferentiation precedes or succeeds cell movement is not yet clear. Transdifferentiation is observed to a certain extent in Ax2 slugs, but only after prolonged migration; triA- slugs show enhanced transdifferentiation even in the absence of migration. To find out the possible reason(s) for the formation of a sub-terminal spore mass in the absence of triA, I have checked whether the defect lies in the ability of the prespore cells to rise up the stalk or in the ability of the upper cup (cells present above the spore mass contributed by a subset of prestalk cells and anterior like-cells) to pull the spore mass to the top. To see which of the two reasons could be responsible for the formation of a sub-terminal spore mass in triA-, I carried out transplantation experiments where the anterior one-fourth region of an Ax2 or triA- slug is grafted to the posterior four-fifth region of a triA- or Ax2 slug and the morphology of the fruiting body is observed. My studies show that the sub-terminal position of the spore mass in triA- is not due to an inability of the prespore cells to rise to the top but to a defect in the upper cup. The upper cup in triA- remains motile but is unable to remain attached to the prespore mass during culmination. It detaches, rises up the stalk and is present at the tip of the stalk. Mixing a minority of triA- cells (20%) with an excess of Ax2 (80%) results in an upper up formed by Ax2 alone. In this situation, the wild type upper cup is able to lift the triA- prespore mass to the top. Thus, the presence of triA (a prespore-specific gene) is essential for the proper functioning of the upper cup cells (which belong to the prestalk class) in order to enable prespore cells to ascend to the top of the stalk.

During zebrafish gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Recent studies have identified silberblick (slb/wnt11) as a key molecule that regulates gastrulation movement through a conserved pathway, which shares significant similarity with a signalling pathway that establishes epithelial planar cell polarity (PCP) in Drosophila (Heisenberg et al., 2000; Veeman et al., 2003), suggesting a role for cell polarity in regulating gastrulation movements. However, the cellular and molecular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still not fully understood. In the first part of the thesis, the three-dimensional movement and morphology of individual cells in living embryos during the course of gastrulation were recorded and analysed using high resolution confocal microscopy. It was shown that in slb/wnt11 mutant embryos, hypoblast cells within the forming germ ring display slower, less directed migratory movements at the onset of gastrulation, which are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. The net movement direction of the cells is not changed, suggesting that slb/wnt11-mediated orientation of cellular processes serves to facilitate and stabilize cell movements during gastrulation. By using an in vitro reaggregation assay on mesendodermal cells, combined with an analysis of the endogenous expression levels and distribution of E-cadherin in zebrafish embryos at the onset of gastrulation, E-cadherin mediated adhesion was found to be a downstream mechanism regulating slb/wnt11 function during gastrulation. Interestingly, the effects of slb/wnt11 on cell adhesion appear to be dependent on Rab5-mediated endocytosis, suggesting endocytic turnover of cell-cell contacts as one possible mechanism through which slb/wnt11 mediates its effects on gastrulation movements. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 23 MB)- Übersicht über Inhalte siehe Dissertation S. 92 - 93"

Wnt11 is a key signalling molecule that regulates cell polarity/migration during vertebrate development and also promotes the invasive behaviour of adult cancer cells. It is therefore essential to understand the mechanisms by which Wnt11 signalling regulates cell behaviour. The process of vertebrate gastrulation provides an excellent developmental system to study Wnt11 function in vivo. It is known that Wnt11 mediates coordinated cell migration during gastrulation via the non-canonical Wnt pathway that shares several components with a the planar cell polarity pathway (PCP) in Drosophila. However, the mechanisms by which these PCP components facilitate Wnt11 function in vertebrates is still unclear. While in Drosophila, the asymmetric localization of PCP components is crucial for the establishment of cell polarity, no asymmetric localization of Wnt11 pathway components have so far been observed in vertebrates. To shed light on the cellular and molecular mechanisms underlying Wnt11 signalling, I developed an assay to visualize Wnt11 activity in vivo using live imaging of Wnt11 pathway components tagged to fluorescent proteins. This allowed me to determine the sub-cellular distribution of these components and to correlate the effect of Wnt11 activity with the behaviour of living embryonic cells. I found that Wnt11 locally accumulates together with its receptor Frizzled7 (Fz7) at sites of cell-cell contacts and locally recruits the intra-cellular signalling mediator Dishevelled (Dsh) to those sites. Monitoring these apparent Wnt11 signalling centres through time-lapse confocal microscopy revealed, that Wnt11 activity locally increases the persistency of cell-cell contacts. In addition, I found that the atypical cadherin Flamingo (Fmi) is required for this process. Fmi accumulates together with Wnt11/Fz7 at sites of cell-cell contact and locally increased cell adhesion, via a mechanism that appears to be independent of known downstream effectors of Wnt11 signalling such as RhoA and Rok2. This study indicates that Wnt11 locally interacts with Fmi and Fz7 to control cell-contact persistency and to facilitate coherent and coordinated cell migration. This provides a novel mechanism of non-canonical Wnt signalling in mediating cell behaviour, which is likely relevant to other developmental systems. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 50 MB: Movies - Nutzung: Referat Informationsvermittlung der SLUB)

