Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The insulin-like growth factor (IGF) system plays an important role in a number of disease states, such as cancer, and has also been implicated in wound and burn healing processes. Two IGF receptors, the type-1 IGF and type-2 IGF receptors, as well as six insulin-like growth factor binding proteins (IGFBP-1 to 6), have well established roles in mediating IGF activity. Earlier studies in this laboratory demonstrated that IGF-II binds to the extracellular matrix (ECM) protein vitronectin (VN), and although IGF-I does not bind directly to VN it can bind indirectly via specific IGFBPs. Therefore the aim of the research described in this thesis was to determine whether binary and ternary complexes of IGF-I/II, IGFBPs and VN affect human keratinocyte cell function. The strategy of pre-binding these complexes to the culture dishes was adopted in this study in an attempt to more accurately reflect the extracellular environment in vivo. These studies demonstrated that the binary complex of IGF-II and VN and the ternary complexes comprised of IGF-I, IGFBP-2, or 3, or 4, or 5 and VN significantly stimulated HaCaT de novo cell protein synthesis in the human keratinocyte cell line. Interestingly, these latter experiments demonstrated that although large increases in protein synthesis were observed using the ternary complexes, IGF-I/IGFBP complexes alone were responsible for the significant increases in protein synthesis and these responses are mediated via the MAPK signaling pathway. In addition, both the dimeric and trimeric complexes significantly enhanced cell migration through 12 μm TranswellsTM. Unlike the protein synthesis assays, VN was critically important in these migratory responses and highlighting the important role that integrins play in cell migration. Cell attachment assays on the other hand demonstrated that the interactions of IGFs with IGFBPs and VN did not affect cell attachment. The data encompassed within this thesis represent the first studies to provide a functional role for the interaction between IGFs, IGFBPs and VN in human keratinocytes. Taken together these results suggest that IGF/IGFBP/VN complexes may hold great potential in situations where enhanced keratinocyte cell migration and proliferation is required, such as in wound healing and skin engineering applications.

[Truncated abstract] Melanoma cell adhesion molecule (MCAM) is highly expressed in more than 70% of metastatic melanoma and is correlated with invasive potential. However, the specific contribution MCAM makes to invasion and metastasis in melanoma is not clear. In this study, I have demonstrated that transfection of MCAM into MCAM-negative melanoma and CHO cells leads to changes in cell shape, and the modulation of cell-to-cell and cell-matrix interactions. MCAM positive cells were slower to spread on collagen type I, collagen type IV and laminin 1 than MCAM negative cells, although these differences were not apparent on vitronectin, fibronectin and laminin 10. In contrast, MCAM expression had little effect on cell adhesion to any of the matrices tested. MCAM positive (compared to negative) cells also showed morphological changes and a rearrangement of the actin cytoskeleton when plated on a matrix containing laminin 5. Taken together, these data suggest that MCAM expression modulates β1-integrinmediated spreading on matrix, but has little effect on αvβ3-mediated cell-matrix interactions. As this study provided little evidence to suggest that MCAM transfection altered β1 integrin expression levels on melanoma cells, it is proposed that a competitive interaction between the cytoplasmic domains of MCAM and β1 integrin may affect mature focal adhesion assembly. MCAM expression in melanoma cells was also associated with decreased cell movement over matrix into a scratch-wound site and an increased tendency to form cell cords on Matrigel. These two assays gauge the propensity of a cell to engage in cell-cell versus cell-matrix interactions, and suggest that MCAM positive cells favour cell-cell adhesion. Interestingly, MCAM transfection was also associated with an increased ability of melanoma cells to migrate through a basement membrane towards a chemoattractant. ... Analysis of the intracellular domain of MCAM revealed the presence of tyrosine and dileucine endocytosis signals. Interestingly, disruption of these two motifs did not seem to impair the internalization of MCAM from the cell surface. The di-leucine motif, however, was necessary for the recycling of MCAM back to the surface following endocytosis. Lastly, MCAM was found to exists as dimers within the cell membrane in the absence of ligand, although the exact location of the dimerization motif is not yet clearly defined. Collectively, findings from my study suggest: MCAM expression in melanoma cells facilitates cell-cell interactions, whilst concomitantly modulating cell-matrix interactions. MCAM transfection also leads to enhanced migration of melanoma cells through a basement membrane. Thus, MCAM expression may increase the ability of melanoma cells to migrate as a collective, a feature of highly invasive cancer. The intracellular domain of MCAM interacts with ApxL2, a novel member of the Shroom family of actin-binding proteins. It is likely that ApxL2 links a proportion of MCAM within the cell to the actin cytoskeleton, contributing to cell shape determination and other processes, such as migration. MCAM exists as dimers on the cell surface and is internalized at least partially by a clathrin-mediated mechanism.

