The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Protein adsorption is established as an early biological response to implanted materials that influences biocompatibility, hence an understanding of how to direct specific protein and cellular responses is critical for the development of future biomaterials. The effects of protein adsorption and subsequent cellular interactions on a variety of surfaces are investigated. Acrylic-based hydrogels are used as a model system in which to investigate both tear and serum protein adsorption from simple and complex solutions. The effect of surface topography, created by colloidal silica, on serum protein adsorption and conformation as well as cell adhesion is also investigated. Tantalum (Ta) and oxidised polystyrene (PSox) are investigated for their ability to support cell adhesion when precoated with various serum proteins. Protein interactions are examined using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), dual polarisation interferometry (DPI) and enzyme-linked immunosorbent assay (ELISA) while cellular interactions are analysed using QCM-D, microscopy and adhesion assays. The QCM-D technique was evaluated for its ability to provide new insight into cell-surface interactions. Most tear and serum proteins were found to adsorb onto the acrylic hydrogels, however, lysozyme was found to absorb into the hydrogel matrix and decrease the hydration, which may lead to an adverse biological response. Fibronectin adsorbed onto nanotextured colloidal silica surfaces was found to be conformationally changed compared to flat controls which is likely to correlate with the reduced endothelial cell adhesion observed on these textured surfaces. Ta and PSox precoated with either serum or fibronectin were shown to support cell adhesion and spreading, while surfaces precoated with albumin were not. QCM-D responses varied between underlying surfaces, protein precoating, ECM deposition, cytoskeletal activity and length of exposure indicating that alterations in cell-material responses are reflected in QCM-D measurements. QCM-D parameters were found to correlate with adhered cell numbers, cell contact area and cytoskeletal activity. The results highlight that characterisation of interfacial interactions with a wide range of analytical techniques is necessary to gain insight into cell-protein-material interactions which can then be utilised in the development of new generations of biomaterials with improved properties designed for specific applications.

<p><b>PAPER 1</b></p><p>In the first paper we show that reducing the average molecular weight from ~350 kDa to <6kDa by acid hydrolysis diminished the cell-stimulating activity of poly-M, measured as TNFproduction from human monocytes. However, the activity of the resulting oligomers (M-blocks) was greatly enhanced when covalently attached to particles (plastic beads or biodegradable albumin particles). Similar results were obtained with detoxified/deacylated LPS (DLPS) and glucuronic acid polymers (C6OXY), but not with G-blocks that by themselves are not active. These results suggest that the supramolecular structure affects the potency of polysaccharide stimuli, and that M-blocks attached to biodegradable albumin particles could possibly be exploited as an immunostimulant for protection against various diseases.</p><p><b>PAPER 2</b></p><p>In paper 2, according to the reviewers suggestion, the designation M-polymers of different molecular size was used in place of poly-M (~350 kDa) and M-blocks (~3 kDa). In this study we demonstrated that M-blocks and DLPS attached to particles engaged different receptors than soluble poly-M and DLPS in activation of monocytes. By using blocking mAbs to CD14, CD11b and CD18, we found that particulate stimuli employed the β2- integrin CD11b/CD18 in addition to the shared CD14 for signaling TNF-production. Moreover, whereas poly-M only bound to CD14-expressing CHO-cells, M-particles preferentially bound to CHO-cells expressing β2-integrins. However, the DLPS- and M-particles failed to activate NF-κB-translocation in CHO-cells co-transfected with CD14 and β2-integrins, suggesting that additional molecules are required for activation of CHO-cells. The major conclusion drawn from this work is that the supramolecular structure, in addition to influence the potency, affects the cellular receptor engagement by carbohydrates like poly-M and DLPS. This points to the importance of comparing the mechanisms involved in activation of immune cells by soluble bacterial components and whole bacteria to achieve a better understanding of inflammatory diseases like sepsis.