Diversos estudos têm demonstrado que os implantes de titânio são altamente biocompatíveis e passíveis de osseointegrar, contudo os mecanismos moleculares que atuam por trás da osseointegração permanecem largamente inexplorados. Uma das possibilidades é que o implante exposto ao sangue do paciente durante a cirurgia absorve proteínas relacionadas com a adesão celular, como a fibronectina e vitronectina presentes no plasma. Células como osteoblastos podem então aderir a estas proteínas através dos mecanismos mediadas pelas integrinas. No presente estudo, utilizamos a imunofluorescência para marcação dos anticorpos contra integrinas α2, α3, α5, α6, αv e β1 em células OsA-CL cultivadas sobre lamínulas de vidro e superfície de titânio modificada por radiação à laser no período de 1h à 24 horas. As células aderidas na superfície lisa das lamínulas de vidro tiveram um maior espalhamento durante as primeiras 3 horas, porém nos outros períodos estudados o espalhamento ocorreu de maneira similar em ambas as superfícies. A expressão de integrinas na superfície rugosa dos implantes manteve-se uniforme em todos os períodos estudados, contudo no grupo controle a expressão de integrinas diminuiu na avaliação de 24 horas. Concluiu-se que as características de superfície dos diferentes biomateriais podem afetar o comportamento celular durante a interação inicial das células com a superfície de material utilizado como substrato. / Many studies have been demonstrate that titanium implants are highly biocompatible, however the molecular mechanisms that are behind of the osseointegration process are still largely unexplored. One of the possibilities is that the implant exposed to the patient blood during the surgery, adsorb proteins related to the cellular adhesion. Cells like the osteoblasts can adhere to these proteins trough the mechanisms mediated by integrins. In the present study, we used the immunofluorescence for marking antibodies against α2, α3, α5, α6,, αv and β1 integrins on culture of OsA-CL cells on laminulas of glass and modified titanium surface modified by laser radiation in the period of 1h to 24 hs. Cells adhered in the smooth surface of laminulas of glass had a bigger spreading during the first 3 hours, however in the other studied periods the spreading occurred in a similar way in both surfaces. The expression of integrins in the roughness surface of the implants remained uniform in all the studied periods, but on the control group the integrins expression decreased in the 24-hours evaluation. We concluded that the characteristics of surface of the different biomaterials can affect the cellular behavior during the initial interaction of the cells with the surface of the used material as substratum.

Accumulating evidence highlights the importance of studying the migration of cells within the context of their natural environment as manipulating the substrate on which a cell is migrating can have a dramatic impact on the mode/mechanisms employed by cells during migration. Central to this phenomenon is the requirement of adhesion to the ECM in order to gain traction during migration. Integrins constitute the main family of cell receptors involved in mediating cell-ECM interactions during motility. Whilst traditionally two-dimensional cell culture studies have placed emphasis on the importance of these receptors for spreading and migration, it has become evident that within more confined environments these receptors, at least for some cell types, are less crucial. In this research we utilise Drosophila embryonic hemocytes as an in vivo model for cell migration. We show that whilst hemocytes migrate within confined environments in vivo, these cells depend on integrins for powering both developmental and inflammatory migrations. Given the close association between these receptors and the actin cytoskeleton we were surprised to discover that removal of the main β integrin subunit, Myospheroid, did not affect cell spreading in vivo and had only a small impact on lamellipodial structure and dynamics. Furthermore we discovered that, in contrast to other cell types previously analysed, removal of this integrin subunit in hemocytes was not accompanied by an increase in the rate of actin retrograde flow within the protrusions, which we believe could reflect abrogation of a positive feedback between Rho, ROCK and Myosin II contraction. Instead, we discover a key role for integrins in regulating the microtubule cytoskeleton, in the maintenance of a polarised microtubule bundle, termed a ‘microtubule-arm’. Although the molecular mechanisms by which this stabilisation is coordinated have yet to be identified, this provides important insight into the co-regulation of adhesion and microtubule cytoskeleton important for the migratory behaviour of these cells. Cell migration reflects the complex and integrated regulation of the actin cytoskeleton by diverse families of actin regulatory proteins. Using hemocytes as a model system, we also explore the regulatory interactions between two main actin regulatory proteins, Diaphanous and Enabled, in vivo. Whilst the function of these proteins in the formation of filopodial protrusions is overlapping, recent research has highlighted the ability of these proteins to regulate the activity of one another. We find that co-expression of Enabled in hemocytes is able to rescue the morphological and migratory defects resulting from overexpression of active Diaphanous. Thus, data here presents Enabled as a negative regulator of Diaphanous, which may play an important role in the migration of hemocytes in vivo.