A movement protein Potato virus X (PVX) chimera virus (PVX.GFP(CH3)) bearing the grapevine virus Grapevine rupestris stem pitting-associated virus (GRSPaV) triple gene block proteins (TGB) (denoted P1, P2 and P3) instead of the PVX TGB was delivered into N. benthamiana and other related species by agro-inoculation. This movement protein PVX chimera virus was found to be unable to support the local and systemic movement of PVX in cis. Local and systemic movement of this PVX chimera virus was restored in trans by the dianthovirus Red clover necrotic mosaic virus (RCNMV) movement protein and by a PVX TGB rescue virus that replaced the GRSPaV TGB with the PVX TGB (PVX.GFP(Rescue)). However, a PVX TGB hybrid chimera virus (PVX.GFP(HY2)) containing PVX P1 and the GRSPaV TGB had limited cell-to-cell, but not systemic, movement.

The main focus of my PhD studies was aimed at understanding the general mechanism of nuclear translocation and isolating novel components of the nuclear translocation pathway in neurons. Using the Drosophila visual system as an in vivo model to study nuclear motility in developing photoreceptor cells (R-cells), I have identified a novel role for the Ser/Thr kinase Misshapen (Msn) and the endocytic trafficking pathway in regulating the nuclear translocation process. / The development of R-cells in the Drosophila eye disc is an excellent model system for the study of nuclear motility owing to its monolayer organization and the stereotypical translocation of its differentiating R-cell nuclei along the apical-basal plane. Prior to my thesis work, several laboratories had identified dynein and its associating proteins in R-cell nuclear translocation, however nothing was known about the signalling pathway that controlled their function in nuclear migration. Thus, one of my thesis goals was to elucidate the signalling mechanism controlling nuclear translocation in R-cells. / Using a combination of molecular and genetic approaches, I identified Msn as a key component of a novel signalling pathway regulating R-cell nuclear translocation. Loss of msn causes a failure of R-cell nuclei to migrate apically. Msn appears to control R-cell nuclear translocation by regulating the localization of dynein and Bicaudal-D (Bic-D). My results also show that Msn enhances Bic-D phosphorylation in cultured cells, suggesting that Msn regulates R-cell nuclear migration by modulating the phosphorylation state of Bic-D. Consistently, my results show that a Bic-D-phosphorylation-defective mutation disrupted the apical localization of both Bic-D and dynein. I propose a model in which Msn induces the phosphorylation of Bic-D, which in turn modulates the activity and/or subcellular localization of dynein leading to the apical migration of R-cell nuclei. / In addition to studying Msn, I have also searched for additional players in R-cell nuclear migration. From a gain-of-function approach, I found that the misexpression of the GTPase-activating-protein (GAP) RN-Tre caused a severe defect in R-cell nuclear migration. Since mammalian RN-Tre is involved in negatively regulating Rab protein activity, I speculated that the RN-Tre misexpression phenotype reflected a role for Rab-mediated vesicular transport in regulating R-cell nuclear migration. I systematically examined the potential role of Rab family proteins in R-cell nuclear migration and found that interfering with the function of Rab5, Rab11 or Shibire caused a similar nuclear migration phenotype. I propose that an endocytic pathway involving these GTPases is required for the targeting of determinants to specific subcellular locations, which in turn drive the apical migration of R-cell nuclei during development.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

