Testicular germ cell cancer (TGCC) has been increasing in incidence over recent decades, and is currently the most common malignancy amongst young men resulting in significant morbidity. These tumours are believed to arise from premalignant germ cell neoplasia in situ (GCNIS) cells, which originate from the aberrant germ cell differentiation from gonocyte to spermatogonia during fetal/early postnatal life. GCNIS cells remain dormant in the testis until puberty when they are activated to become tumours. Therefore, GCNIS cells remain in a pre-invasive stage during early childhood and early adulthood prior to the development of a seminoma or non-seminoma TGCC. GCNIS cells are phenotypically similar to gonocytes with expression of stem cell/early germ cell markers including OCT4, PLAP and LIN28. Furthermore, proteins which are expressed in more mature germ cells (spermatogonia) such as MAGE-A4 have also been shown to be expressed in GCNIS cells and these studies have indicated that GCNIS cells are a heterogeneous population in terms of protein expression profile. The relationship between the protein expression profile of individual GCNIS cells populations and their oncogenic potential has not been fully explored. GCNIS cells are located in the seminiferous tubules supported by somatic Sertoli cells. These cells have been previously reported to exhibit an immature protein expression profile in GCNIS tubules from patients with testis cancer, suggesting that the germ stem cell niche in GCNIS tubules resembles that of a fetal one. Associations between Sertoli cell maturation and GCNIS progression into tumour formation has not been fully investigated. Oncogenes are key players in the regulation of oncogenic potential of cancer cells. Gankyrin is an oncogene that has been shown to down-regulate OCT4, and interact with MAGE-A4 in hepatocellular carcinoma and colorectal cancer, where Gankyrin interaction with MAGE-A4 reduces the oncogenic potential of tumour cells. In this study I aimed to investigate the heterogeneity of GCNIS in relation to disease stage and Sertoli cell development. We also aimed to determine the role of Gankyrin in TGCC cell survival and invasion. The co-expression of early germ cells proteins such as OCT4, LIN28 and PLAP was characterized in GCNIS cells during childhood and adulthood pre-invasive TGCC and in invasive disease characterized by the presence of a testicular tumour. These results show that LIN28 was expressed in 95% of OCT4 GCNIS cells, whereas PLAP expression in GCNIS cells increased as the disease progressed from childhood pre-invasive disease to invasive seminoma (32.3% v 76%; p < 0.05). In contrast there was a reduction in the proportion of MAGE-A4 expressing GCNIS cells with disease progression. The MAGE-A4 expressing population was also less proliferative than the MAGE-A4 negative GCNIS population. The methylation status of GCNIS cells was then investigated. EZH2 a methyltransferase previously reported to be important for TGCC development, was expressed in GCNIS cells at all stages of disease, however the histone 3 modification H3K27me3 (mediated by EZH2) was expressed in a significantly higher percentage of the proliferative OCT4+/MAGE-A4- GCNIS cells compared with the OCT4+/MAGEA4+ population (11.7% v 1.1%; p < 0.01) which could indicate a repressive role for H3K27me3 over MAGE-A4 expression. Next, it was determined whether an association between Sertoli cell maturation status and progression of TGCC could be observed. The maturation status of Sertoli cells was studied using proteins indicative of immature (desmin, cytokeratin, fibronectin and AMH) and mature (vimentin and androgen receptor) Sertoli cells. These studies demonstrated heterogeneity of Sertoli cells maturation in GCNIS-containing tubules. Desmin, fibronectin, AMH and vimentin expression did not show any association with TGCC progression. Cytokeratin was expressed in Sertoli cells of human fetal testis up to second trimester of fetal life, absent in tubules with active spermatogenesis but heterogeneously present in GCNIS, demonstrating that cytokeratin expression is indicative of the presence of GCNIS. Androgen receptor was weakly present in Sertoli cells from human fetal testis and pre-pubertal pre-invasive TGCC testis whereas in GCNIS of adult pre-invasive testis and invasive samples, androgen receptor was abundantly expressed in Sertoli cells of GCNIS-containing tubules. These combined results for cytokeratin and androgen receptor suggest that Sertoli cells from GCNIS-containing tubules, in pre-invasive and invasive TGCC patients are partially differentiated. Gankyrin expression was characterised in fetal germ cells, GCNIS cells and TGCC tissue. In fetal testis nuclear Gankyrin was absent in OCT4+/MAGE-A4- (gonocyte) population whereas it was present in a subpopulation of OCT4-/MAGE-A4+ (spermatogonia) germ cells. In GCNIS cells from TGCC patients nuclear Gankyrin was expressed in 87%, 63.3%, 91.5% and 79% in childhood pre-invasive, adult pre-invasive, seminoma and non-seminoma GCNIS cells respectively. Finally, in seminoma cells, Gankyrin