</p><p><b>PAPER 3</b></p><p>Poly-M activates cells in a CD14-dependent manner, but CD14 is linked to the membrane with a GPI-anchor and mediates activation by interaction with other, signal-transducing molecules, like the TLRs. By using blocking mAbs to TLR2 (generated in our lab, paper 5) and TLR4, we found that both receptors were involved in mediating TNF-production from human monocytes in response to poly-M. Furthermore, TLR4 mutant (C3H/HeJ) and knockout (TLR4-/-) murine macrophages were completely non-responsive to poly-M, whereas TLR2-deficient macrophages showed reduced TNF-responses. These findings indicate that CD14, TLR2 and TLR4 on primary cells all participate in cytokine-induction by poly-M, and that TLR4 may be necessary for activation.</p><p><b>PAPER 4</b></p><p>In addition to CD14, β2-integrins have been implicated in LPS-induced cellular activation, and in this study we compared the involvement of CD14 and β2-integrins in TNF-production and NF-κB-activation induced by LPS and GBS cell wall fragments. With blocking mAbs to CD14 and CD18 we found that LPS and GBS cell walls shared CD14, but in addition the cell walls employed CD11/CD18 in mediating TNF-production from human monocytes. Both stimuli specifically induced NF-κB-translocation in CD14-transfected CHO-cells, but only LPS could activate cells transfected with CD11/CD18. The lack of response to GBS cell walls in CD11/CD18-transfected CHO-cells indicated that the cell walls need CD14 for cell activation. Further in paper 4 we demonstrate the ability of GBS cell walls to activate LPS-hyporesponsiv C3H/HeJ mouse macrophages, suggesting that LPS and GBS cell walls employ different receptors/signaling mechanisms in murine macrophages.</p><p><b>PAPER 5</b></p><p>When it was discovered that human TLR2 and TLR4 are involved in microbial recognition, we started to generate a mouse mAb to human TLR2, and in paper 5 we report the production and characterization of the mAb TL2.1. We subsequently used this mAb to evaluate the role of TLR2 in mediating activation by heat-killed GBS and <i> L monocytogenes</i>.<i> L. monocytogenes</i>, but not GBS, activated TLR2-transfected CHO-cells to IL-6-production, and the response was inhibited by TL2.1. A CD14 mAb and TL2.1 both inhibited TNF-production from monocytes induced by <i>L. monocytogenes</i>, but neither mAb affected the TNF-response triggered by GBS. Our results suggest that CD14 and TLR2 are engaged in cell activation by <i>L. monocytogenes</i>, but that neither receptor seem to be involved in activation by GBS. This study was the first to show that human TLR2 can discriminate between two G+ bacteria.</p><p><b>PAPER 6</b></p><p>In paper 6 we report the generation of a new TLR2 mAb, TL2.3, that stained with the same specificity as TL2.1 (anti-TLR2, paper 5). We used these mAbs to investigate the expression of TLR2 protein in human cells. We found that TLR2 was highly expressed in blood monocytes, less in granulocytes, and not present in lymphocytes. The protein level was measured on quiescent and activated cells by extra- and intracellular flow cytometry, and by immunoprecipitation of TLR2 from metabolic S35-labeled cells. Surprisingly, TLR2 protein was detected in activated B-cells located in lymphoid germinal centers, indicating that subsets of lymphocytes may express TLR2. We further show that TLR2 protein was differentially regulated on monocytes and granulocytes after exposure to LPS, pro- or anti-inflammatory cytokines. However, we could not correlate the regulation of TLR2 to cellular responses, as for instance the three anti-inflammatory cytokines TGFβ, IL-4 and IL-10 all inhibited lipopeptideinduced TNF-production, but either did not affect, reduced, or increased the level of surface TLR2, respectively. Thus, the biological significance of TLR2-regulation remains to be found.</p>

Meiosis is a complex process involving one round of DNA replication followed by two rounds of cell divisions. The proper segregation of homologs at meiosis I and sister chromatids during meiosis II is essential for the survival of the offspring. Aberrant chromosome segregation at any stage of meiosis can lead to aneuploidy. Meiotic chromosome segregation without crossing over or chiasmata is a widespread but poorly understand chromosome segregation pathway. In male Drosophila meiosis the absence of recombination in chromosomes makes it easier to identify mutations which influence homologous chromosome pairing and segregation. Modifier of Mdg4 in Meiosis (MNM), a protein encoded by modifier of mdg4, is required for integrity of chromosome territories and stability of achiasmatic bivalents and for normal homolog segregation in male Drosophila meiosis I. MNM localizes to clusters of nucleolar and autosomal foci during meiotic prophase I (PI) and to a novel, compact structure associated with the X-Y bivalent during prometaphase I (PMI) and metaphase I (MI). Stromalin in Meiosis (SNM), a member of the SCC3/STAG