The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure. A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions. This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding.

Enteropathogenic Escherichia coli (EPEC) is one of many bacterial and viral pathogens that can exploit the eukaryotic actin cytoskeleton for its own purposes. EPEC injects its own receptor, Tir, into the host cell plasma membrane where, upon binding the bacterial adhesin, intimin, can trigger actin assembly beneath bound bacteria resulting in characteristic actin "pedestals". The formation of these lesions is thought to be critical for bacterial colonization; and can also provide insight into actin dynamics of mammalian cells. EPEC Tir stimulates multiple signaling pathways converging on a central actin nucleation promoting factor, N-WASP. The best-characterized pathway of actin pedestal formation also involves the eukaroytic adaptor protein, Nck, but at least two Nck-independent signaling cascades have also been identified. Multiple aspects of Tir-mediated signaling cascades remain unclear. For example, although Nck can directly bind and activate N-WASP, current models of Tir-mediated, Nck-dependent actin signaling postulate an indirect interaction between Nck and N-WASP mediated by one or more unidentified host factors. Additionally, the relationship of this pathway to the Nck-independent pathways is unknown. To better understand Tir-mediated actin assembly, a detailed and quantitative analysis of the domain requirements of Nck and N-WASP for pedestal formation was conducted. The results indicate that, contrary to previously favored models, Nck is unlikely to require additional host factors to bind N-WASP during pedestal formation, but apparently directly stimulates this nucleation promoting factor. In addition, the results show that the Nck-dependent and -independent pathways target distinct regulatory domains of N-WASP.

Modulating chromatin structure is an important step in maintaining control over the eukaryotic genome. SWI/SNF, one of the complexes belonging to the growing family of ATP-dependent chromatin remodeling enzymes, is involved in controlling the expression of a number of inducible genes whose proper regulation is vital for metabolism and progression through mitosis. The mechanism by which SWI/SNF modulates chromatin structure at the nucleosome level is an important aspect of this regulation. The work in this dissertation focuses on how the Saccharomyces cerevisiae SWI/SNF complex uses the energy of ATP-hydrolysis to alter DNA-histone contacts in nucleosomes. This has been approached in a two part fashion. First, the three-dimensional structure and subunit composition of SWI/SNF complex has been determined. From this study we have identified a potential region of the SWI/SNF complex that might [be] a site for nucleosomal interaction. Second, functional analysis of the ATPase domain of Swi2p, the catalytic subunit of SWI/SNF, has revealed that a specific conserved motif is involved in coupling ATP hydrolysis to the mechanism of chromatin remodeling. These results provide a potential model for the function of the SWI/SNF chromatin remodeling complex at the nucleosome level.

Definitive evidence that natural killer (NK) cells mediate an antiviral effect in vivo was obtained using murine cytomegalovirus (MCMV) as a model system. Adoptive transfer studies using a variety of physical and immunochemical techniques to enrich and deplete NK cell activity showed that the cell population capable of mediating resistance (as assayed by enhanced survival and reduction in spleen virus titers) had the phenotype of an NK cell: a nylon wool nonadherent, asialo GM1+, NK 1.2+, ly 5+, Thy-1-, Ia-, low-density lymphocyte. Adoptive transfer of IL-2-dependent cloned NK cells (but not T cells) also provided resistance. NK cells did not provide resistance to lymphocytic choriomeningitis virus (LCMV). Selective depletion of NK cell activity by injection of mice with antibody to anti-asialo GM1 lowered resistance to MCMV, mouse hepatitis virus, and vaccinia virus but not to LCMV. NK cell depletion resulted in up to 1000-fold increases in spleen and liver virus titers, correlating with more severe pathology in these organs. NK cells were found to have antiviral effects early (0-3 days) but not late (6-9 days) postinfection. NK cell depletion resulted in markedly increased MCMV-induced suppression of T cell function, which is probably responsible for the delayed clearance of virus seen in these mice. NK cell depletion resulted in increased virus synthesis during persistent MCMV infection, but had no effect on the course of persistent LCMV infection, despite elevated NK cell and interferon (IFN) levels found in these LCMV-infected mice. The reason why NK cells play a role against MCMV but not LCMV infection was not due to differences in NK cells induced by these 2 viruses, but more likely due to target cell susceptibility. IFN pretreatment of MCMV-infected cells failed to protect them against NK cell-mediated lysis, whereas uninfected and LCMV-infected cells were almost totally protected. These IFN-pretreated, LCMV-infected cells were not resistant to cell-mediated lysis in general, as this treatment increased their sensitivity to virus-specific T cell-mediated lysis by 2- to 3-fold. This enhanced sensitivity to lysis correlated with increased surface expression of H-2 antigens, but not viral antigens. In summary, these studies provide compelling evidence that NK cells can mediate antiviral effects in vivo, and provide some insights into their mode of action and consequences of their disfunction.