was expressed in the cytoplasm indicating a change in localisation as the GCNIS cells become invasive. We used siRNA to knockdown Gankyrin in NT2 (a TGCC cell line) cells in-vitro and demonstrated a decrease in cell number, suggesting that Gankyrin might play a role in TGCC progression and invasiveness. Gankyrin down-regulation also resulted in an increase in p53 and p21 mRNA level. Given the role of P53 and p21 in cisplatin cytotoxic effect in TGCC we went on to investigate the role of Gankyrin in cisplatin resistance using NT2 cells. We demonstrate that Gankyrin mediated cisplatin resistance through the p53/p21 pathway, upregulating apoptosis rates through BAX and FAS, whilst there was no effect on cell proliferation, cell cycle or cell migration. In conclusion, we have shown that GCNIS cells are heterogeneous and their phenotype can determine their oncogenic potential. We also show that Sertoli cells from GCNIS-containing tubules undergo partial differentiation displaying markers of immature and mature Sertoli cells, with a heterogeneous association of cytokeratin with GCNIS presence. We also demonstrate that the oncogene Gankyrin has a role in NT2 cells survival and cisplatin resistance indicating that manipulation of Gankyrin may have a role in the treatment of TGCC.

A live birth from a somatic cell nuclear transfer (SCNT) embryo represents a small percentage of donor cells that survived the reprogramming gauntlet. The inability to reprogram histone modifications in the donor cell line could add to the reprogramming deficiencies associated with SCNT. The effects of two histone modifications associated with transcriptional activation (H3K4m3 and H4K16ac) and two histone modifications associated with repressing transcription (H3K9m2 and H3K27me3) were evaluated in the context of their association to three genes known to contribute to maintaining totipotency: Nanog, Oct4, and Sox2. A µChIP assay was utilized using antibodies specific for each histone modification followed by real time PCR (qPCR) analysis to quantify the percentage of each gene associated with each particular histone modification. Gene expression analysis was followed by immunofluorescence and protein analysis. Results of these analyses suggest that gene association to certain histone modifications did not accurately predict gene expression in bovine blastocyst embryos. Of the three genes studied, only Oct4 expression differed significantly between in vitro fertilized (IVF; control) and SCNT blastocysts. Protein levels detected through immunofluorescence correlated directly with the gene expression analysis. Nanog and Sox2 expression profiles of IVF and SCNT bovine blastocysts are similar, yet the histone modification profiles associated with all three genes differ significantly. Altered expression levels in developmentally important genes will likely result in abnormal activity of the associated cellular pathway. Aberrant histone modifications, along with abnormal Oct4 expression, may contribute to the low percentage of SCNT embryos that result in live offspring.

Matrin3 (MATR3) is an RNA binding protein involved in many roles in the nucleus, such as chromatin architecture and gene expression regulation, modulating transcriptional and post-transcriptional processes as RNA splicing and mRNA stabilization. Nevertheless, some functions of MATR3 within the cells are not entirely clear. MATR3 has been associated with Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that damages motor neuron (MN) cells and leads to progressive muscle paralysis and respiratory failure. A better understanding of MATR3 activity within cell physiology could represent an essential breakthrough for studying MATR3-associated pathologies. Using MATR3-silenced human pluripotent stem cell (hiPSC) line model, we collected data on the MATR3 role in the pluripotency and in the neural induction and differentiation. We found that the downregulation of MATR3 alters the expression level of crucial self-renewal factors such as OCT4, NANOG, KLF4, and LIN28A. We observed MATR3 acts at multiple levels of the gene expression, i.e. regulating YTHDF1 expression, and in RNA metabolism, having a role in mRNA stabilization and translation. The reduction of stemness potential caused by MATR3 downregulation creates a defect during the neurodifferentiation process, which does not arrest motor neurons formation but induces selective alterations that may affect motor neurons functionality. Indeed, several morphological and molecular abnormalities were observed during the neuronal differentiation, such as the alterations of the formation of neuroepithelial rosettes that arise in a reduction of neurite lengths and arborization in neuronal cells. On this basis, we investigated neuronal differentiation in the brain organoids grown from iPSCs derived from ALS patients fibroblasts. We show, for the first time, that MATR3 is a critical factor in orchestrating the stemness network through transcriptional, post-transcriptional, and translational regulation, therefore affecting the differentiation of mature neurons.

