Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis. Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

The Dorsal Raphe Nucleus (DRN) is a complex brain region that has been implicated in disorders such as anxiety and depression. The DRN is divided into subregions through its rostrocaudal and mediolateral axis. It has been reported that after a single restraint session there is differential spatial activation of nitric oxide synthase (NOS) across the DRN. The temporal profile of NOS activity during acute stress is not known but it is important because duration of acute stress is associated with different general responses. In this report rats were restrained for 1, 3, or 6 hours and nicotinamide adenine phosphate diaphorase (NADPH-d) was stained as an index to NOS activity to determine the spatio-temporal profile of NOS throughout a 6 hour restraint. Astrocyte reactivity was also measured to determine whether NOS activation correlated with GFAP expression since astrocytes react to neural activity and store and release l-arginine, the precursor for nitric oxide production. The results showed that the DRN had a dynamic response to acute restraint stress, most notably in the caudal lateral wings where activation increased after 3 hours of restraint (p = > 0.001) but neuron count decreased after 6 hours (p = 0.040). Astrocytes did not correlate with NOS activation but they showed spatio-temporal differences as well whereas they were more active in the rostral half of the DRN. In conclusion, the present study suggests that NOS produced in the DRN may have a role in prolonged exposure to acute stress and that subregions show differential NOS activation.

Hyaluronan is a ubiquitous glycosaminoglycan which is an important constituent of the extracellular matrix (ECM). In addition to organizing the extracellular matrix and regulating tissue homeostasis, hyaluronan, by binding to its main cell surface receptor CD44, is involved in intracellular signaling pathways regulating major cellular processes during development, wound healing, inflammation and cancer. Accumulation of hyaluronan in cancer promotes progression of the disease and correlates with poor prognosis. This thesis focuses on the regulation of hyaluronan synthesis and its signaling in normal and cancer cells. Cancer cells in solid tumors are surrounded by stroma, which has an essential role in the growth and metastasis of tumors. Prominent members of the tumor stroma are fibroblasts, which synthesize ECM components, such as hyaluronan, and secrete growth factors, and activate intracellular signaling pathways. We demonstrate a cross-talk between the receptors for platelet-derived growth factor BB (PDGF-BB), transforming growth factor β (TGFβ) and CD44 in dermal fibroblasts. We found that PDGF-BB can activate the Smad signaling pathway downstream of the TGFβ receptor I (TβRI), and that PDGF-BB-induced migration depends on TβRI. CD44 forms a ternary complex with the receptors for PDGF-BB and TGFβ, and negatively regulates their signaling. Furthermore, we demonstrate that TGFβ stimulation of mammary epithelial cells transcriptionally upregulates hyaluronan synthase 2 (HAS2), which is essential for TGFβ-induced epithelial-mesenchymal transition (EMT); in this process, polarized epithelial cells adapt a mesenchymal phenotype which facilitates migration and invasion. HAS2 protein activity and stability is regulated by posttranslational modifications, including ubiquitination. We investigated the ubiquitination of HAS2 in aggressive breast cancer cells, whose metastasizing capability depends on HAS2-synthesized hyaluronan. We identified two deubiquitinating enzymes, USP4 and USP17, which target HAS2 and affect its activity and stability. In summary, these studies increase the knowledge about the regulation of hyaluronan production and its role in cancer progression.

Stress associated psychiatric disorders such as depression, anxiety, and post-traumatic stress disorder affect a large proportion of the population. Reductions in the complexity of neuronal morphology and reduced neurogenesis are commonly observed outcomes following stress exposure in rodent models and may represent a mechanism for the reduced brain volume in stress sensitive regions such as the hippocampus observed in individuals diagnosed with stress associated disorders. Multiple lines of evidence suggest that glycogen synthase kinase (GSK)-B may play a role in the neurodegenerative phenotype observed following stress exposure. GSK3B is atypical in that it is inhibited by phosphorylation. This inhibitory phosphorylation has typically been studied by examining the phosphorylation state of the serine 9 (S9) site. Inhibition of GSK3B is implicated in synaptic stabilization, increased expression of trophic factors that support dendritic complexity and neurogenesis, reduced apoptosis, and the antidepressive effects of currently implemented therapeutics. It is surprising then that little research has examined the regulation of GSK3B by stress. A novel GSK3B phosphorylation site, serine 389 (S389), has recently been described that is regulated by p38 mitogen activated protein kinase (MAPK) and is independent of S9 phosphorylation by AKT. p38 MAPK is implicated in the behavioral effects of stress exposure making an understanding of its interaction with GSK3B S389 phosphorylation during stress a compelling research target. The current studies examine GSK3B regulation following variate stress exposure in stress reactive brain regions, describe the anatomical specificity of GSK3B S389 phosphorylation in the brain, and detail the behavioral phenotype of a novel mutant mouse that cannot inhibit GSK3B by S389 phosphorylation (GSK3B KI). Region specific changes in GSK3B phosphorylation were observed following stress exposure, as well as voluntary exercise, a behavior that confers stress resistance. Elevated GSK3B S389 phosphorylation was associated with increased levels of phosphorylated p38 MAPK. This pathway is implicated in the response to DNA damage, and, surprisingly, we observed that histone H2A-variant-X (H2A.X), a marker of DNA damage, was elevated following stress and exercise. Accumulated DNA damage is a proposed driver of neurodegeneration suggesting that the pathway activated by stress may be engaged to protect against such decline. Consistent with a role in the response to DNA damage, we observed a primarily nuclear localization of GSK3B S389 phosphorylation in the brain while S9 phosphorylation was found in nuclear and cytosolic compartments. Further, we observed neurodegeneration in hippocampal and cortical regions of GSK3B KI mice supporting the idea that the inhibition of GSK3B by S389 phosphorylation observed following stress and exercise may be protective. Though largely similar to wild type mice in behavioral tests, increased auditory fear conditioning was evident in GSK3B KI mice. Contextual and cued freezing was prolonged in GSK3B KI mice, a phenotype that is commonly observed in stress models. Together these findings suggest that GSK3B S389 phosphorylation is playing a critical role in neuronal integrity that is independent of GSK3B S9 phosphorylation, and that the subset of neurons protected by GSK3B S389 phosphorylation may play an important role in preventing a portion of the maladaptive behavioral changes observed following stress exposure.