The KIL gene of vaccinia virus encodes for a host range protein; in the absence of which, the virus is unable to grow in certain cell lines (RK-13 and some human cell lines). KIL function can be complemented in RK-13 cells by the cowpox host range gene product CP77 despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats ofthe K1L gene in the host-range restriction of growth in RK-13 cells. The growth of a recombinant vaccinia virus, with the K1L gene mutated in the most conserved ankyrin repeat, was severely impaired as evidenced by lack of plaque formation and reduction in viral titers. Infection of RK-I3 cells with the mutant recombinant vaccinia virus resulted in total shutdown of both cellular and viral protein synthesis early in infection, indicating that the host restriction mediated by the ankyrin repeat is due to a translational block. A comparison of the cellular localization of the K1L wild type and mutated forms showed no difference, as both localized exclusively in the cytoplasm of RK-I3 cells. We also investigated the interaction of the vaccinia virus K1L protein with cellular proteins in RK-13 cells and co-immunoprecipitated a 90 kDa protein identified as the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein which may be interacting with K1L.

The first type of dynein identified, axonemel dynein (Gibbons and Rowe, 1965), slides adjacent microtubules within the axoneme, generating the force necessary for ciliary and flagellar beating. The outer dynein arm is an important component of the flagellar axoneme, providing up to 60% of the force for flagellar motility. In the absence of the outer arm, cells swim with a slow-jerky motion at about 1/3rd the speed of wild-type cells, and the flagellar beat frequency is markedly reduced. Sixteen genes (ODA1-ODA16) have been identified that are required for outer arm assembly in Chlamydomonas reinhardtii. In addition, PF13, PF22, and FLA14 are required for outer dynein arm assembly, but their phenotypes are pleiotropic, suggesting that they affect additional flagellar components. Of the uncloned genes, ODA5, ODA8, and ODA10 are of particular interest because they do not encode subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC). Mutant alleles of these genes are unable to complement in temporary dikaryons, suggesting that the gene products interact with each other (Kamiya, 1988). Since the genes encoding all of the known components of the outer dynein arm and the ODA-DC have been characterized, it is of great interest to identify the gene products of these additional, uncloned ODA alleles. The first chapter provides an introduction to the Chlamydomonasflagellum, the dyneins in general, the outer dynein arm in particular, and mutations that impinge on the assembly and regulation of this important axonemal structure. The second chapter addresses the identification and isolation of genomic DNA containing the ODA5 gene. Utilizing a NIT1-tagged oda5-insertional mutant, I identified sequences flanking the site of the inserted NIT1 gene. These sequences were used to isolate wild-type genomic clones spanning the ODA5 gene. When transformed into the oda5 mutant, the wild-type clones rescued the mutant phenotype. These results demonstrated the successful isolation of the ODA5 gene. The third chapter describes the identification of the ODA5 gene and its corresponding cDNA. The rescuing genomic fragments were sequenced. Gene modeling was used to predict intron-exon splice sites. Primers to predicted exons were designed and used to obtain the ODA5 cDNA. The gene structure of Oda5 was analyzed and its predicted amino acid sequence deduced. Secondary structure predictions indicate that Oda5p is likely to contain a series of coiled-coil domains, followed by a poly-glycine sequence and a short, highly charged region. Northern analysis demonstrated that ODA5 gene expression is upregulated by deflagellation, a hallmark of many flagellar mRNAs. Data in CHAPTER IV further characterize the Oda5 protein and its association with the axoneme. Oda5p localizes to the flagellum, consistent with the enhancement in mRNA levels in response to deflagellation. Within the flagellum, Oda5p is an axonemal component that is released from the axoneme upon high salt extraction, as are the ODA-DC and the outer dynein arm. However, Oda5p does not associate with this super-complex in the high salt extract as determined by sucrose gradient sedimentation. Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC,demonstrating it does not require these complexes for localization. Furthermore, Oda5p assembles onto the axoneme in the oda8, but not the oda10 mutant, demonstrating a role for the Oda10 protein in localization of Oda5p. These data provide the first biochemical evidence for an interaction between Oda5p and Oda10p. CHAPTER V reveals the discovery of a previously unrecognized phenotype exhibited in both oda5 and oda10 mutant strains: a defect in the assembly of a previously unknown flagellar adenylate kinase (AK). The protein levels of this flagellar AK are reduced in oda5 mutant axonemes, as determined by quantitative mass spectrometry. Direct enzymatic assays confirmed a reduction in AK activity in both oda5 and oda10 mutant axonemes, providing a second line of biochemical evidence supporting a complex containing Oda5p and OdalOp. The sequence of the flagellar AK gene and its cDNA were determined. CHAPTER VI details our efforts to identify the ODA10 gene. Genomic clones were isolated, which contain sequences at, or near, the ODA10 locus. Analysis of the genomic clones yielded no insights into the identity of the ODA10 gene. The inability of these clones to rescue the Oda10-motility phenotype indicates that these clones most likely do not contain an intact ODA10 gene. And lastly, CHAPTER VII discusses future experimentation that can be done based on the data provided by the current study.

