INTRODUÇÃO: O carcinoma espinocelular de origem bucal (CEC) apresenta uma alta taxa de mortalidade devido à invasividade das células tumorais. A migração celular, principal evento da invasão e metástase, pode ser regulada tanto por fatores intrínsecos, como adesão e contratilidade celular, quanto extrínsecos, como composição, densidade e remodelagem da matriz extracelular (MEC). OBJETIVO: Avaliar o papel de elementos intrínsecos e extrínsecos sobre o processo invasivo do carcinoma espinocelular de origem bucal. MÉTODOS: Foi realizada imuno-histoquímica para as proteínas: Miosina II (isoformas A, B e C), metaloproteinases de matriz (1, 2, 9 e 14); imunofluorescência as proteínas: e-caderina, n-caderina, FAK, paxilina, vinculina e fibronectina em amostras de CEC oral. Foi realizado ensaio de migração nas seguintes condições: 1 – matriz 2D com o substrato de fibronectina, ou laminina ou matrigel; 2 – matriz 3D com colágeno na presença ou não de fibronectina ou laminina; 3 – matriz 3D com diferentes concentrações de colágeno (0,6; 1,2 e 1,8 mg/ml) + fibronectina na presença ou não de um inibidor de MMP. Foi realizado análise de adesão celular utilizando-se o microscópio TIRF e o microscópio confocal, tanto em matrizes 2D quanto 3D. Foram realizados esferoides celulares para avaliar a contratilidade celular, através do plaqueamento das células em gel de agarose e a utilização de drogas que inibem ou que induzem a contratilidade, bem como a partir de células transfectadas com versões fosfomiméticas para a cadeia leve de miosina. Foi realizado ainda western blotting para proteínas: e-caderina, FAK, vinculina, paxilina, N-caderina, integrinas e as isoformas de miosina II, bem como foi avaliado os níveis de ativação das proteínas da família RhoGTPase, as quais estão envolvidas no controle da migração celular. RESULTADOS: A expressão das MMPs analisadas e das isoformas de miosinas foi maior nas zonas de invasão tumoral, sendo que o CEC oral também apresenta uma maior expressão de proteínas associadas à adesão com a MEC. A migração celular foi afetada pela densidade e a composição da MEC, bem como pela atividade das MMPs. Adicionalmente, a modulação das proteínas de adesão célula-matriz altera a velocidade de migração, a direcionalidade dessa migração e também a forma de migração, mudando de uma migração coletiva para uma migração individual. O aumento na contratilidade células resulta numa dispersão celular enquanto que a diminuição da contratilidade resulta numa melhor adesão célula – célula. CONCLUSÕES: O comportamento das células tumorais pode ser modulado através de fatores extrínsecos como, por exemplo, a alteração no microambiente tumoral, seja ela por mudança no substrato ou na densidade da matriz, e também dos fatores intrínsecos como a alteração nos níveis de miosina. / INTRODUCTION: Oral squamous cell carcinoma (OSCC) presents high mortality index due to the invasive phenotype of tumor cells. Cell migration is the main event in cell invasion and metastasis and it can be regulated by intrinsic factor, such as adhesion and cell contractility, and extrinsic factors, such as density and extracellular matrix (EMC) remodeling. OBJECTIVE: Analyze the role of intrinsic and extrinsic factor during the invasive process of oral squamous cell carcinoma. METHODS: We performed immunostaining in OSCC samples for the following proteins: myosin II (isoforms A, B and C), matrix metalloproteinase (1, 2, 9 and 14) e-cadherin, n-cadherin, FAK, paxillin, vinculin and fibronectin. We also performed migration assays with OSCC cell line in the following conditions 1 – 2D matrix with fibronectin or laminin or matrigel; 2 – 3D matrix with collagen in the presence or not of fibronectin or laminin; 3 – 3D matrix with different collagen concentration (0,6; 1,2 e 1,8 mg/ml) with fibronectin in the presence or not of the MMP inhibitor. In order to analyze cell adhesion, it was performed Total Internal Reflectance Fluorescence and Confocal microscopy, in 2D and 3D matrix. To analyze cell contractility, cells were plated in agarose gel in order to produce spheroids, which were treated with drugs that inhibit or induce cell contractility or cells were previously transfected with Myosin Light Chain phosphomimetics mutants. It was also performed western blotting to: e-cadherin, n-cadherin, FAK, paxillin, vinculin and myosin II isoforms, as well as it was analyze the levels in RhoGTPase family, which are involved in cell migration control. RESULTS: The expression to MMPs and myosin II isoforms were higher at invasion zone of the tumor, and the OSCC presented higher expression of proteins associated to adhesion to ECM. Cell migration was affected by the EMC composition and density and by MMP activity. Also, the modulation of cell-matrix adhesion proteins altered migration speed, cell directionality as well as influenced the switch between collective and single cell migration. The increase in cell contractility resulted in cell dispersion while the decrease in cell contractility resulted in a better cell-cell adhesion. CONCLUSIONS: The behavior of cell tumor can be modulate by extrinsic factors, for example, the change in tumor microenvironment, by the change in the EMC substrate or density and by intrinsic factors such as the alteration in myosin levels.

