<p>The presented work describes construction and analysis of recombinantly produced forms of Aleuria aurantia lectin (AAL). The binding properties of the produced AAL forms were studied using techniques such as tryptophan fluorescence, hemagglutination analysis, ELISA and surface plasmon resonance analysis.</p><p>Lectins are proteins that are ubiquitous in nature with the ability to bind specifically to different types of carbohydrates. The physiological function of different lectins is not always known, but they are involved in many recognition events at molecular and cellular levels. In research, lectins are widely used for structural and functional studies of complex carbohydrates, and they are also used to detect changes in the carbohydrate pattern on glycoproteins in different diseases.</p><p>With the use of recombinant technology it is now possible to refine properties of lectins such as decreasing the valency and alter specificity and affinity. This may be a way of constructing more suitable reagents for use in diagnostic glycosylation analysis assays.</p><p>AAL has been extensively used in different types of research for its ability to bind the monosaccharide fucose and to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. AAL was expressed recombinantly (rAAL) and its properties was investigated. These studies reveled that one of the binding sites in rAAL had unusually high affinities towards fucose and fucosecontaining oligosaccharides with Kd-values in the nanomolar range. This binding site is not detected in AAL that have been exposed to fucose during its purification, and therefore we proposed that this site may be blocked with free fucose in commercial preparations of AAL.</p><p>Normally lectin-oligosaccharide interactions are considered to be of weak affinity, so the finding of a high affinity site was interesting for the future study of recombinant forms of AAL. The next step was to produce recombinant AAL forms with decreased valency. This was done using site-directed mutagenesis. First a monomeric form of AAL (mAAL) was constructed and then a monovalent form of AAL, containing only one fucose-binding site (S2-AAL) was constructed. Both of these forms had retained ability to bind fucose. The binding characteristics of mAAL were similar to that of rAAL, but mAAL showed decreased hemagglutinating activity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and did not bind to sialylated fuco-oligosaccharides such as sialyl-LewisX. This study shows that molecular engineering techniques could be important tools for development of reliable and specific diagnostic and biological assays for carbohydrate analysis.</p>

<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>

<p>The goal of this project is to express both the normal CYP26 B1 and the spliced CYP26 B1 from human in <em>Escherichia coli </em>(E.coli) cells for further crystallization. This will be achieved by cloning in the DNA fragments into the Champion pET SUMO vector that is later transformed into <em>E.coli </em>cells. The CYP26 B1 contains a hydrophobic helix at the N-terminal of the protein, making both protein expression and crystallization difficult. Two variants of both full-length CYP26 B1 and the spliced variant will therefore be made, one with the trans-membrane helix present and one without the helix. The SUMO-vector will produce a fusion protein that will make CYP26 B1 more hydrophilic and improve the purification of the two proteins.</p>

<p>Flaviviruses, family flaviviridae, are often associated with severe diseases and many of the viruses have been shown to affect the immune response. Interferons confer important ways of defending the host against viral infections because they provide a link between the innate and the adaptive immunity. Langat virus (LGTV), belonging to the Tick-Borne Encephalitis (TBE) complex of viruses, has recently been shown to provide interactions with interferon receptors and inhibit the interferon-mediated response. Similarly, it has been demonstrated that the TBE virus NS5 protein affects type 1 interferon signaling. In this study we have analyzed the interaction between the TBE NS5 protein and interferon receptors by using the yeast two-hybrid system. Our results support the idea that the inhibition of interferon signaling does not involve a direct interaction between TBEV NS5 and the interferon receptors IFNAR2, IFNGR1 and IFNGR2.</p>

<p>Infection by the parasite Bonamia ostreae has infected and caused major mortality of the flat oyster, Ostrea edulis, over the last 25 years throughout the coasts of Europe and the United States of America. The conventional techniques for the diagnosis of infection with Bonamia ostreae are typically by histology and cytology. Both have a low sensitivity and Bonamia ostreae in weekly infected oysters can remain undetected when analyzed by such techniques. Molecular methods like the Polymerase Chain Reaction have recently been applied for a more reliable and sensitive detection of Bonamia ostreae.</p><p>The aim of this project was to optimize a PCR for the specific detection of the 18S Small Ribosomal subunit rDNA gene of Bonamia ostreae in formalin fixed Ostrea edulis tissues. While the PCR was successfully optimized for purified oyster DNA from fresh tissue it was difficult to apply on formalin fixed oyster tissues due to poor quality DNA from the fixed tissues. Ethanol fixed tissues were also tested for Bonamia ostreae, however, the primers were not specific for Bonamia ostreae and uninfected oysters also tested positive which led to the conclusion that the PCR could not be used as a reliable detection method for Bonamia ostreae in oysters. Despite using alternative primers which were designed to amplify other components of the Bonamia ostreae genome no consistent results were achieved to reliably use the PCR method for the accurate detection of Bonamia ostreae in oysters. The conclusion of this project is that other genomic sites in Bonamia ostreae must be identified as a target for PCR for this test to be specific.</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.