<p>Immunodeficient-causing diseases such as HIV and leukemia have no cures, often require meticulous treatments and result in high morbidity or mortality. Although bone marrow transplants are an option for a subset of leukemia patients, the shortage of donors and the requirement for donor matching restricts the efficacy of this treatment option. Therefore there is a prominent clinical need for alternative sources of hematopoietic stem/progentior cells with lymphopoietic potential. Recently we described the direct conversion of human dermal fibroblasts to multilineage hematopoietic progenitors by ectopic expression of OCT4. This direct conversion method was used to assess whether OCT4-transduced fibroblasts had the capacity to derive cells of the lymphoid lineage. This work shows the transient co-expression of CD34 and CD45 of fibroblasts within 7 days of OCT4 transduction followed by stable expression of CD45 on fibroblasts by day 15. The acquisition of hematopoietic markers, however, did not coincide with colony formation as previously described. Furthermore, CD45+ cells that were enriched and cultured in hematopoietic conducive conditions did not acquire co-expression of CD34 as previously shown. Interestingly, CD34 expression was shown to be inversely correlated with OCT4 expression. Therefore the constitutive expression of OCT4 may have (1) inhibited the acquisition of CD34 expression on CD45+ cells (2) downregulated the expression of CD34 on the day 7 CD34+CD45+ fibroblasts, thereby resulting in the transient expression of these markers. Furthermore, this work shows that expression of CD45 on OCT4-transduced fibroblasts is required for survival on the MS5 stromal cell line used to support hematopoietic progenitors with lymphopoietic potential, while supplementation of CD45+ fibroblasts with hematopoietic progenitor supportive conditions resulting in co-expression of CD34 and CD45 is required for acquisition of CD19, a pan-B cell marker on CD45+ fibroblasts. These findings suggest OCT4-transduced fibroblasts have lymphopoietic potential.</p> / Master of Science (MSc)

Experimentos anteriores realizados em nosso laboratório indicam que o sucesso gestacional é afetado pela poluição atmosférica. O presente estudo teve como objetivo avaliar os efeitos biológicos associados a uma curva dose resposta das partículas de exaustão do diesel (PED) sobre o desenvolvimento embrionário inicial e o potencial de implantação, utilizando-se como modelo a fertilização in vitro e o cultivo embrionário de camundongos. No Experimento 1, encontrou-se um efeito negativo dose-dependente sobre o desenvolvimento embrionário inicial, o processo de eclosão, a alocação das células e a morfologia da massa celular interna (MCI) dos blastocistos. A análise post-hoc revelou que o desenvolvimento precoce do embrião não foi afetado pelas concentrações de 0,2 µg/cm2 ou 2 µg/cm2, mas foi significativamente afetado pela concentração de 20 µg/cm2 de PED. O processo de eclosão foi prejudicado pelas concentrações de 2 µg/cm2 e 20 µg/cm2. A alocação das células da MCI e a relação entre as células da MCI e do trofectoderma foram significativamente afetadas por todas as concentrações. Adicionalmente, observou-se um efeito negativo sobre a morfologia da MCI para as concentrações de 2 µg/cm2 e 20 µg/cm2. O Experimento 2, apesar de não mostrar efeito significativo sobre o potencial de implantação, evidenciado pela capacidade de adesão dos blastocistos e crescimento trofoblástico, revelou que a morfologia da MCI no dia 8 de cultivo, as taxas de viabilidade e de apoptose celular e a expressão de Oct4 e Cdx2 foram significativamente afetadas. O teste HSD-Tukey demonstrou que a presença de PED (0,2 µg/cm2 e 2 µg/cm2) durante o desenvolvimento embrionário aumentou significativamente a taxa de células em apoptose dos embriões tanto no dia 5 quanto no dia 8 de cultivo e, embora a proporção de células viáveis no dia 8 tenha sido prejudicada por ambas as concentrações, apenas a exposição a 2 µg/cm2 de PED diminuiu a viabilidade celular no dia 5. Por outro lado, tanto a concentração de 0,2 µg/cm2 como a de 2 µg/cm2 tiveram um efeito negativo significativo sobre a qualidade da MCI no dia 8 e a taxa de