Vital cellular processes such as nutrient uptake, receptor signaling, and cell migration are controlled by a balance between cell surface receptor internalization and recycling. Clathrin-mediated endocytosis is the major mechanism of receptor internalization in which cargo-enriched endocytic vesicles form at, and are released from, the plasma membrane before maturing into early endosomes. The receptors can then be sorted into fast and slow recycling pathways that replenish receptor levels at the cell surface. A major fast recycling pathway is controlled by the small GTPase Rab4a, which plays a central role in cell migration and cancer cell invasion through regulation of integrin αvβ3 recycling. Recent studies have discovered a family of clathrin-coated vesicle proteins, known as adaptin-ear-binding coat-associated proteins (NECAPs), that consists of two family members, NECAP1 and NECAP2. NECAP1 functions in endocytosis and cooperates with the clathrin adaptor AP-2 to control endocytic vesicle size, number and cargo. Importantly, NECAP2 did not rescue the knock-down phenotype of NECAP1, revealing that NECAPs are not functionally redundant. The studies described in this dissertation show that NECAP2 controls the fast recycling of epidermal growth factor receptor and transferrin receptor. Furthermore, NECAP2 specifically functions in Rab4a-mediated fast recycling together with the clathrin adaptor AP-1. In contrast, NECAP2 has no effect on AP-1-mediated transport from the Golgi or on other Rab4a-dependent sorting events that utilize additional clathrin adaptors and effector proteins. Thus, NECAP2 regulates a sub-route within the Rab4a recycling pathway and, in fact, is the first protein known to date to show this level of specificity. NECAP2 knock-down revealed that this sub-route controls cell migration and cancer cell invasion. Specifically, NECAP2 knock-down impaired the recycling of integrin αvβ3 to the cell surface, leading to decreased Rac1 activation and integrin αvβ3-dependent persistent cell migration. NECAP2 depletion also alleviated the inhibitory effect on integrin α5β1 recycling, switching cells to integrin α5β1-dependent cell migration. Notably, loss of NECAP2 function in breast cancer cells inhibited invasive migration in a 3D invasion model system. Therefore, the NECAP2 pathway may provide a therapeutic target, in particular for the 25% of breast cancers with amplification of Rab4a.

The PI3K-Akt signalling pathway is a well-established driver of cancer progression. One key process promoted by Akt phosphorylation is tumour cell motility; however the mechanism of VEGF-induced Akt phosphorylation leading to motility remains poorly understood. Previously, it has been shown that Akt phosphorylation, induced by different factors, causes both stimulation and inhibition of motility in different cell types. However, differential phosphorylation of Akt at T308 and S473 residues by VEGF and its role in head and neck cancer cell motility and progression is unknown. The cell lines investigated in this study exhibited a change in phosphorylation of Akt in response to VEGF. However, in terms of motility, VEGF stimulated oral cancer and its associated cell lines, but not normal keratinocytes or oral mucosal fibroblasts. The addition of a PI3 kinase and mTOR inhibitor, inhibited the phosphorylation of Akt and also effectively blocked VEGF-induced oral cancer cell motility, whereas only the PI3 kinase inhibitor blocked oral cancer associated fibroblast cell motility. This study therefore discloses that two different mechanisms of Akt phosphorylation control the motility potential of different cell lines. Akt phosphorylated at both residues controls oral cancer cell motility. Tobacco, alcohol and HPV infection are associated with increased risk of HNSCC. However, little is known about the underlying signalling events influencing risk. It was also aimed to investigate the relationship between these risk factors and Akt phosphorylation, to determine prognostic value. VEGF-positive HNSCC biopsies, with known HPV status, were analysed by immunohistochemistry (IHC) for Akt, phosphorylated at residues S473 and T308. Comparisons between the tissues were carried out using a Mann-Whitney U test. Associations between the variables and continuous immunohistochemical parameters were evaluated with general linear models. Patient characteristics and pAkt IHC score were analysed for possible association with overall survival by Cox proportional hazard models. Immunohistochemistry revealed that cancer patients had significantly higher levels of pAkt T308 than S473 (P < 0.001). Smoking and alcohol were found to be independent risk factors for Akt phosphorylation at T308 (P = 0.022 and 0.027, respectively). Patients with tumours positive for HPV or pAkt S473 had a poorer prognosis (P = 0.005, and 0.004, respectively). Patients who were heavy drinkers were more likely to die than non-drinkers (P = 0.003). Patients with low pAkt T308 were more likely to be HPV positive (P = 0.028). Non-drinkers were also found to have lower levels of pAkt T308 and were more likely to have tumours positive for HPV than heavy drinkers (P = 0.044 and 0.007, respectively). This study suggests different mechanisms of carcinogenesis are initiated by smoking, alcohol and HPV. The resultant data propose higher phosphorylation of Akt at T308 as a reliable biomarker for smoking and alcohol induced HNSCC progression and higher phosphorylation of Akt at S473 as a prognostic factor for HNSCC.

