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Precise spatial and temporal control of cellular adhesion and signal transduction events are necessary for accurate animal development. Given the necessity for cell communication in carrying out processes like cell fate specification, growth, cell migration and differentiation, it is not surprising that signaling transduction pathways, such as EGFR, BMP, Notch, Wingless and Hippo, are intimately involved. All these pathways encompass a cascade of molecular events over which there is exquisite spatial and temporal control. A wide array of mechanisms, involving a diverse set of molecules, acts to provide this regulatory control. One such molecule implicated in the BMP signaling pathway in Drosophila development is Kek5, a Leucine rich repeat and Immunoglobulin domain (LIG) family member. Here I show that Kek5 modulates both BMP signaling and adherens junctions. For these functions, I further demonstrate that structural elements in both extracellular and intracellular region of Kek5 are critical, providing new insight into the LIG family and their roles in signaling pathways.

The acquisition of cellular adhesion machinery likely represented a key factor in the evolutionary transition from unicellular to multicellular organisms. Within metazoa, cellular adhesion is an integral aspect of organismal integrity through its regulation of a wide range of processes, including tissue patterning, cellular proliferation, and migration. As such, dysregulation of adhesion has been linked to diverse pathologies including cancers and neurodegenerative diseases. At the molecular level, adhesion is mediated by specific transmembrane cell adhesion molecules (CAMs) and intracellular complexes that create a dynamic link between the extracellular milieu and the intracellular cytoskeleton. At the sequence level, immunoglobulin domains act to mediate homo- and heterophilic interactions among CAMs and thus adhesion between neighboring cells. LIGs, a family of Ig-containing proteins that contain Leucine-rich repeats, represent candidates for novel CAMs with functions in axonal regeneration and synaptic pathfinding - all of which are highly dependent on cellular adhesion. In Drosophila, two LIG family members, Kekkon1 (Kek1) and Kekkon5 (Kek5) have been been implicated in EGF signaling, and Bone Morphogenetic Protein signaling as well as cellular adhesion, respectively. To investigate the putative role of Kek5 as a CAM, characterization of Kek5 activity was carried out at the cellular and molecular level. From this it was discovered that Kek5 is able to induce a dramatic upregulation of the adherens junction component Armadillo, in addition to epithelial extrusion and cell enlargement. Together, the studies presented within support a model in which Kek5 acts in a homophilic fashion to upregulate Arm and that this activity is functionally separable from other observed effects (epithelial extrusion and cell enlargement).

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Environmental response as determined by the cell-polymer interaction stands as the greatest restriction to the implementation of new polymeric materials. Cell-polymer interactions are most influenced by substrate surface free energy, surface chemistry, topography, and rigidity[1]. Alteration of these properties through surface treatment has become a common approach to attain the desired cellular interaction. This study investigates Kr-F excimer laser(248 nm) surface modification of poly(methyl methacrylate), glycol-modified poly(ethylene terephthalate), and polytetrafluoroethylene and its effect on the adhesion of Escherichia Coli K-12 bacteria. These three polymers were chosen for their very different mechanisms of ablation as well as their range of surface free energies and bacterial responses[2-4]. Polymers were ablated using a pulsed Kr-F excimer laser with a dose of 3.3x 10-9 W/cm2 per pulse. This high level of UV radiation was sufficient to cause significant surface damage on both PMMA and PTFE. PETG showed some signs of wavering in the surface and material removal was confirmed through optical microscopy. Due to the extensive damage associated with ablation, a much lower-powered, continuous beam Kr-F laser was used for contact angle samples. It delivered a dose of 1.27 W/cm2. Contact angle measurements were then taken which showed dose-dependent surface free energy in all three polymers. Following ablation, bacterial adhesion to PETG was improved two-fold, while it decreased in both PTFE and PMMA. Surface chemistry analysis supported the idea that the ablation occurred through chain scission, since there were no new surface groups created. There were significan texture modifications observed in PTFE and PMMA whicle PETG demonstrated the rolling structure characteristic of polyesters following laser ablation described in Wefers et al [4] and Hopp et al [5]. Contact angle measurements showed a correlation between radiation dose and surface free energy of all three polymers. / Master of Science