Although it is unclear which cellular factor(s) is responsible for the genetic instability associated with initiating and sustaining cell transformation, it is known that most, if not all, cancers have mutations that inactivate the Rb-mediated growth control pathway. We show here that acute inactivation of Rb by RNA interference or expression of the E7 viral oncoprotein from human papillomavirus (HPV), and the resultant deregulation of one E2F family member, E2F1, leads to DNA double strand break (DSB) accumulation. These DSBs occur independent of apoptosis induction, and activation of ATM, NBS1, p53, or MAD2, and generation of reactive oxygen species (ROS). Although ROS accumulation is associated with DSBs generated from the mis-expression of another nuclear oncoprotein, c-MYC, we find that E2F1 does not contribute to c-MYC associated DSBs, indicating that the DSBs associated with these oncoproteins arise through distinct pathways. However, we find that small changes in E2F1 levels by inhibition of c-MYC transactivated microRNAs known to limit E2F1 protein expression, lead to DSB accumulation. These results suggest that despite the DSBs arising by different mechanisms, c-MYC assists in the regulation of E2F1-associated DSBs. We also find elevated levels of E2F1-associated DSBs in Rb mutated cancer cell lines in the absence of an exogenous DSB stimulus. These basal, E2F1-associated DSBs are substantially lower in Rb wildtype cancer cell lines that have p16ink4 inactivated or express HPV E7. However, we show that we can manipulate DSB levels in these cancer cell lines by modulating Rb and E2F1 activity and suggest that these results may be extended to breast tumor organ culture. Thus, Rb status is key to regulating both the proliferation promoting functions associated with E2F and for preventing DNA damage accumulation if E2F1 becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2F1 might contribute to the genetic instability associated with transformation and tumorigenesis.

The focus of this thesis is the role of dengue virus in the induction of chemokines. Dengue virus (DENV) occurs as four distinct serotypes, called DENV 1,2,3,and 4. Symptomatic DENV infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. Many factors including T cell cross reactivity, viral burden, antibody dependent enhancement and induction of chemokines and cytokines have been reported in DHF and may play a role in the pathogenesis of DENV infection. Cytokines have been shown to modulate endothelial cell permeability [1-3]. Recent studies have shown that DENV-infected endothelial cells secrete the chemokine, interleukin (IL)-8 in vitro [4]. In addition, the permeability of an endothelial cell monolayer was found to be increased by interleukin-8 (IL-8) in vitro[5]. This thesis examines the effects of DEN2V infection on the induction of chemokines, and specifically, which DEN2V viral protein(s) are involved in the induction of IL-8. The chemokine induction profile following DEN2V infection was initially assessed in various cell lines that may represent potential targets in vivo, including monocytes, liver cells and endothelial cells. We hypothesized that distinct profiles of chemokine secretion can be induced by DEN2V infection of various cell types in vitro. We found RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) and IL-8 were induced in two of the five cell lines. DEN2V infection of primary monocyte-derived dendritic cells induced RANTES and IL-8 along with macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monocyte chemoattractant protein-1 (MCP-1) but at an earlier time post infection than in the cell lines. These results showed that DEN2V infection induces distinct chemokine profiles in many cell types. In addition, monocytic-derived DCs can secrete chemokines upon infection with DEN2V. Characterization of the signaling pathways induced by DEN2V revealed that DEN2V induction of chemokines in human embryonic kidney (HEK293A) cells is mainly through the nuclear factor kappaB (NFκB) pathway, as previously reported for endothelial cells and 293T cells [4,6]. Alternatively, the liver cell line (HepG2) activated mainly activator protein (AP)-l. In addition, DENV infection can induce the activation of the interferon-stimulated response element (ISRE) driven promoter. IL-8 has been shown to have multiple effects on the immune system ranging from recruiting cells to the site of infection to countering the antiviral effects of type I interferon (IFN) [7,8]. Previous reports have shown that viral proteins can induce chemokines such as seen with IL-8 induction with the nonstructural protein 5A (NS5A) and core proteins from hepatitis C virus [9,10]. We hypothesized that protein(s) from DENV could induce chemokine production. The expression of DENV proteins was analyzed for effects on IL-8 and RANTES production in HEK293A cells. The effects of viral replication on IL-8 and RANTES induction were also analyzed using a DENV replicon that contains genes for the capsid protein and the nonstructural proteins. Transfection of plasmids expressing NS5 or the DEN2V replicon induced the expression and secretion of IL-8 but not RANTES. We attributed the lack of RANTES induction to the inability of NS5 or the DEN2V replicon to induce transcription from the ISRE driven promoter. We also found that NS5 and the DEN2V replicon induced IL-8 mainly through the CCAAT/enhancer binding protein (c/EBP) and AP-1 pathways. The profile of transcription factor activation is different from what was seen with DENV infection of HEK293A cells and suggests that the transient expression of the NS5 protein and the replication and/or translation of the DEN2V genome use different pathways than viral infection to induce IL-8. In addition, we found that the expression of prM-E, known to produce virus-like particles, could induce IL-8 secretion and activate transcription from the IL-8 promoter. As with the expression of NS5, RANTES was not induced. Analysis of the transcription factors involved in IL-8 induction using luciferase reporter constructs indicated that expression of prM-E induced transcription of IL-8 through NFκB, AP-1 and c/EBP, similar to what was seen with DEN2V infection of HEK293A cells. These results suggest that production of virions or virus-like particles induce IL-8 but that another mechanism in the viral life cycle is responsible for the induction of RANTES expression by DEN2V infection. We were also interested in the effects of drugs that have been used previously to inhibit cytokine or chemokine production on chemokine induction during DEN2V infection. We hypothesized that pharmacological inhibitors of cytokines will inhibit secretion of chemokines in DEN2V infected cells. We found that the pharmacological inhibitors SB203580 and rolipram enhanced chemokine production in a DEN2V infected liver cell line (HepG2), whereas dexamethasone had the same effect in a kidney epithelial cell line (HEK293A). We conclude that drugs that inhibit signaling pathways involved in cytokine production in other experimental systems can have variable effects on chemokine induction in different cell types during DEN2V infection. The data generated in this thesis extend our understanding of how DEN2V manipulates the host cell during viral infection to produce chemokines and perhaps enhance viral propagation and dissemination through the induction of IL-8. In addition, this study provides insight into the variable effects pharmacological drug treatment may have on disease progression during DENV infection. These results increase our understanding of DENV pathogenesis and may be helpful in finding better strategies for treatment and prevention.