A paracoccidioidomicose é uma enfermidade sistêmica causada principalmente pelo fungo Paracoccidioides brasiliensis. Recentemente, observou-se que a capacidade fagocítica dos macrófagos alveolares (MA) em animais diabéticos está diminuída em comparação aos MA de animais sadios, e que a insulina estimula a atividade fagocítica em MA oriundos de animais diabéticos e sadios por mecanismos diferentes. Neste projeto, usando um modelo de carência relativa de insulina (diabetes mellitus experimental), estudamos a intervenção da insulina em um modelo de infecção sistêmica. Após aprovação do comitê de ética (protocolo CEUA/FCF/421), camundongos machos da linhagem C57BL/6 (diabéticos, 60 mg/kg aloxana/10 dias, e seus respectivos controles) receberam injeção intratraqueal contendo suspensão de leveduras de P. brasiliensis ou volume equivalente de PBS estéril. Animais dos grupos de tratamento receberam insulina pela via subcutânea diariamente por 12 dias. Nas amostras, foram avaliados: a) o número de células dos lavados peritoneal (LPe) e broncoalveolar (LBA), o leucograma, e a glicemia (monitor de glicose); b) os níveis séricos de insulina no soro pela técnica de ELISA; c) as concentrações de citocinas (TNF-&#945;, IL-6, IL-4, IL-10, IL-12, CINC-1, CINC-2, CINC-3) nos LPe e LBA e nos homogenatos (ELISA). Após incubação de 55 dias, comparados aos controles (2.9±0,4g e 192±7.5 mg/dL), animais tornados diabéticos (0.87 ± 0,25 g e 570,1 ± 9,27mg/dL) apresentaram redução no ganho de massa corpórea durante o período de experimentação e elevados níveis de glicose sanguínea. O tratamento de insulina reduziu os níveis de glicose (547±36,8mg/dL vs.323,6±36,9mg/dL), embora não o suficiente para tornar os animais normoglicêmicos. Comparados aos controles, animais diabéticos apresentaram número reduzido de leucócitos no LPe (2.2 x106 ± 0.2cells/mm3 vs 1.3 x106 ± 0.1cells/mm3) e, no LBA, reduzidas concentrações de CINC-2 (662,3±73,8pg/mL vs 312,7±114,7pg/mL), CINC-1 (115,5.0±25,5pg/mL vs 88,3±24,7pg/mL) e IL-10(320,9±58,4pg/mL vs 161,0±59,4pg/mL) depois da infecção. O tratamento com insulina restaurou a concentração de leucócitos nos LPe de animais diabéticos, mas não no LBA. Os dados sugerem que a insulina modula a produção/liberação de citocinas, sem alterar a migração de leucócitos para LBA e restaurando a migração destes para LPe durante o curso da paracoccidioidomicose. / Paracoccidioidomycosis is a systemic disease mainly caused by Paracoccidioides brasiliensis fungus that interact with antigen-presenting cells, changing its main biological functions. Recently it was observed that the phagocytic capacity of these cells in diabetic animals for IgG opsonized targets is decreased compared to healthy animals, and that insulin stimulates the phagocytic activity in alveolar macrophages in from diabetic and healthy animals, by different mechanisms. In this project, using a model of relative lack of insulin (experimental diabetes mellitus), we studied the intervention of insulin in a model of systemic infection. After approval by the committee of ethics (protocol CEUA/FCF/421), C57BL/6 male diabetic (60mg/kg aloxan/10days) mice and their respective controls were subjected to intratracheal injection of suspension of P. brasiliensis or an equivalent volume of TBS sterile. In the forty-third day, insulin-treated mice were treated subcutaneously daily with insulin for 12 days at 6 P.M. We evaluated: a) the number of cells of the peritoneal(PeL) and bronchoalveolar (BAL) fluids, the leucogram and the glucose levels; b) the levels of insulin on the serum by the technique of ELISA; c) the levels of cytokines (TNF-&#945;, IL-6, IL-4, IL-10, IL-12, CINC-1, CINC-2, CINC-3) in the BAL and PeL fluid, and on organ homogenates. After 55 days, relative to controls (2.9±0,4g and 192±7.5 mg/dL)), mice rendered diabetic (0.87 ± 0,25 g and 570,1 ± 9,27mg/dL) exhibited a reduction in body weight gain during the experimental period and sharply elevated blood glucose levels. Treatment of diabetic animals with insulin induced a reduction in blood glucose levels (547±36,8mg/dL vs.323,6±36,9mg/dL), but it was not sufficient to reduce glycemia to control values. In addition, relative to controls, infected diabetic mice exhibited a reduction in the number of leukocytes into the PeL fluid ((2.2 x106 ± 0.2cells/mm3 vs 1.3 x106 ± 0.1cells/mm3) and reduced BAL concentrations of CINC-2 (662,3±73,8pg/mL vs 312,7±114,7pg/mL), CINC-1 (115,5.0±25,5pg/mL vs 88,3±24,7pg/mL) and IL-10 (320,9±58,4pg/mL vs 161,0±59,4pg/mL) after P. brasiliensis infection. Treatment of diabetic mice with insulin restored concentrations of leukocytes in the PeL fluid but not in the BAL. Data presented suggest that insulin modulates the production/release of cytokines but not leukocyte migration to the BAL while restoring this paramether on the PeL during the course of P. brasiliensis fungus-induced PCM.