Thesis (PhD.(Physiology)--Faculty of Health Sciences)-University of Pretoria, 2008. / Summary in English and Afrikaans. Includes bibliographical references.

<p>Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development. </p><p> The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.</p><p> This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control. </p><p>In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.</p><p> In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.</p><p> The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.</p> / Dissertation

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics.

abstract: Thousands of high-resolution images are generated each day. Segmenting, classifying, and analyzing the contents of these images are the key steps in image understanding. This thesis focuses on image segmentation and classification and its applications in synthetic, texture, natural, biomedical, and industrial images. A robust level-set-based multi-region and texture image segmentation approach is proposed in this thesis to tackle most of the challenges in the existing multi-region segmentation methods, including computational complexity and sensitivity to initialization. Medical image analysis helps in understanding biological processes and disease pathologies. In this thesis, two cell evolution analysis schemes are proposed for cell cluster extraction in order to analyze cell migration, cell proliferation, and cell dispersion in different cancer cell images. The proposed schemes accurately segment both the cell cluster area and the individual cells inside and outside the cell cluster area. The method is currently used by different cell biology labs to study the behavior of cancer cells, which helps in drug discovery. Defects can cause failure to motherboards, processors, and semiconductor units. An automatic defect detection and classification methodology is very desirable in many industrial applications. This helps in producing consistent results, facilitating the processing, speeding up the processing time, and reducing the cost. In this thesis, three defect detection and classification schemes are proposed to automatically detect and classify different defects related to semiconductor unit images. The first proposed defect detection scheme is used to detect and classify the solder balls in the processor sockets as either defective (Non-Wet) or non-defective. The method produces a 96% classification rate and saves 89% of the time used by the operator. The second proposed defect detection scheme is used for detecting and measuring voids inside solder balls of different boards and products. The third proposed defect detection scheme is used to detect different defects in the die area of semiconductor unit images such as cracks, scratches, foreign materials, fingerprints, and stains. The three proposed defect detection schemes give high accuracy and are inexpensive to implement compared to the existing high cost state-of-the-art machines. / Dissertation/Thesis / Ph.D. Electrical Engineering 2010

Le peptide antimicrobien LL-37 a été retrouvé surexprimé dans différents types de cancer et plus particulièrement dans le cancer du sein dans lequel il est associé au développement des métastases. Nous avons observé, in vitro, que la migration de trois lignées cancéreuses mammaires est augmentée par le peptide LL-37 et son énantiomère (D)-LL-37, excluant la fixation du peptide à un récepteur protéique. Sur les cellules cancéreuses mammaires MDA-MB-435s, le peptide se fixe à la membrane plasmique et diminue sa fluidité. La microscopie électronique localise LL-37 dans les cavéoles et à la surface de structures impliquées dans la migration cellulaire, les pseudopodes. LL-37 induit une entrée de calcium via le canal TRPV2 dont l’activité est augmentée par son recrutement dans les pseudopodes. Ce recrutement est dépendant de l’activation de la voie de signalisation PI3K/AKT induite par LL-37. L’entrée de calcium via TRPV2 est potentialisée par l’activation du canal potassique BKCa, localisé aussi dans les pseudopodes. Des ARN interférents contre TRPV2 inhibent à 70% la migration induite par LL-37, donnant un rôle prépondérant à ce canal dans les effets pro-migratoire du peptide. La fixation du peptide LL-37 aux membranes des cellules cancéreuses et