cohesion family, is required for homolog pairing in male Drosophila but not for meiotic sister chromatid cohesin. SNM protein co-localizes with MNM to the nucleolus throughout PI and to a prominent focus on the X-Y bivalent during PMI and MI. Mutations of snm and mnm exhibit similar homolog pairing failure during meiosis I. Consequently we used the Yeast Two-Hybrid System to determine whether SNM and MNM can interact with each other. We concluded that MNM can interact with itself and SNM. We also found that SNM interacts with the BTB domain of MNM and that the FLYWCH domain in the C-terminus of the MNM protein may play a role in the interaction between MNM and SNM. Sister chromatid cohesion (SCC) is required for proper chromosome segregation during mitosis and meiosis. The protein complex cohesin is a major component of SCC and links sister chromatids together from the time of their replication until their segregation. sisters unbound (sun) is a novel gene required in male and female Drosophila for meiotic SCC. Mutations in sun cause premature sister chromatid segregation (PSCS) and nondisjunction (NDJ) of both homologous and sister chromatids, and also disrupt normal recombination and synapsis in female meiosis. The four chromatids in each bivalent exhibit random segregation at meiosis I. We found that centromeric cohesion is lost in the absence of SUN during mid-prophase (S4). Surprisingly, cytological analysis shows chromosome behavior appears relatively normal during meiosis I. Double mutations sun snm and sun mnm impair the integrity of chromosome territories. In addition we found that SNM, but not MNM, is required for centromere pairing in mid-prophase (S3) and simultaneous loss of SNM and SUN proteins causes PSCS at mid-prophase I (S3), which is earlier than in single mutants in snm or sun. These findings indicated that these two proteins play complementary roles in meiotic cohesion.

Thesis (Ph. D.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

<b>PAPER 1</b> In the first paper we show that reducing the average molecular weight from ~350 kDa to &lt;6kDa by acid hydrolysis diminished the cell-stimulating activity of poly-M, measured as TNFproduction from human monocytes. However, the activity of the resulting oligomers (M-blocks) was greatly enhanced when covalently attached to particles (plastic beads or biodegradable albumin particles). Similar results were obtained with detoxified/deacylated LPS (DLPS) and glucuronic acid polymers (C6OXY), but not with G-blocks that by themselves are not active. These results suggest that the supramolecular structure affects the potency of polysaccharide stimuli, and that M-blocks attached to biodegradable albumin particles could possibly be exploited as an immunostimulant for protection against various diseases. <b>PAPER 2</b> In paper 2, according to the reviewers suggestion, the designation M-polymers of different molecular size was used in place of poly-M (~350 kDa) and M-blocks (~3 kDa). In this study we demonstrated that M-blocks and DLPS attached to particles engaged different receptors than soluble poly-M and DLPS in activation of monocytes. By using blocking mAbs to CD14, CD11b and CD18, we found that particulate stimuli employed the β2- integrin CD11b/CD18 in addition to the shared CD14 for signaling TNF-production. Moreover, whereas poly-M only bound to CD14-expressing CHO-cells, M-particles preferentially bound to CHO-cells expressing β2-integrins. However, the DLPS- and M-particles failed to activate NF-κB-translocation in CHO-cells co-transfected with CD14 and β2-integrins, suggesting that additional molecules are required for activation of CHO-cells. The major conclusion drawn from this work is that the supramolecular structure, in addition to influence the potency, affects the cellular receptor engagement by carbohydrates like poly-M and DLPS. This points to the importance of comparing the mechanisms involved in activation of immune cells by soluble bacterial components and whole bacteria to achieve a better understanding of inflammatory diseases like sepsis. <b>PAPER 3</b> Poly-M activates cells in a CD14-dependent manner, but CD14 is linked to the membrane with a GPI-anchor and mediates activation by interaction with other, signal-transducing molecules, like the TLRs. By using blocking mAbs to TLR2 (generated in our lab, paper 5) and TLR4, we found that both receptors were involved in mediating TNF-production from human monocytes in response to poly-M. Furthermore, TLR4 mutant (C3H/HeJ) and knockout (TLR4-/-) murine macrophages were completely non-responsive to poly-M, whereas TLR2-deficient macrophages showed reduced TNF-responses. These findings indicate that CD14, TLR2 and TLR4 on primary cells all participate in cytokine-induction by poly-M, and that TLR4 may be