expressão de Oct4 nos blastocistos e aumentaram a porcentagem de células desses blastocistos expressando Cdx2, adicionalmente, a razão Oct4/Cdx2 dos embriões expostos a 0,2 µg/cm2 e 2 µg/cm2 foi significativamente menor. Frente a esses resultados, presumi-se que as PED poderiam estar envolvidas nos mecanismos que levariam à diminuição do sucesso reprodutivo observado em camundongos expostos à poluição atmosférica ambiental / Previous experiments conducted in our laboratory demonstrate that successful pregnancy is affected by air pollution. The aim of this study was to evaluate the biological effects associated with a dose-response curve of the diesel exhaust particles (DEP) on early embryonic development and implantation potential, using mice in vitro fertilization and culture embryo as model. In Experiment 1, we found a negative dose-dependent effect on the embryonic development, hatching process, cell allocation and morphology of inner cell mass (ICM) of blastocysts. A post-hoc analysis revealed that the early development of the embryo was not affected by concentrations of 0.2 g/cm2 or 2g/cm2, but was significantly affected by the concentration of 20 g/cm2 of DEP. The hatching process was impaired by concentrations of 2 g/cm2 and 20 g/cm2. Cell allocation of ICM and the ratio between cells of ICM and trophectoderm were significantly affected by all concentrations. Addicionaly, we observed a negative effect on ICM morphology was observed for the 2 µg/cm2 and the 20 µg/cm2 concentrations. Experiment 2, despite showing no significant effect on implantation potential, as evidenced by the adhesion ability and trophoblast outgrowth, revealed that ICM morphology on day 8 of culture, rates of cell viability and apoptosis, and expression of Oct4 and Cdx2 were significantly affected. The Tukey HSD test showed that presence of DEP (0.2 g/cm2 and 2 g/cm2) during embryonic development increased significantly the rate of apoptotic cells in embryos as on day 5 as on day 8 of culture, although the proportion of viable cells on day 8 was impaired by both concentrations, only exposure to 2 g/cm2 PED decreased cell viability on day 5. On the other hand, both the concentration of 0.2 g/cm2 such as 2 g/cm2 had a significant negative effect on the quality of ICM on the day 8 and the rate of expression of Oct4 on blastocysts, and increased the percentage of cells from these embryos expressing Cdx2, also, Oct4/Cdx2 ratio were significantly lower in the blastocysts derived from embryos exposed to 0.2 g/cm2 and 2 g/cm2¬ concentrations. Given these results, the suggestion is that DEP could be involved in the mechanisms that lead to decreased reproductive success observed in mice exposed to environmental pollution

Experimentos anteriores realizados em nosso laboratório indicam que o sucesso gestacional é afetado pela poluição atmosférica. O presente estudo teve como objetivo avaliar os efeitos biológicos associados a uma curva dose resposta das partículas de exaustão do diesel (PED) sobre o desenvolvimento embrionário inicial e o potencial de implantação, utilizando-se como modelo a fertilização in vitro e o cultivo embrionário de camundongos. No Experimento 1, encontrou-se um efeito negativo dose-dependente sobre o desenvolvimento embrionário inicial, o processo de eclosão, a alocação das células e a morfologia da massa celular interna (MCI) dos blastocistos. A análise post-hoc revelou que o desenvolvimento precoce do embrião não foi afetado pelas concentrações de 0,2 µg/cm2 ou 2 µg/cm2, mas foi significativamente afetado pela concentração de 20 µg/cm2 de PED. O processo de eclosão foi prejudicado pelas concentrações de 2 µg/cm2 e 20 µg/cm2. A alocação das células da MCI e a relação entre as células da MCI e do trofectoderma foram significativamente afetadas por todas as concentrações. Adicionalmente, observou-se um efeito negativo sobre a morfologia da MCI para as concentrações de 2 µg/cm2 e 20 µg/cm2. O Experimento 2, apesar de não mostrar efeito significativo sobre o potencial de implantação, evidenciado pela capacidade de adesão dos blastocistos e crescimento trofoblástico, revelou que a morfologia da MCI no dia 8 de cultivo, as taxas de viabilidade e de apoptose