Integrin recycling has been generally assumed to be important for cell migration but the trafficking pathways and the molecules regulating integrin trafficking remain poorly characterized. Furthermore, little is known about the activation status of endocytosed integrins and how it affects the recycling of these receptors. It is likely that FA-engaged integrins will follow different trafficking pathways than bulk integrins and here I sought to study the endocytic fate of this particular integrin pool using the MT-induced FA disassembly assay. I found that integrins previously resident at FAs travel through different Rab compartments after FA disassembly and that their return to the plasma membrane is Rab11- and Src-dependent. Strikingly, I unveiled new functions for FAK and Src family kinases in this process by showing that these kinases are critical to keep integrins active during endocytic trafficking. This finding is unprecedented since it was not known whether endocytosed integrins were kept active during their trafficking. Interestingly, reassembly of FAs from endocytosed integrin occurred preferentially at the leading edge of migrating cells suggesting that integrins are trafficked in a polarized fashion. Furthermore, the recycling of integrins from the Rab11-positive compartment to the plasma membrane is a long-range transport implying the existence of a MT motor committed to this task. Consistently, I identified that a kinesin-II motor, Kif3AC, is engaged in this process. My work establishes a FAK- and Src family kinases-based mechanism for integrin "adhesion memory" during endocytic trafficking and identifies a direct link between FA disassembly and reassembly through an endocytic recycling pathway involving Rab5 and Rab11 and a kinesin-II family member.

Diversos estudos têm demonstrado que os implantes de titânio são altamente biocompatíveis e passíveis de osseointegrar, contudo os mecanismos moleculares que atuam por trás da osseointegração permanecem largamente inexplorados. Uma das possibilidades é que o implante exposto ao sangue do paciente durante a cirurgia absorve proteínas relacionadas com a adesão celular, como a fibronectina e vitronectina presentes no plasma. Células como osteoblastos podem então aderir a estas proteínas através dos mecanismos mediadas pelas integrinas. No presente estudo, utilizamos a imunofluorescência para marcação dos anticorpos contra integrinas α2, α3, α5, α6, αv e β1 em células OsA-CL cultivadas sobre lamínulas de vidro e superfície de titânio modificada por radiação à laser no período de 1h à 24 horas. As células aderidas na superfície lisa das lamínulas de vidro tiveram um maior espalhamento durante as primeiras 3 horas, porém nos outros períodos estudados o espalhamento ocorreu de maneira similar em ambas as superfícies. A expressão de integrinas na superfície rugosa dos implantes manteve-se uniforme em todos os períodos estudados, contudo no grupo controle a expressão de integrinas diminuiu na avaliação de 24 horas. Concluiu-se que as características de superfície dos diferentes biomateriais podem afetar o comportamento celular durante a interação inicial das células com a superfície de material utilizado como substrato. / Many studies have been demonstrate that titanium implants are highly biocompatible, however the molecular mechanisms that are behind of the osseointegration process are still largely unexplored. One of the possibilities is that the implant exposed to the patient blood during the surgery, adsorb proteins related to the cellular adhesion. Cells like the osteoblasts can adhere to these proteins trough the mechanisms mediated by integrins. In the present study, we used the immunofluorescence for marking antibodies against α2, α3, α5, α6,, αv and β1 integrins on culture of OsA-CL cells on laminulas of glass and modified titanium surface modified by laser radiation in the period of 1h to 24 hs. Cells adhered in the smooth surface of laminulas of glass had a bigger spreading during the first 3 hours, however in the other studied periods the spreading occurred in a similar way in both surfaces. The expression of integrins in the roughness surface of the implants remained uniform in all the studied periods, but on the control group the integrins expression decreased in the 24-hours evaluation. We concluded that the characteristics of surface of the different biomaterials can affect the cellular behavior during the initial interaction of the cells with the surface of the used material as substratum.