l’activation de canaux ioniques constituent un nouvel axe de recherche pour comprendre le rôle du peptide dans la progression tumorale. / The antimicrobial peptide LL-37 is overexpressed in several types of cancer, among which breast cancer were it is associated with metastasis development. Our experiments on three mammary cancer cell lines have shown that LL-37 increases cell migration. Both its natural (L)-form and its (D)-enantiomer are equally active, excluding a specific binding to a protein receptor. On the MDA-MB-435s cell line, LL-37 attaches to plasma membrane and reduces its fluidity. Electron microscopy localized LL-37 on the surface of pseudopodia, structures implicated in cell migration, and in caveolae. LL-37 induces calcium entry via the TRPV2 channel, which is recruited to pseudopodia. Recruitment depends on activation of PI3K/AKT signaling induced by LL-37. Calcium entry via TRPV2 is potentiated by activation of the BKCa potassium channel also located in pseudopodia. TRPV2 suppression by RNA interference results in 70% reduction of cell migration induced by LL-37, attributing a crucial role of this channel to the promigratory effects of the peptide. Binding of LL-37 to cancer cell membranes and in consequence the activation of ion channels constitutes a novel research field to understand its role in tumor progression.

Astrocytoma is defined as the neoplasia of astrocytes, the most abundant non-neuronal glial cells in brain. According to the WHO classification, the different grades of astrocytoma are- gradeI/pilocytic astrocytoma (benign form), grade II/diffuse astrocytoma (DA), grade III/anaplastic astrocytoma (AA) and grade IV/Glioblastoma (GBM). Patients with grade II astrocytoma have median survival time of 6-8 years after surgical intervention. While the more aggressive grade III has a median survival of 2-3 years. Grade IV is the most malignant form and has a median survival of 15 months approximately. In spite of all the progress in the fields of diagnosis and therapy, the prognosis of GBM still remains very poor. The aggressiveness and poor survival of GBM is due to the recurrence which is primarily because of intratumoral heterogeneity, presence of glioma stem cells and infiltration of the tumor cells into the normal brain parenchyma. Apart from the role of genetic mechanisms in triggering tumorigenesis, epigenetic modifications particularly the DNA methylation and histone modifications, are now recognized as frequent alterations playing a crucial role in the development and progression of human malignancies. There are two distinct DNA methylation abnormalities. The first is the reduction in genome-wide DNA methylation levels (global hypomethylation) and the second is the hypermethylation in the CpG island of specific gene promoters. Hypomethylation is believed to induce proto-oncogene activation and chromosomal instability, whereas hypermethylation is strongly associated with silencing of tumor suppressor genes. Thus, DNA methylation can function as a “switch” to activate or repress gene transcription, providing an essential mechanism for overexpressed or silenced genes involved in the regulation of cell cycle, DNA repair, growth signalling, angiogenesis, apoptosis, migration, invasion and thus in the initiation and progression of astrocytoma. Recent studies have identified biomarkers with prognostic impact which would include promoter methylation of O⁶-methyl guanine-DNA methyltransferase methylation (MGMT), isocitrate dehydrogenase 1(IDH1) mutation and a glioma CpG-island methylator (G-CIMP) phenotype. In the current study, we have characterized the DNA methylation profile for the different grades of astrocytoma and analysed the significance of methylation events occurring commonly in all the grades or uniquely only in grade IV. One of the GBM-specific hypomethylated and upregulated genes, Fibromodulin (FMOD), was extensively investigated in terms of its role in glioma pathogenesis and its regulation. FMOD was found to induce F-actin stress fibre formation and promote glioma cell migration. We also found that FMOD-mediated glioma cell migration is dependent on Integrin/FAK/Src/Small Rho GTPases signalling cascade. We further found that TGFβ pathway regulates FMOD expression through a process involving active demethylation and chromatin state