celular e a expressão de Oct4 e Cdx2 foram significativamente afetadas. O teste HSD-Tukey demonstrou que a presença de PED (0,2 µg/cm2 e 2 µg/cm2) durante o desenvolvimento embrionário aumentou significativamente a taxa de células em apoptose dos embriões tanto no dia 5 quanto no dia 8 de cultivo e, embora a proporção de células viáveis no dia 8 tenha sido prejudicada por ambas as concentrações, apenas a exposição a 2 µg/cm2 de PED diminuiu a viabilidade celular no dia 5. Por outro lado, tanto a concentração de 0,2 µg/cm2 como a de 2 µg/cm2 tiveram um efeito negativo significativo sobre a qualidade da MCI no dia 8 e a taxa de expressão de Oct4 nos blastocistos e aumentaram a porcentagem de células desses blastocistos expressando Cdx2, adicionalmente, a razão Oct4/Cdx2 dos embriões expostos a 0,2 µg/cm2 e 2 µg/cm2 foi significativamente menor. Frente a esses resultados, presumi-se que as PED poderiam estar envolvidas nos mecanismos que levariam à diminuição do sucesso reprodutivo observado em camundongos expostos à poluição atmosférica ambiental / Previous experiments conducted in our laboratory demonstrate that successful pregnancy is affected by air pollution. The aim of this study was to evaluate the biological effects associated with a dose-response curve of the diesel exhaust particles (DEP) on early embryonic development and implantation potential, using mice in vitro fertilization and culture embryo as model. In Experiment 1, we found a negative dose-dependent effect on the embryonic development, hatching process, cell allocation and morphology of inner cell mass (ICM) of blastocysts. A post-hoc analysis revealed that the early development of the embryo was not affected by concentrations of 0.2 g/cm2 or 2g/cm2, but was significantly affected by the concentration of 20 g/cm2 of DEP. The hatching process was impaired by concentrations of 2 g/cm2 and 20 g/cm2. Cell allocation of ICM and the ratio between cells of ICM and trophectoderm were significantly affected by all concentrations. Addicionaly, we observed a negative effect on ICM morphology was observed for the 2 µg/cm2 and the 20 µg/cm2 concentrations. Experiment 2, despite showing no significant effect on implantation potential, as evidenced by the adhesion ability and trophoblast outgrowth, revealed that ICM morphology on day 8 of culture, rates of cell viability and apoptosis, and expression of Oct4 and Cdx2 were significantly affected. The Tukey HSD test showed that presence of DEP (0.2 g/cm2 and 2 g/cm2) during embryonic development increased significantly the rate of apoptotic cells in embryos as on day 5 as on day 8 of culture, although the proportion of viable cells on day 8 was impaired by both concentrations, only exposure to 2 g/cm2 PED decreased cell viability on day 5. On the other hand, both the concentration of 0.2 g/cm2 such as 2 g/cm2 had a significant negative effect on the quality of ICM on the day 8 and the rate of expression of Oct4 on blastocysts, and increased the percentage of cells from these embryos expressing Cdx2, also, Oct4/Cdx2 ratio were significantly lower in the blastocysts derived from embryos exposed to 0.2 g/cm2 and 2 g/cm2¬ concentrations. Given these results, the suggestion is that DEP could be involved in the mechanisms that lead to decreased reproductive success observed in mice exposed to environmental pollution

Primitive extraembryonale Endoderm (pXEN) Stam-Zelllinien der Ratte repraesentieren wahrscheinlich die festgelegten Vorläufer des extraembryonalen. Die im mesenchymalen Zustand gehaltenen pXEN-Zellen können in vitro weiter zu parietalen und viszeralen Endoderm-ähnlichen Zellen differenzieren. pXEN-Zellen zusätzlich halten moderate Konzentrationen des ICM-Markers Oct4 aufrecht. Die Bedeutung von Oct4 in pXEN-Zellen ist jedoch unbekannt. Bei höheren Zelldichten, beobachteten wir eine erhöhte Oct4-Expression und gleichzeitig eine Tendenz zu Epithelialisierung (MET) und viszeral endodermaler (VE) Differenzierung. Um zu klären, ob die Oct4-Expression kausal beteiligt ist, modulierten wir die Oct4-Konzentration. Transienter Knockdown von Oct4 reduzierte tendenziell die Expression von MET / VE-assoziierten Genen; umgekehrt förderte die Doxycycline-induzierte