Cancer metastasis is the critical event leading to 90% of cancer related death. Although significant improvement in our understanding on cancer metastasis has been made through years of research, the fundamental mechanism behind this process is still not fully elucidated. For cancer researchers, the “gold standard” for metastasis studies has traditionally been the use of tissue culture and mouse models. Tissue culture offers the simplest system and ease of control but is not able to recapitulate many of the features found in an in vivo tumor microenvironment. On the other hand, mouse model systems offer the most sophisticated and physiologically relevant platforms for studying cancer. However, the lack of control over the in vivo environment in these mouse models and inherent discrepancies from human physiology make results from these models difficult to be translated to clinical trials. The advancement in microfabrication techniques and cancer models developed based on these techniques has shown potential in addressing the gap between in vitro tissue culture and mouse models. Microscopic tumor microenvironments could be built in these in vitro systems to study behavior of human cancer cells. However, the expertise involved in and extra instrumentation needed for implementing these systems have prevented their widespread use by general cancer researchers. In this dissertation, we developed two simple microfabricated systems and demonstrated their application in two aspects of cancer research. The first system is a microfabricated cell patterning stencil, where paracrine signaling can be established and its impact can be measured based on cell migration. Using this tool, we investigated the interaction between melanoma and microenvironmental cells from their common metastasis target organ. Through these simple patterning techniques, we observed significant effects that a given microenvironmental cell line had on the two different melanoma lines, as well as how melanoma affected different microenvironmental cell lines. The second system, a microfluidic device, is able to present individual soluble factors to cancer cells in order to test the response of cancer cells to these physiologically relevant factors. Through this stand-alone system, we found that breast cancer metastasis is influenced by the protein molecules secreted by themselves as well as the local glucose level. Through these findings we believe that our microfabricated systems can benefit the general cancer research community in which a complicated problem can be broken down into manageable pieces and studied on a simple platform in a controlled way. Observation made through these systems can inspire general cancer researchers to form new hypotheses and eventually lead to new findings. / 2017-02-17T00:00:00Z

The ability of cells to sense oxygen is a highly evolved process that facilitates adaptations to the local oxygen environment and is critical to energy homeostasis. In vertebrates, this process is largely controlled by three intracellular prolyl-4-hydroxylases (PHD 1-3). These related enzymes share the ability to hydroxylate the hypoxia-inducible transcription factor (HIF), and therefore control the transcription of genes involved in metabolism and vascular recruitment. However, it is becoming increasingly apparent that proline-4-hydroxylation controls much more than HIF signaling, with PHD3 emerging as the most unique and functionally diverse of the PHD isoforms. In fact, PHD3-mediated hydroxylation has recently been purported to function in such diverse roles as sympathetic neuronal and muscle development, sepsis, glycolytic metabolism, and cell fate. PHD3 expression is also highly distinct from that of the other PHD enzymes, and varies considerably between different cell types and oxygen concentrations. This thesis will specifically examine the role of PHD3 expression in cancer cells, with a focus on the mechanisms of PHD3 gene silencing. In the final chapters, I will examine the consequences of this silencing in cancer, and discuss the discovery of a novel role for PHD3 in epithelial-to-mesenchymal-like transition and cell migration.