transitions on FMOD promoter. This work has been divided into three parts: Part I: Characterization of DNA methylome during progression of Astrocytoma To investigate the aberrant methylation pattern on a genome-wide scale, 17 Grade II, 16 Grade III and 36 Grade IV tumor samples as well as 9 control brain tissues were analysed using Infinium Human Methylation 450K Bead Array on Illumina platform. The analysis was carried out in two parts. Firstly, we validated the dataset with already existing TCGA dataset. Upon comparison, the methylation profile of our dataset was highly correlated to the TCGA dataset with correlation coefficient of 0.99. In addition, we also checked the methylation status of few known hypermethylated and hypomethylated genes which showed the similar type of differential methylation. Then, we characterized the differentially methylated CpGs based on their spatial distribution in the human genome, for different grades of astrocytoma. CpG-rich regions show more of hypermethylation while the non-CpG rich regions, like open sea or gene body, are observed to be hypomethylated. Secondly, we also analysed the differentially methylated genes which contribute to physiological events in gliomagenesis. We hypothesized that the methylation specific events that occur in grade II and remain similarly methylated in grade IV are the ones probably contributing to the initial astrocyte transformation. However, the methylation specific events responsible for the aggressive nature of grade IV may occur as differentially methylated genes only in grade IV (and not in grade II). In this analysis, we have identified differentially methylated genes that play a role in initial transformation process (293 genes hypermethylated and downregulated while 23 genes were hypomethylated and upregulated) and also those that play a role in tumor aggressiveness (459 genes hypermethylated and downregulated while 350 genes were hypomethylated and upregulated). The differentially methylated genes that were common in both grade II and grade IV showed an enrichment of cell proliferation pathways while the differentially methylated genes uniquely present in grade IV showed enrichment in pathways related to the aggressiveness phenotype of tumorigenesis like cell motility and angiogenesis. Part II: Fibromodulin (FMOD), a GBM-specific hypomethylated and upregulated gene, is essential for glioma cell migration Among differentially methylated genes specifically in GBM, fibromodulin (FMOD) is one of the top most hypomethylated genes. FMOD is a member of leucine – rich repeat proteoglycan that is widely distributed in interstitial connective tissues. We found that FMOD is hypomethylated and upregulated only in grade IV/GBM, not in the grade II. FMOD promoter methylation status is significantly negatively correlated to its transcript levels.Towards identifying functions of FMOD in glioma cells, total RNA derived from U251 cells transfected with either non-targeting siRNA or FMOD siRNA was subjected to transcriptome profiling. There were 872 genes upregulated and 299 genes downregulated in FMOD silenced cells than in control cells. PANTHER pathway analysis using the differentially regulated genes identified several pathways to be associated with FMOD. Cytoskeleton regulation by Rho GTPase, which is known to be involved in cell motility and migration, is enriched with highest significance. In coherence with the pathway analysis, modulating FMOD levels in glioma cells affected in glioma cell migration. Upon FMOD overexpression, there was significant increase in migration than in control cells. Conversely, when FMOD is silenced, there was delay in migration than in control cells and the delayed migration was rescued by the addition of recombinant purified FMOD protein. Prior neutralization with FMOD specific antibody inhibited cell migration suggesting that secreted FMOD promotes glioma cell migration. Overexpression of FMOD in glioma cells induced actin stress fibre formation required for the migration of cells. On the contrary, FMOD silencing resulted in the loss of F-actin stress fibres which was restored upon addition of FMOD purified protein exogenously to the media. To investigate further the role and the requirement of specific Rho GTPase in FMOD-mediated migration, each of members of Rho GTPase family