Transcription of the osteocalcin gene, which encodes a bone-specific 10 kDa protein, is controlled by the coordinated utilization of modularly organized basal and hormone-responsive enhancer elements. Activation of these sequences involves the interaction of specific transcription factors to these promoter elements. It is becoming increasingly accepted that nuclear architecture provides a basis for support of tightly regulated modulation of cell growth and tissue-specific transcription which is required for the onset and progression of differentiation. Thus packaging of DNA as chromatin can facilitate the cooperative interaction between activities of independent regulatory elements that contribute to the level of transcription. Here, we show that a specific nucleosomal organization supports the constitutive expression of the osteocalcin gene in ROS 17/2.8 rat osteosarcoma cells and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of this gene in normal diploid osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNase I hypersensitive sites (proximal: -170 to -70, and distal: -600 to -400) and a selective nucleosome positioning over the osteocalcin gene promoter are directly associated with developmentally stage-specific transcriptional activation in bone-derived cells. In addition, we found that chromatin hyperacetylation prevents a key transition in the chromatin structure which is required for the formation of the distal DNase I hypersensitive site. This transition involves the interaction of specific nuclear factors and is necessary for the subsequent ligand-dependent binding of the vitamin D receptor complex. Finally, we have established a requirement for sequences residing in the proximal region of the osteocalcin gene promoter for both formation of the proximal hypersensitive site and basal transcriptional activity. Our approach was to assay nuclease accessibility in ROS 17/2.8 cell lines stably transfected with promoter deletion constructs driving expression of a CAT reporter gene.

The DNA replication checkpoint transcriptionally up-regulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF (aka DSC1), the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitrophosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Substitution of two potential phosphorylation sites with phospho-mimetic amino acids suffice to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggest that checkpoint regulation of the MBF transcription factor may be a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. Our result shows that both the replication checkpoint and the S-phase DNA damage checkpoint are involved in activating MBF regulated S-phase gene transcription and that this coordinated transcriptional response is beneficial for survival during replication stress. I demonstrate that the beneficial role of the transcriptional response during checkpoint activation is mediated by three major MBF transcripts: cdc22, mrc1 and mik1. Mrc1 dependent stabilization of stalled fork is important during S phase arrest. However, cells ability to prevent mitosis (Mik1 dependent) along with stable fork (Mrc1 dependent) both are crucial for survival. Our data also suggest that the level of Cdc22 is a determining factor for replication checkpoint activation and when over-expressed can alleviate the effects not only in HU but also in MMS.