Obstructive sleep apnea syndrome (OSAS) is a complex disorder characterized by repetitive bouts of upper airway collapse during sleep, causing subsequent intermittent hypoxia, hypercapnia, and fragmented sleep and is also associated with significant morbidity including daytime sleepiness, hypertension, and elevated cardiovascular risk. OSAS affects at least 4% of men and 2% of women; unfortunately, it is estimated that 80% to 90% of adults with OSAS remain undiagnosed. Both clinical characteristics and complex genetic and environmental interactions have made it difficult to understand OSAS disease etiology and identifying patients at risk is still elusive. A pattern of gene expression in cells or tissues related to a disease state for OSAS would provide beneficial information to be most effective in screening or diagnosing this disease. Objectives: The objectives of this study were to: 1) map out the study design and bench assay strategies by which to investigate this issue; 2) find out if there are specific differences in the global gene expression profiles of adult females with OSAS compared to those without OSAS, under conditions in which subjects were clinically similar (BMI, diabetes, cardiovascular disease, etc.); and 3) assess the protein expression differences that could potentially be linked via well-established molecular pathways associated with any differences found in global gene expression profiles in the presence and absence of OSAS. Methods: Subjects were overweight premenopausal Caucasian women with untreated OSAS (n=6; age = 40.7 ± 3.4; BMI = 49.04 ± 6.97; apnea-hypopnea index = 27.3 ± 16.02), and control subjects (n=10) (age = 38.2 ± 7.6; BMI = 47.94 ± 6.15; apnea-hypopnea index < 5), and matched for other clinical characteristics (diabetes, cardiovascular disease status, medications, etc.) recruited from either Carilion Clinic Pulmonary/Sleep Medicine or Carilion Clinic Bariatric Surgery practices. Subjects provided a fasting blood sample in which the monocytes were isolated from whole blood. The RNA was extracted from the monocytes, assessed for purity and quantity, frozen and shipped to collaborators at Dana-Farber Cancer Institute and hybridized to Affymetrix whole human genome chips on a gene chip. The initial computational evaluation and interpretation generated the hypothesis. Two-step quantitative real time polymerase chain reaction (qPCR) was performed to verify the results from the microarray analysis. The laminin enzyme immunoassay (EIA), and cellular adhesion assays were performed to determine if genomic changes resulted in proteomic and phenotypic assessments. Results: OSAS subjects had nine aberrantly regulated genes, of which three genes (LAMC-1, CDC42, and TACSTD2) showed a pattern in segregation between OSAS and controls subjects based on expression patterns. In addition, qPCR indicated a 2.1 fold increase in LAMC-1 and a 1.1 fold increase CDC42 expression unique to the tissue samples of patients with OSAS. Though the serum laminin EIA did not differ between groups, a statistically significant increase in peripheral blood mononuclear cells (PBMC) cellular adhesion in OSAS patients versus control subjects was found. The OSAS subjects had a well cell count of 9.27 ± 1.54 cells vs. controls 5.75 ± 0.78 cells (p Ë‚ 0.05), which is relative to the 103 cells/field that were plated. Conclusions: Cells isolated from women with moderate-severe OSAS show an abnormality in cellular adhesion, a process driven in part by the gene LAMC-1, which was also aberrantly expressed in these subjects. This suggests that inflammation may be linked to the pathogenesis of OSAS. This pilot study has provided the framework and preliminary data needed to propose a larger study with extramural research funding. / Ph. D.

INTRODUÇÃO: Superfícies de titânio modificadas por diferentes métodos foram estudadas com base nos parâmetros físicos e químicos de caracterização superficial e sua influência no comportamento de células pré-osteoblásticas (MC3T3) in vitro. MÉTODOS: Discos de titânio comercialmente puro grau II foram submetidos a três métodos de modificação de superfície (polimento, nitretados em plasma em configuração planar e gaiola catódica). As diferentes superfícies foram caracterizadas para observar o efeito do processamento na estrutura da camada superficial, na rugosidade e molhabilidade. Ensaios de adesão e proliferação celular usando linhagens de células pré-osteoblásticas MC3T3 foram realizados para avaliar o efeito das novas superfícies no comportamento celular in vitro. RESULTADOS: Os resultados demonstraram que a nitretação em plasma na configuração de gaiola catódica produz superfícies mais rugosas (p<0,02) e com menores ângulos de contato com a água. CONCLUSÕES: A adesão celular é maior nas superfícies mais rugosas do que nas superfícies polidas (p<0,05) e reagem de modo diferente a composição química do substrato e à topografia da superfície. / PURPOSE: The aim of this study was to evaluated the physico-chemical properties of different titanium surfaces modified by means of low temperature plasma nitridind on rat osteoblast cell adhesion and proliferation. METHODS: Pure Titanium discs grade II was submitted to three different surface preparations (polishing, glow discharge plasma nitriding in planar and cathodic cage configurations). Surface parameters as roughness, wettability and chemichal composition was determined to compare influency of gas mixture on the modified surface material properties. Cellular morphology was observed by scanning electron microscopy. To evaluate the effect of the surface on cellular response, osteoblast cells (MC3T3) adhesion and proliferation was quantified and data analised by Kruskal-Wallis and Friedman statistical tests. RESULTS: plasma nitriding discs shows rougher surfaces( p<0,02) in cathodic cage configuration and lower contact angle values. MC3T3 cells attached on rough surfaces produced by cathodic cage configuration was statistically significant p<0,05 compared to polished discs. CONCLUSIONS: Glow discharge plasma nitriding improve titanium surface roughness and wettability. MC3T3 cell adhesion behavior is related to substrate chemical composition and topography.

The chemokine, Monocyte Chemoattractant Protein (MCP-1), enhances integrin mediated monocyte adhesion to the vascular endothelium during inflammation. In this study, we demonstrate that MCP-1 promotes rapid sub-second adhesion of THP-1 cells to Vascular Cell Adhesion Molecule-1 (VCAM-1), but not to Intercellular Cell Adhesion Molecule-1 (ICAM-1). MCP-1 activates membrane tethered Very Late Antigen 4 (VLA-4, α4β1), but not necessarily cytoskeleton anchored VLA-4. Activated tethered VLA-4 bonds tremendously increased the period of time monocytes remain bound from hundreds of milliseconds to several seconds and also increased the distance over which immunologic surveillance occurs from several microns up to 20 microns along the endothelium. Lastly at the single molecule level, MCP-1 stimulated tethered VLA-4 bonds exhibit increased resistance to pulling force. In conclusion MCP-1 increased tethered VLA-4 bond resistance to force providing a mechanism for monocyte recruitment to the endothelium.

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