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The inflammatory nature of the tumor microenvironment provides a cytokine and chemokine rich proliferative environment. Much of the responsibility of this environment is due to the production of Reactive Oxygen Species (ROS). These studies examined the proliferative rich tumor environment from a new perspective of Nitric Oxide Synthase (NOS) dysregulation. NOS’s have the ability to become uncoupled and generate superoxide in lieu of nitric oxide (NO). A requirement of NOS for the production of NO is the cofactor tetrahydrobiopterin (BH4) and when it is missing NOS becomes uncoupled and turns into a peroxynitrite synthase. Here I demonstrate that NOS is uncoupled in tumor cells due to depleted BH4 levels. This uncoupling leads to decreased NO signaling and increased pro-inflammatory, pro-survival, signaling as a result of the increased generation of ROS/RNS from uncoupled NOS activity. I was able to recouple NOS through exogenous BH4 both in vitro and in vivo, reducing ROS/RNS and reestablishing NO signaling through cGMP protein associated kinase. Reduction of ROS/RNS resulted in the reduced activity of two major constitutively active transcription factors in breast cancer cells, NFκB and STAT3. In MCF-7 and MDA231 cells I found that increased NO-dependent PKG signaling led to tumor cell toxicity mediated by downregulation of β-catenin. Downregulation of β-catenin led to increased protein levels of p21 in MCF-7 and p27 in MDA 231cells, ultimately resulting in cell death. These results suggest that there is potential for BH4 as a therapeutic agent since exogenous dietary BH4 ameliorates chemically induced colitis, and reduced azoxymethane (AOM) induced colon and spontaneously developing mammary carcinogenesis.

DNA methylation is the most recognizable epigenetic mechanism. In general, DNA hypomethylation is associated with increased gene expression whereas DNA hypermethylation is associated with decreased gene expression. To date, little is known about the role of DNA methylation in the pathophysiology of preeclampsia. In this study, we examined the differences in DNA methylation in omental arteries of normal pregnant and preeclamptic women using the high throughput Illumina HumanMethylation27 BeadChip assay. We found 1,685 genes with a significant difference in DNA methylation at a false discovery rate of < 10% with many inflammatory genes having reduced methylation. The thromboxane synthase gene was the most hypomethylated gene in preeclamptic women as compared to normal pregnant women. When we examined the expression of thromboxane synthase in omental arteries of normal pregnant and preeclamptic women we found it to be significantly increased in preeclamptic women. The increased expression was observed in vascular smooth muscle cells, endothelial cells and infiltrating neutrophils. Experimentally induced DNA hypomethylation increased the expression of thromboxane synthase in the neutrophil-like HL-60 cell line, whereas tumor necrosis factor α (TNFα), a neutrophil product, increased its expression in cultured human vascular smooth muscle cells (VSMC). These finding suggest that DNA methylation and release of TNFα by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in systemic blood vessels of preeclamptic women, contributing to the hypertension and coagulation abnormalities. We also explored the possible contribution of DNA methylation to the altered expression of genes involved in collagen metabolism in preeclampsia. Several matrix metalloproteinase (MMP) genes, including MMP1 and MMP8, were significantly less methylated in preeclamptic women, whereas TIMP and COL genes were either significantly more methylated or had no significant change in their DNA methylation status. Experimentally induced DNA hypomethylation increased the expression of MMP-1, but not TIMP-1 or COL1A1, in cultured VSMCs and increased the expression of MMP-1 and MMP-8 in HL-60 cells. These findings suggest that DNA methylation contributes to the imbalance in genes involved in collagen metabolism in blood vessels of preeclamptic women.

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