was silenced and their effect on FMOD-induced silencing was studied. FMOD mediated glioma cell migration was delayed when RhoA, Rac1 and Cdc42 were silenced. In order to understand whether FMOD activates Integrin mediated signalling pathway, we performed western blot analysis to check the levels of phospho-FAK in either FMOD overexpressing or knockdown condition. We observed phospho-FAK levels increased upon FMOD overexpression and decreased upon FMOD silencing compared to the respective controls. Additional experiments revealed that inhibitors to Integrin, FAK and Src were able to abrogate the FMOD induced glioma cell migration. These results suggest that FMOD utilizes a pathway that involves Integrins, FAK, Src and Rho GTPases in promoting glioma cell migration. To comprehend the effect of FMOD promoter methylation status and its expression in GBM patient scenario, we stratified the patients into either high or low FMOD expression and promoter hypermethylation or hypomethylation. The GBM patients with low FMOD transcript levels and promoter hypermethylation showed better survival than the other group. Part III: Regulation of FMOD expression through TGFβ-dependent epigenetic remodelling in glioma To study how FMOD is regulated in glioma, we investigated the promoter sequence of FMOD by MatInspector. Several Smad-binding sites were located in FMOD promoter which indicated that FMOD might be regulated via TGFβ signalling pathway. Firstly, we checked active TGFβ signalling in glioma cell lines – LN229, U87 and U251. TGFβ-dependent signalling was active in U251 and U87 cells compared to LN229 cells as seen by the levels of phospho-Smad2. Moreover, FMOD transcript level was found to be high in U251 compared to LN229 cells. Further, TGFβ treatment increased FMOD promoter luciferase activity as well as FMOD transcript level in LN229 cells. In contrast, U251 cells that were treated with TGFβ RI inhibitor showed a significant decrease in FMOD promoter luciferase activity as well as FMOD transcript level. We correlated these findings with Smad2 occupancy at FMOD promoter by chromatin immunoprecipitation (ChIP). Smad2 association at FMOD promoter is found to be relatively higher in U251 cells than in LN229 cells which suggested that TGFβ induced transcription factor, Smad2, drives FMOD expression in U251 cells. Next, we investigated the role of TGFβ in FMOD promoter demethylation and chromatin state transition. Upon TGFβ treatment in LN229 cells, we found that there was gradual demethylation of FMOD promoter in a time-dependent manner. TGFβ treatment also altered the chromatin state by increasing the active marks (H3K4me3 and H3K9Ac) and decreasing the repressive mark (H3K27me3) with a simultaneous increase in Smad2 occupancy in the FMOD promoter. In contrast, TGFβ RI inhibitor treatment of U251 cells resulted in methylation of FMOD promoter in a time-dependent manner. Further, we observed a significant enrichment of repressive histone marks (H3K27me3) and loss of active chromatin marks (H3K4me3 and H3K9Ac) with a concomitant decrease in Smad2 occupancy at FMOD promoter. DNMT3A/B and EZH2 enzymes play a key role in DNA methylation and H3K27 trimethylation respectively. Accordingly, we examined the transcript levels of DNMT3A/B and EZH2 in LN229 cells treated with TGFβ as well as U251 cells treated with TGFβ RI inhibitor. In presence of TGFβ, DNMT3A/B and EZH2 transcript levels were significantly downregulated than in untreated cells in a time-dependent manner. Conversely, in U251 cells treated with TGFβ RI inhibitor, there was a significant increase in DNMT3A/B and EZH2 transcript levels when compared to untreated cells. TGFβ is known to promote glioma cell migration. In order to understand whether TGFβ-mediated glioma cell migration occurs via FMOD, we performed migration assay in U251 cells with or without TGFβ RI inhibitor followed by addition of either BSA control or FMOD purified protein. Upon TGFβ RI inhibitor treatment, there was delay in the migration of U251 cells than in untreated control cells which was rescued when purified FMOD protein was added, indicating that FMOD is essential for TGFβ signalling cascade to induce glioma cell migration. Therefore, we conclude from these results that epigenetically regulated FMOD is essential for TGFβ mediated glioma cell migration.