Microsporidia infect invertebrate and vertebrate animals. Human pathogenic microsporidia are associated with severe disease in immunocompromised individuals, and mostly asymptomatic infection in the immunocompetent. Treatment options for microsporidiosis are limited, incompletely effective, and associated with toxicity. Furthermore, microsporidia infection of healthy individuals is poorly understood, and the consequences of asymptomatic infection have not been determined. Little is known about the molecular mechanisms of microsporidia infection, but such information is essential for the development of new therapies. Spores adhere to host cell surfaces in vitro. Our laboratory has focused on determining specific host cell and microsporidia spore surface participants in spore adherence. Our previous studies have shown that host cell sulfated glycosaminoglycans and the spore surface protein EnP1 participate in spore adherence to host cells. Additionally, in vitro inhibition or augmentation of spore adherence decreased or increased host cell infection, respectively. These studies demonstrated the importance of spore adherence in host cell infection and began to characterize the host cell and spore determinants of adherence. The goal of this research was to further characterize host cell and spore participants in microsporidia adherence and infection of host cells in vitro. We characterized an intracellular microsporidia protein and related antibodies for analyses of microsporidia spore surface proteins; characterized a spore surface protein, MsADAM, involved in spore adherence to and infection of host cells in vitro; and suggested a role for host cell integrins in microsporidia adherence to and infection of host cells in vitro.

Metastatic melanoma is unusually lethal with a ten year survival rate of less than 10%. Conventional DNA-damaging agents produce little improvement in patient survival. Vemurafenib (Zelboraf), a targeted therapeutic that inhibits the oncogenic BRAF demonstrates significant survival benefit. Unfortunately, it is now evident that there is both intrinsic and acquired resistance. Consequently, new strategies for sensitizing melanomas to vemurafenib are needed. Melanoma resistance to therapy is fueled in part by the integrins, the major cell surface adhesion receptors which are highly over-expressed in melanoma. Both integrin antagonists and agents that engage defective integrins increase the sensitivity of melanomas to chemotherapy. Our laboratory has identified a novel peptide, denoted vinculin activating peptide or VAP that targets integrins from within the cell and brings aberrant integrin function intact. VAP sensitizes melanoma to dacarbazine in vitro and in vivo. The effect VAP has on overcoming resistance to targeted therapies like vemurafenib, as well as the mechanism for its effects are not well understood. The goals of this project are to determine if VAP can be employed to improve sensitivity and/or overcome resistance to vemurafenib and to identify the cell surface target of VAP. Our results show that VAP not only improves melanoma sensitivity to vemurafenib but also decreases intrinsic resistance to this promising drug. In addition, we present evidence that β1 and β3 integrins are the target of VAP's effects. Since peptide-based therapies are not stable in the clinic, we explored another integrin binding partner, kindlin-2. We found that kindlin-2 is over expressed in resistant melanomas. The inhibition of kindlin-2 increases β1 integrin activation and decreases β3 integrin functions. Agents that bring aberrant β1 and β3 integrin function intact can be employed to improve sensitivity and overcome resistance to vemurafenib suggesting that combinatorial therapies that employ vemurafenib and integrin-based agents might be efficacious in combatting resistance in melanoma patients.