O glioblastoma (GBM) é o tumor cerebral maligno mais comum e agressivo em adultos. Uma combinação de terapia padrão com outras terapias baseadas no conhecimento de sua biologia é necessária para melhorar a sobrevida de pacientes com GBM. Muitos estudos foram desenvolvidos em busca de proteínas de membrana expressas em GBM, pois são potenciais alvos para imunoterapia. A proteína transmembrânica CD99 foi descrita como altamente expressa em astrocitomas de diferentes graus de malignidade. Embora seu mecanismo de ação ainda não seja totalmente compreendido, CD99 está envolvido na adesão e migração celular em diferentes tipos de tumores. O gene CD99 codifica duas proteínas distintas, denominadas isoforma 1, maior, de 32 kDa, e isoforma 2, gerada por splicing alternativo e menor, de 28 kDa. No presente estudo, foi demonstrada a expressão predominante da isoforma 1 em astrocitomas de diferentes graus de malignidade em comparação com o cérebro normal, bem como na linhagem celular de GBM humano U87MG. O transcriptoma das células U87MG transfectadas com siRNA para CD99 foi analisado em relação ao controle. Um total de 2.670 genes diferencialmente expressos foi identificado. Uma análise de enriquecimento no banco de dados DAVID revelou os seguintes processos como os mais significativos: junções aderentes célula-célula; adesão célula-célula envolvendo ligação de caderina e adesão celular. Ensaios funcionais baseados nestes achados (migração, invasão e adesão) foram realizados com células U87MG após o silenciamento de CD99 com dois shRNAs diferentes. A eficiência de silenciamento foi de 80 e 97%, para o shCD991 e 2, respectivamente, confirmada a nível de expressão do gene e da proteína. O silenciamento de CD99 reduziu a migração e invasão para ambos os shRNAs, com diminuição mais acentuada da migração para o shCD99 2, com maior nível de silenciamento de CD99. No ensaio de adesão, a linhagem U87MG shCD99 1 apresentou propriedades adesivas mais baixas que o controle, enquanto o shCD99 2 apresentou resultado oposto, com maior adesão celular do que seu controle. Provavelmente o silenciamento de CD99 afetou a redução da adesão celular em um padrão distinto, sugerindo que o resultado pode ser dependente do nível de expressão remanescente de CD99. Além disso, o CD99 e a faloidina colocalizaram nos lamelipódios e filopódios, sugerindo um papel importante no rearranjo do citoesqueleto. Foi demostrado, ainda, que o silenciamento de CD99 levou à redução da proliferação celular in vitro e diminuição do tumor in vivo. Camundongos imunodeficientes nos quais foram implantadas células silenciadas no cérebro apresentaram uma maior sobrevida que os animais que receberam células controle. A via de sinalização pela qual CD99 modula a proliferação no GBM ainda precisa ser elucidada. Migração, invasão e proliferação são as principais características do GBM que limitam uma ressecção cirúrgica completa e, consequentemente, levam frequentemente à recorrência. Portanto, análises posteriores das vias ativadoras do CD99 no contexto da migração, invasão, proliferação celular e apoptose são válidas para revelar novas estratégias terapêuticas para limitar a progressão do GBM / Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. A combination of standard therapy with other biologically based therapies is necessary to improve the survival of patients with GBM. Many studies have been developed in pursuit of expressed membrane proteins in GBM, which are potential targets for immunotherapy. The transmembrane protein CD99 is highly expressed in different malignant grades of astrocytomas. Although its mechanism of action is not still fully understood, CD99 is involved in cell adhesion and migration in different type of tumors. The CD99 gene encodes two distinct transmembrane proteins, named isoform 1, longer with 32 kDa, and isoform 2, generated by alternative splicing, shorter with 28 kDa. In the present study, we demonstrated predominant expression of isoform 1 in astrocytomas of different malignant grades compared to normal brain, and in the human GBM cell line U87MG. The transcriptome of U87MG cell line transfected with siRNA for CD99 was analyzed in relation to control. A total of 2.670 differentially expressed genes were identified. An enrichment analysis by DAVID Bioinformatics Database revealed the following processes as the most significant: cell-cell adherens junction; cadherin binding involved in cell-cell adhesion and cell-cell adhesion. Functional assays based on these findings (migration, invasion and adhesion) were performed with U87MG cells after knocking down CD99 with two different shRNAs. The CD99 silencing efficiency was 80 and 97%, for shCD99 1 and 2, respectively, confirmed at gene and protein level. The CD99 knockdown reduced migration and invasion for both shRNA, with the highest decrease of migration observed in the higher CD99 knocked down cells. In adhesion assay, shCD99 1 U87MG showed lower adhesive properties than the control, whereas shCD99 2 cells presented opposite results, with higher cell adhesion than control. Probably CD99 knockdown affected in the reduction of cell adhesion in a distinct pattern, suggesting that the result is dependent on CD99 remaining expression level. Additionally, CD99 and phalloidin colocalized at lamellipodia and filopodia, sugesting that CD99 plays an important role to cytoskeleton rearrangement. It has also been demonstrated that CD99 silencing caused reduction of cell proliferation in vitro and decreased tumor in vivo. Immunodeficient mice in which knocked down cells were implanted in the brain had a longer survival than animals that received control cells. The signaling pathway by which CD99 modulates proliferation in GBM still needs to be elucidated. Migration, invasion and proliferation are major characteristics of GBM, which limits the complete surgical tumor resection, and consequently leads to tumor recurrence. Therefore, further analysis of CD99 activating pathways in the context of cell migration, invasion, proliferation and apoptosis is worthwhile to unveil new therapeutic strategies to halt GBM progression