Approximately 10% of all gastric cancer cases worldwide are associated with Epstein-Barr virus (EBV) infection. This subgroup differs from non-infected equivalents through a distinct mutational and epigenetic signature, which is crucial for the establishment and maintenance of the viral latent state. Disruption of the latent state and subsequent induction of the lytic cycle, called reactivation, can be used as a therapeutic strategy for this cancer type through either combination with antiviral agents or by induction of an EBV-specific immune response. In this study, epigenetic inhibitors were tested for their potential to reactivate EBV using a novel screening method based on the expression of Zebra, a key regulator of EBV reactivation. Histone deacetylase inhibitors (HDACis), more specifically benzamide-based HDACis, were the most potent group of reactivating agents in this screen. Those HDACis induced the expression of lytic cycle genes as well as targeted cell killing in combination with antiviral agents. Moreover, the reactivation through those agents was sufficient to activate cytotoxic T-cells and thereby ensured a supportive role of the immune system in targeted cell killing. In an additional screen, topoisomerase inhibitors and other chemotherapeutic agents showed synergistic effects on Zebra expression together with HDACis, which was linked to the induction of p53 expression through those compounds. While p53 is known to be involved in the expression of Zebra, the viral DNA processivity factor Ea-D and the viral DNA binding protein DNBI were identified as a potential novel binding protein of p53 and could thus point towards a novel role of p53 in EBV lytic replication. Taken together, this study identifies potential novel therapeutic compound combinations for the treatment of EBV-associated epithelial cancers that could be translated into the clinic after further assessment.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial-host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa.

Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial-host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa.

No description available.

Cytokines are known to be involved in mastitis, yet their production by bovine epithelial cells has so far not been reported. In this research a bovine mammary alveolar cell line (MAC-T) was used as a simplified model for mammary glands. Cultured MAC-T cells were stimulated with 1, 10 and 20 mug/ml of a bacterial cell wall component (lipopolysaccharide) to verify the production of epithelially derived bovine cytokines. Cytokine mRNA production was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) using RNA samples isolated during the first 24 hrs of lipopolysaccharide stimulation. Interleukin-1alpha/beta, interleukin-6 and interleukin-8 mRNA production seemed to be time and dose dependent. Restriction enzyme analysis of PCR products confirmed the identity of the cytokines. Enzyme linked immunosorbent assays for interleukin-1 and interleukin-8 showed that the respective mRNA's were translated into proteins. Interleukin-8 protein was detectable 6 hrs after lipopolysaccharide stimulation; maximal levels of approximately 55 pg/ml were reached at 48 hrs post stimulation. Interleukin-1 was detected 1hr after stimulation; concentrations peaked between 500 and 600 pg/ml at 2, 5 and 12 hrs for 20, 10 and 1 mug/ml of lipopolysaccharide respectively. The amount of protein produced by the MAC-T cell line was relatively low and required high concentrations of lipopolysaccharide. Nevertheless, this model demonstrated a time and dose dependent cytokine production. These results suggest that bovine epithelial cells could be a source of cytokine production during mammary infection.

Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 5 uppsatser.