microRNAs are single-stranded small RNAs that function as post-transcriptional regulators of gene expression. We are studying the mir-44 family, specifically mir-44 and mir-45, which have identical sequence. Loss of mir-44 and mir-45 results in defects that suggest that the mir-44 family acts to negatively regulate the MAPK pathway. The MAPK pathway regulates sex myoblast migration, a process which is required for normal egg laying. We hypothesized that the mir-44 family of microRNAs is necessary for normal sex myoblast migration and subsequent formation of the functional egg laying structure in the hermaphrodite. We created a mutant that had mutations in both mir-44 and mir-45 and a transgene that expresses GFP in the sex myoblast cells. Then we observed the migration and division of the sex myoblasts in wild-type and mutant worms using fluorescence microscopy. In all cases, the mutant worms displayed a greater percent difference from average sex myoblast migration and division. However, a two-tailed two-proportions z-test found no significant difference between wild type and mutant sex myoblast migration (p=0.9148), nor in mutant sex myoblast division along the axial (p=0.4205) and sagittal (p=0.3583) planes of the body. This allows us to conclude that mir-44 and mir-45 are unlikely to be responsible for the migration nor division of the sex myoblasts, and the defects are likely due to interference with a different biological mechanism.

Problem investigated: Chronic wounds are not only a major burden to the patient arising from general pain and discomfort but also generate economic costs to both these individuals and the health care system. Various electrical stimulation regimes have been employed to study the effects of electrical stimulation on wound healing both in vivo and in vitro. In was hypothesised that electrical stimulation using various waveforms can modulate cell function, particularly cell migration. The aim of this thesis was to study the effects of electrical stimulation on cellular migration, in particular endothelial cells and fibroblasts, key cell types involved in wound healing. The impact of collagen matrix on cell migration was also assessed. Methods: Cells were seeded on either glass or collagen I substrate and stimulated with various electrical regimes via platinum electrodes connected to a constant current source. Cell migration was accessed by manual tracking of cell nuclei over a period of 3 hours from digital time-lapse images acquired during stimulation. Data from cell tracking were analysed for directional migration, migration rates and mean square displacement. Results: No directional cell migration for both endothelial cells and fibroblasts were observed when stimulated with either alternating or biphasic currents. However, surface substrate had impacted on cell motility with opposite effects being observed for the two cell types. Endothelial cells tended to migrate at a faster rate on collagen I substrate than on glass, compared with fibroblasts, which displayed a slower rate of migration on collagen I substrate. Significant changes in mean square displacement of biphasic current stimulated cells on collagen I substrate compared to unstimulated cells were also observed. Conclusion: This thesis has illustrated cell migration can be modulated by electrical stimulation, in particular asymmetric biphasic current. It has also been demonstrated surface substrate can impact cell migration.

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

Lysophosphatidic acid (LPA) is a potent bioactive lipid component of oxidized low density lipoproteins (oxLDL). High concentrations of LPA have been detected in human atherosclerotic plaques. Our data has shown that LPA highly induces smooth muscle cell (SMC) migration. Cyr61, a matricellular protein, which also accumulates in human atherosclerotic plaques, has been implicated in the injury-induced neointimal formation. Smooth muscle cell migration is a key event in the development of atherosclerosis, and it contributes to the progressive growth of atherosclerotic lesions. Data generated by this study demonstrate that LPA markedly induces Cyr61 expression in mouse aortic smooth muscle cells (MASMC). We hypothesized that LPA-induced matricellular Cyr61 mediates LPA-induced MASMC migration. To date, little is known about the relationship between LPA and Cyr61 in smooth muscle cells; the signaling pathway leading to LPA-induced Cyr61 is unknown. Furthermore, whether Cyr61 contributes to LPA-induced cell migration is unrevealed. Our study demonstrates that LPA, by binding to LPA1 receptor, activates the intracellular signaling pathway leading to the activation of PKCdelta which in turn contributes to the increased expression of Cyr61 in MASMCs. Interestingly, we found that after LPA-induced Cyr61 mRNA has been translated into its protein intracellularly, the de novo synthesized proteins promptly accumulate in the Golgi apparatus and then translocalize to the extracellular matrix. Importantly, our data reveal a novel LPA/Cyr61 pathway in controlling MASMC migration. Understanding the mechanism underlying LPA induction of Cyr61 provides new insight into pathogenesis of atherosclerosis.