Very few cancer patients are cured through drug therapy alone, with the majority exhibiting acquired resistance. To date, most studies of therapeutic escape have focused upon tumor-intrinsic mechanisms of drug resistance with little attention paid to the role of normal host cells in preventing complete tumor eradication. In the present study we implicate co-operative bi-directional signaling between melanoma cells and fibroblasts in the generation of a pro-survival niche that mediates drug resistance. Mass-spectrometry based phosphoproteomics was used to show that BRAF inhibition and chemotherapy drugs enhanced the survival of both melanoma cells and fibroblasts through the induction of fibronectin (FN)/integrin α5β1 signaling. Immunohistochemical staining confirmed the induction of FN in mouse xenografts and human melanoma specimens following BRAF inhibitor treatment. Adhesion to FN amplified the adaptive EGFR, HER3 and c-MET receptor tyrosine kinase (RTK) signals required for PI3K/AKT/Mcl-1-mediated melanoma cell survival when BRAF was inhibited. At the same time, BRAF inhibition led, directly and indirectly, to the paracrine release of HGF and neuregulin from fibroblasts, with TGF-β release from the melanoma cells increasing both fibroblast differentiation and survival. Although dual inhibition of RTKs and BRAF did not reverse host-mediated resistance, therapeutic escape was overcome through combined BRAF/PI3K inhibition, suggesting the PI3K/AKT pathway to be a common signaling vulnerability in microenvironment-mediated drug resistance. Our work suggests that durable responses to targeted therapies will only be achieved through dual targeting of the tumor and the adaptive host responses to therapy. These findings are especially important for a cancer such as melanoma, where as few as one cell can repopulate the entire tumor in vivo.

Abstract not available

Circulating neutrophil activation occurs during cardiac surgery with extracorporeal circulation (ECC) and is implicated in the pathophysiology of inflammatory tissue injury and peri-operative organ dysfunction. However, neutrophil directed antiinflammatory strategies have failed to demonstrate consistent therapeutic benefit indicating that the nature and significance of peri-operative circulating neutrophil activation remains incompletely defined. In particular, conformational activation of the b2 integrin Mac-1 (CD11b/CD18), which is required for neutrophil adhesion competence and facilitation of effector functions, has not previously been investigated during cardiac surgery, and the relative contribution of cellular activation and bone marrow neutrophil recruitment to peri-operative changes in circulating neutrophil phenotype and function is unknown. A novel whole blood flow cytometric technique was used to analyze circulating neutrophil phenotype (total Mac-1, conformationally-active CD11b, CD10, CD16, L-selectin and P-selectin glycoprotein ligand-1) and function in cardiac surgery patients to characterize the nature of changes in Mac-1 expression and activation status, and the effects of relative neutrophil immaturity on circulating neutrophil phenotype and function. The effect of heparin, a known CD11b ligand, on Mac-1 epitope expression was also investigated. Circulating neutrophil numbers observed during ECC were mathematically modeled to determine the acute response of the bone marrow neutrophil reserve to an inflammatory stimulus. Plasma cytokine, chemokine and acute phase mediators were measured in cardiac and lung surgery patients to determine potential regulators of systemic neutrophil recruitment. Neutrophils newlyemergent from the bone marrow were characterized as CD10-/CD16low and exhibited distinct changes in cell surface markers and enhanced functional responses, relative to their more mature CD10+ counterparts. Conformational activation of CD11b occurred peri-operatively and provided a more sensitive measure of circulating neutrophil activation status than changes in total Mac-1 or L-selectin expression, although detection of Mac-1 epitopes was reduced in the presence of heparin. Modeling of circulating neutrophil numbers predicted that post-mitotic maturation time was acutely abbreviated by 8.4 hours during 71 minutes of ECC. Systemic chemokine release occurred with cardiac but not non-cardiac thoracic surgery indicating some specificity of the acute inflammatory response. These findings expand the understanding of peri-operative circulating neutrophil activation and recruitment, and identify potential therapeutic targets to limit neutrophil injurious potential during cardiac surgery with ECC.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.