O carcinoma espinocelular (CEC) de cabeça e pescoço é uma neoplasia maligna que implica em baixas taxas de sobrevida e prognóstico desfavorável. A perda de adesão do epitélio e a migração celular são processos biológicos envolvidos na carcinogênese. O objetivo desse estudo foi descrever o padrão de expressão da molécula cinase de adesão focal (FAK) e suas formas fosforiladas, FAK-Tyr576 e FAK-Tyr925, em amostras de CEC no centro do tumor (CT) e zona de invasão (ZI) e no tecido epitelial não neoplásico adjacente a estes tumores (EA). De acordo com a graduação histopatológica, 48,1% do total das amostras de CEC são de grau moderado. Segundo o sistema pTNM, 62,9% eram T1 e T2, 64,8% não apresentaram metástase regional e não houve metástase à distância. O tempo máximo de acompanhamento foi de 5,9 anos e obteve-se a proservação final de 46 dos 54 pacientes, onde foi observado que 58,1% apresentaram boa evolução e 41,9% exibiram evolução ruim. A média em anos para que os pacientes obtivessem o status de evolução ruim foi de 5,01. Quanto ao estadiamento clínico, houve maior imunomarcação no estádio IVa quando comparado ao estádio II no EA da FAK Tyr-576 e ocorreu maior expressão no estádio IVb quando comparado ao estádio I no CT da FAK Tyr-576. Os pacientes que apresentaram evolução ruim após o período máximo de acompanhamento tiveram alta expressão no EA e na ZI da FAK Tyr-576 e no CT da FAK Tyr-925. Na ZI, o aumento de expressão da FAK está associado ao aumento da expressão da FAK Tyr-576. À medida que há aumento da expressão no EA há aumento da expressão na ZI na FAK Tyr-576. E quando há aumento da expressão no EA há aumento da marcação na ZI na FAK Tyr-925. Apesar do papel de todas as FAKs fosforiladas não estar claro, mas sabendo-se que são essas FAKs que estão ativas no câncer, a partir dos dados do presente trabalho pode-se sugerir que a avaliação da marcação FAK Tyr -576 no EA, no CT e na ZI venha a fazer parte da rotina no diagnóstico histopatológico e das margens da peça operatória, como norteadores da conduta terapêutica e do protocolo de acompanhamento. / Squamous cell carcinoma (SCC) of the head and neck is a malignant neoplasm that implies low rates of survival and poor prognosis. The loss of adhesion and cell migration are biological processes involved in carcinogenesis. The aim of this study was to describe the pattern of expression of focal adhesion kinase molecule (FAK) and its phosphorylated forms, FAK-Tyr576, FAK-Tyr925, in SCC samples in the center of the tumor (CT) and invasion zone (ZI) and non-neoplastic epithelial tissue adjacent to these tumors (EA). According to histopathological grading, 48,1% of SCC samples are moderate. According to the system pTNM, 62,9% were T1 and T2, 64.8%, had no regional metastasis and no distant metastasis. The maximum time of follow up was 5,9 years and was obtained as the final proservation 46 of 54 patients, where it was observed that 58,1% of patients had good prognosis and 41,9% of patients had poor prognosis. The average years for patients to obtain the poor prognosis of status was 5.01. As for clinical staging, there was greater immunostaining in the stadium IVa compared to stage II in EA Tyr-576. And FAK expression was higher in stage IVb when compared to CT in stage I of FAK Tyr-576. Patients with poor prognosis after the maximum follow-up period had high expression in EA and ZI of FAK Tyr-576 and CT of FAK Tyr-925. In ZI, increased expression of FAK is associated with increased FAK Tyr-576 expression. As there is increased expression in EA's increased expression in ZI in FAK Tyr-576. And when there is increased expression in EA's increased marking the ZI in FAK Tyr-925. Despite the role of all phosphorylated FAKs not clear, but given that these are FAKs that are active in cancer, from the present data it can be suggested that the evaluation of expression of FAK Tyr 576 in EA, CT and ZI will be part of the routine histopathological diagnosis and surgical specimen margins, as guiding the therapeutic management and monitoring protocol.