Expression eines menschlichen Oct4-Transgens die MET / VE-Differenzierung und verhinderte die Bildung charakteristischer Gang-Strukturen. Im letzteren Fall ging dem MET eine anfängliche Zell-Verlängerung und eine erhöhte Zellmotilität voraus. Da ein GSK3-Inhibitor und Activin A auch den MET / VE-Phänotyp stimulierten, fragten wir uns, ob Oct4 über die Wnt/β-Catenin oder TGFβ Signalwege wirkt. Die verschiedene Schritte der Wnt/β-Catenin Signalgebung hemmen, blockierten die hOct4-induzierte MET- und VE-Expression nicht. Im Gegensatz dazu verhinderte Repsox, ein Inhibitor von Alk5 (TGFBR1), das hOct4-induzierte MET und die Expression von MET- und VE-Genen und stimulierte eher die Expression von parietalen Endoderm (PE) Genen. Zusammengefasst zeigen diese Daten eine Rolle für Oct4 bei der MET / VE-Differenzierung auf, wahrscheinlich durch Stimulation eines TGFβ Signalweges. Weiterführende Experimente sind erforderlich um zu bestimmen, wie die zwei Prozesse der MET- und VE-Differenzierung innerhalb der extraembryonalen Endoderm-Linie unterschieden und in Beziehung gesetzt werden. / Rat primitive extraembryonic endoderm (pXEN) cell lines appear to represent the committed precursors of the extraembryonic endoderm. The pXEN cells maintained in the mesenchymal state can further differentiate to the parietal endoderm and visceral endoderm like-cells in vitro. In addition, pXEN cells maintain moderate levels of the ICM marker Oct4, a transcription factor that plays important roles in pluripotency, plasticity, and differentiation. However, the significance of Oct4 in pXEN cell lineage specification is unknown. We observed that rat pXEN cells show increased Oct4 expression at higher densities, a condition that also promotes their epithelialization (MET) and visceral endodermal (VE) differentiation. In order to elucidate whether the Oct4 expression is causally involved, we modulated the Oct4 levels. Transient knockdown of Oct4 tended to reduce the expression of MET/VE-associated genes; conversely, the doxycycline-induced expression of a human Oct4 transgene promoted MET/VE differentiation and prevented the formation of characteristic duct structures. In the latter case, the MET was preceded by an initial elongation and increased cell motility. Since GSK3 inhibitor and Activin A also stimulated the MET/VE phenotype, we then asked whether Oct4 acts through the Wnt/β-catenin or TGFβ pathways. Wnt inhibitors did not block the hOct4-induced MET and VE expression. By contrast, Repsox, an inhibitor of Alk5 (TGFBR1), prevented the hOct4-induced MET and the expression of MET and VE genes and rather stimulated the expression of parietal endoderm (PE) genes. Taken together, these data indicate a role for Oct4 in MET/VE differentiation via stimulation of TGFβ signaling. Further work is needed to determine how the two MET and VE differentiation processes are distinguished and related within the extraembryonic endoderm lineage.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-25T20:42:57Z No. of bitstreams: 1 JamileMarinhoBezerraDeOliveiraMoura_TESE.pdf: 18433655 bytes, checksum: e3b33cd1bc7a5e8e3f1a6cdd83ebdcbd (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-26T20:02:15Z (GMT) No. of bitstreams: 1 JamileMarinhoBezerraDeOliveiraMoura_TESE.pdf: 18433655 bytes, checksum: e3b33cd1bc7a5e8e3f1a6cdd83ebdcbd (MD5) / Made available in DSpace on 2016-08-26T20:02:15Z (GMT). No. of bitstreams: 1 JamileMarinhoBezerraDeOliveiraMoura_TESE.pdf: 18433655 bytes, checksum: e3b33cd1bc7a5e8e3f1a6cdd83ebdcbd (MD5) Previous issue date: 2016-02-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / As neoplasias de gl?ndulas salivares exibem uma ampla variedade de comportamento biol?gico e grande diversidade morfol?gica, e esta heterogeneidade inerente a este grupo de tumores suscita o interesse em pesquisar estas les?es. As c?lulas-tronco s?o a principal fonte para a gera??o e manuten??o da diversidade celular e homeostase do tecido, dist?rbios na regula??o destas c?lulas podem levar ? produ??o de c?lulas-tronco alteradas, denominadas de c?lulas-tronco tumorais, que possuem potencial proliferativo e capazes de originar e/ou manter o tumor. Pesquisas acerca das c?lulas-tronco tumorais