necessary for activation. <b>PAPER 4</b> In addition to CD14, β2-integrins have been implicated in LPS-induced cellular activation, and in this study we compared the involvement of CD14 and β2-integrins in TNF-production and NF-κB-activation induced by LPS and GBS cell wall fragments. With blocking mAbs to CD14 and CD18 we found that LPS and GBS cell walls shared CD14, but in addition the cell walls employed CD11/CD18 in mediating TNF-production from human monocytes. Both stimuli specifically induced NF-κB-translocation in CD14-transfected CHO-cells, but only LPS could activate cells transfected with CD11/CD18. The lack of response to GBS cell walls in CD11/CD18-transfected CHO-cells indicated that the cell walls need CD14 for cell activation. Further in paper 4 we demonstrate the ability of GBS cell walls to activate LPS-hyporesponsiv C3H/HeJ mouse macrophages, suggesting that LPS and GBS cell walls employ different receptors/signaling mechanisms in murine macrophages. <b>PAPER 5</b> When it was discovered that human TLR2 and TLR4 are involved in microbial recognition, we started to generate a mouse mAb to human TLR2, and in paper 5 we report the production and characterization of the mAb TL2.1. We subsequently used this mAb to evaluate the role of TLR2 in mediating activation by heat-killed GBS and L monocytogenes. L. monocytogenes, but not GBS, activated TLR2-transfected CHO-cells to IL-6-production, and the response was inhibited by TL2.1. A CD14 mAb and TL2.1 both inhibited TNF-production from monocytes induced by L. monocytogenes, but neither mAb affected the TNF-response triggered by GBS. Our results suggest that CD14 and TLR2 are engaged in cell activation by L. monocytogenes, but that neither receptor seem to be involved in activation by GBS. This study was the first to show that human TLR2 can discriminate between two G+ bacteria. <b>PAPER 6</b> In paper 6 we report the generation of a new TLR2 mAb, TL2.3, that stained with the same specificity as TL2.1 (anti-TLR2, paper 5). We used these mAbs to investigate the expression of TLR2 protein in human cells. We found that TLR2 was highly expressed in blood monocytes, less in granulocytes, and not present in lymphocytes. The protein level was measured on quiescent and activated cells by extra- and intracellular flow cytometry, and by immunoprecipitation of TLR2 from metabolic S35-labeled cells. Surprisingly, TLR2 protein was detected in activated B-cells located in lymphoid germinal centers, indicating that subsets of lymphocytes may express TLR2. We further show that TLR2 protein was differentially regulated on monocytes and granulocytes after exposure to LPS, pro- or anti-inflammatory cytokines. However, we could not correlate the regulation of TLR2 to cellular responses, as for instance the three anti-inflammatory cytokines TGFβ, IL-4 and IL-10 all inhibited lipopeptideinduced TNF-production, but either did not affect, reduced, or increased the level of surface TLR2, respectively. Thus, the biological significance of TLR2-regulation remains to be found.

The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis.

Sliding clamps and their cognate clamp loaders facilitate DNA synthesis, DNA repair, and sister chromatid cohesion in eukaryotes. ELG1 (enhanced level of genome instability) encodes a member of the fourth clamp-loader-like complex identified to date, and is important in the maintenance of genome integrity. Like all clamp loaders, Elg1 is a replication factor C (RFC) homologue. I examined the roles of the unique and conserved regions of S.cerevisiae Elg1 in resistance to exogenous DNA damage and suppression of spontaneous DNA damage. The conserved RFC region of Elg1 mediates association with chromatin function. The unique C- terminus of Elg1 mediates oligomerization with Rfc2-5, a core complex present in all clamp loaders, and is essential for Elg1 function. Finally, the N-terminus of Elg1 promotes its nuclear localization and contributes to the maintenance of genome stability. The Elg1-RFC complex most likely functions in collaboration with the sliding clamp PCNA. Combining mutations in ELG1 and PCNA results in endogenous DNA damage, which activates a noncanonical DNA damage response that results in upregulation of dNTP production. Increased dNTP pools allow significant DNA synthesis to occur at hydroxyurea (HU) concentrations that prevent replication in wild type cells. However, consistent with the recognized correlation between dNTP levels and spontaneous mutation, the double mutant exhibits a significant increase in mutation frequency. These phenotypes are also detectable in the single mutants although to a lesser extent. Together, these findings suggest that spontaneous mutagenesis stimulated by endogenous DNA damage may be a general feature of the DNA damage response.