e das prote?nas a elas associadas em algumas neoplasias orais t?m sido desenvolvidas, no entanto, o papel destas em neoplasias de gl?ndulas salivares n?o est? ainda bem estabelecido. Desta forma, o objetivo deste estudo foi identificar c?lulas do par?nquima tumoral que expressam marcadores de c?lulas-tronco tumorais, atrav?s da avalia??o da imunoexpress?o do OCT4 e CD44, em uma s?rie de casos de neoplasias de gl?ndulas salivares. A amostra foi constitu?da por 20 adenomas pleom?rficos, 20 carcinomas mucoepiderm?ides e 20 carcinomas aden?ides c?sticos localizados nas gl?ndulas salivares menores e maiores. Todos os casos estudados exibiram express?o positiva para OCT4 e CD44, sendo observado que para ambos marcadores, as neoplasias localizadas nas gl?ndulas salivares maiores exibiram maior imunomarca??o quando comparada com as les?es das gl?ndulas salivares menores apresentando diferen?a estatisticamente significativa (p=<0,001). Na amostra total e no grupo das gl?ndulas salivares menores, as neoplasias malignas exibiram maior imunorreatividade para OCT4 do que o adenoma pleom?rfico. No entanto, n?o foi encontrada diferen?as estatisticamente significativas de imunoexpress?es entre as les?es e entre suas classifica??es/grada??es histomorfol?gicas. Analisando a correla??o entre as imunoexpress?es de OCT4 e CD44 foi observada uma correla??o positiva moderada (r=0,444) com signific?ncia estat?stica entre os mesmos. A elevada express?o de OCT4 e CD44 pode indicar que estas prote?nas desempenham papel importante na identifica??o de c?lulas-tronco tumorais, permitindo uma previs?o do comportamento biol?gico das neoplasias de gl?ndula salivar, apresentando n?veis menores em tumores benignos e maiores nos tumores malignos. / Salivary gland neoplasms exhibit a wide variety of biological behavior and a high morphological diversity raises the interest in researching these lesions. The stem cells are the main source for the generation and maintenance of cell diversity, disorders in the regulation of these cells can lead to the production of altered stem cells, termed cancer stem cells capable of generate the tumor. Researches on cancer stem cells and associated proteins have been developed in some oral cancers; however, their role in salivary gland neoplasms is not well established. Thus, the aim of this study was to identify the tumor parenchyma cells exhibiting stem cell characteristics, by evaluating the immunoreactivity of OCT4 and CD44, in a number of cases of salivary gland neoplasms. The sample consisted of 20 pleomorphic adenomas, 20 mucoepidermoid carcinomas and 20 adenoid cystic carcinoma located in minor and major salivary glands. The expression of OCT4 and CD44 was evaluated by the percentage of positive cells (PP) and the intensity of expression (IE), it is realized the sum of the scores, resulting in the total score immunostaining (PIT) ranging 0-7. All studied cases showed positive expression of OCT4 and CD44 and higher values than the control groups. It was observed that for OCT4 luminal cells and non-luminal were immunostained in the case of pleomorphic adenomas and adenoid cystic carcinoma. Already the immunoreactivity of CD44 was particularly evident in the non-luminal cells of these lesions. In mucoepidermoid carcinomas for both markers, there was immunoreactivity in squamous and intermediate cells and absence of staining mucous cells. For both markers, a statistically significant higher immunostaining was verified in neoplasms located in the major salivary glands compared with lesions in the minor salivary (p<0.001). At the total sample and in the group of minor salivary glands, malignant neoplasms exhibited higher immunoreactivity for OCT4 than pleomorphic adenoma. However, there was no statistically significant difference between the lesions and between their classifications histomorphologic. Analyzing the correlation between OCT4 and CD44 immunoexpressions, a statistically significant moderate positive correlation (r = 0.444) was observed. The high expression of OCT4 and CD44 may indicate that these proteins play an important role in identifying cancer stem cells, allowing a prediction of biological behavior of salivary gland neoplasms.