Human cytomegalovirus (HCMV) infection is usually asymptomatic in healthy individuals. However, severe disease and/or death can occur in immunocompromised patients. The overall objective of this project was to develop a plant-derived CMV subunit vaccine and determine its effectiveness. Since CMV is species-specific, guinea pig CMV (GPCMV) was used as a model for HCMV. Four gene constructs were engineered to contain either the rice glutelin-1 promoter (Gt1) or the maize ubiquitin promoter, and the gene for either glycoprotein B (gB) or phosphoprotein 65 (pp65) of GPCMV. Agrobacterium tumefaciens were transformed with the Gt1-gB construct and subsequently used to transform Oryza sativa (rice) or Arabidopsis thalina . Due to technical difficulties associated with rice transformations, experimental work was continued with only A. thaliana. PCR analysis of plant tissue confirmed maintenance of the gB transgene for seven generations, and Western blots of selected seed extracts revealed a gB-specific band not found in non-transformed A. thaliana extracts. Guinea pigs (4 per group) were subcutaneously immunized with 20 mg of seed protein extract from GPCMV gB A. thaliana plants (estimated to contain approximately 50 mug of gB), an equal amount of total protein from non-transformed A. thaliana seeds (negative control), or baculovirus-derived GPCMV gB (positive control). Two animals immunized with seed-derived gB demonstrated GPCMV-specific antibody responses when analysed by ELISA. Western blot analysis confirmed that this response was specific for GPCMV gB. Neutralization assays were performed using serum obtained 14 days after the last immunization. Three of the 4 animals immunized with the gB seed extract showed viral neutralizing responses, with titres ranging from 40 to 3240. These results indicate that GPCMV gB expressed in the seeds of A. thaliana can generate a GPCMV-specific immune response. Based on data from GPCMV-specific ELlSAs, Western blots, and viral neutralization assays using serum from immunized animals, I concluded that gB was authentically expressed in A. thaliana seeds with at least some epitopes similar to those found on native GPCMV gB that were able to neutralize viral infectivity.

Mutations in genes encoding chromatin-remodelling proteins, such as the ATRX gene, underlie a number of genetic disorders including X-linked mental retardation syndromes; however knowledge of the role of these proteins in CNS development is limited. In Drosophila, ISWI is essential for development, the protein functions as the ATP-hydrolysing component in several chromatin-remodelling complexes. The two mammalian ISWI orthologs, SNF2H and SNF2L are differentially expressed, suggesting that they possess distinct developmental roles. Prevalent expression of SNF2H occurs during neuroprogenitor proliferation while SNF2L transcripts are increased in maturing neurons. It is well known that an appropriate balance between neuroprogenitor proliferation and neuronal differentiation during the embryonic stages is critical to regulate the size of the developing neocortex. Here, I have analyzed mice ablated for Snf2l chromatin remodelling activity to determine its role in cortical development. The loss of active Snf2l resulted in a 1.4-fold increase in the ratio between brain weight and body weight. This was accompanied by a concomitant increase in cell number in the cerebral cortical strata ranging from 1.3- to 1.6-fold (in embryonic and adult tissues). This increase did not coincide with an observable change in the frequency of apoptotic events. Pulse-labelling experiments with BrdU resulted in similar proportions of labelled cells within the periventricular zone of embryonic (E15.5) cortices from wildtype and mutant mice. We observed a 3-fold increase in mitotic cells by phospho-histone H3 staining. BrdU birthdating and BrdU/Ki67 double-labelling experiments revealed that there was a 3-fold increase in the number of progenitors that re-entered the cell cycle and a concomitant decrease in cell cycle exit. Gene expression analysis of Snf2l-mutants revealed a perturbation in the expression patterns of many genes, including genes encoding neurogenic factors and cell cycle regulators. One such gene identified was Foxg1. Foxg1 is regulated by Snf2l, and limiting Foxg1 mitigated the brain phenotype. Our results suggest loss of Snf2l alters the timing between cell cycle exit and differentiation leading to increased cell numbers in the developing neocortex.

Host-Pathogen interactions are characterized by inflammatory responses, aimed at eliminating invading pathogens. During inflammatory responses, immune cells secrete cytokines and chemokines, with a broad range of immuno-regulatory roles. The role of the signaling protein, SHP-1 in regulating signaling pathways activated by cytokine, antigen and growth factor receptors has been studied for some time. However, the involvement of SHP-1 in the regulation of Lipopolysaccharide (LPS)-activated signal transduction pathways leading to cytokine production is less well understood. Loss of SHP-1 leads to development of complex inflammatory disorder(s). To understand the role of SHP-1 during the inflammation process, the SHP-1-deficient moth-eaten (me/me) mouse model was used to investigate LPS-induced TNF-alpha and IL-10 production, representing pro and anti-inflammatory cytokines, respectively. Results show that me/me splenic macrophages, when challenged with LPS in-vitro , secreted lower levels of IL-10 and concomitantly elevated TNF-alpha levels, compared to normal healthy littermate controls. Deregulated LPS-induced TNF-alpha and IL-10 production in SHP-1 deficient splenic macrophages were observed irrespective of LPS dose and time of stimulation as demonstrated by ELISA. These findings were further confirmed by RT-PCR, SHP-1 antisense experiments in normal splenic macrophages and adenoviral reconstitution of SHP-1 expression in me/me macrophages, suggesting a dual role of SHP-1 as a negative regulator of LPS induced TNF-alpha and a positive regulator of IL-10. To delineate the precise role of SHP-1 in regulating LPS-mediated IL-10 production, LPS-activated signaling proteins, which represent SHP-1 targets were examined. Results obtained established a role of p38 MAPK in LPS-mediated IL--10 secretion, but also demonstrate that P38 involvement is independent of SHP-1. Results of this study further reveals a role for tyrosine kinase Src and an adhesion molecule Pyk2 in SHP-1 dependent LPS-mediated IL-10 production and infer that optimal production of LPS-induced IL-10 requires an assembly of a signalosome, consisting of Src-Pyk2-SHP-1 proteins. In conclusion, we show for the first time that: SHP-1 is a positive regulator of LPS-induced IL-10 production in splenic macrophages and, LPS-mediated IL-10 production is governed by two distinct signaling pathways, only one of which is SHP-1 dependent.

Post-translational modification of proteins via the covalent attachment of Ubiquitin plays an important role in the regulation of protein stability and function in eukaryotic cells. In the second chapter of my thesis, a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is described. The applicability of this approach is demonstrated by the identification of seventy ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. In the third chapter of my thesis, an application of a microfluidic protein processing device, termed the Proteomic Reactor, for enzymatic digestion of proteins for subsequent LC-MS/MS analysis is described. Use of the Proteomic Reactor following immunoaffinity purification enabled the identification of numerous low abundance ubiquitinated proteins from the human 1299 lung cancer cell line expressing reduced amounts of the ubiquitin-dependent chaperone known as the Valosin-Containing Protein. In the fourth chapter of my thesis, a method for determining peptide ion score cutoffs that permit the confident identification of ubiquitinated proteins by MS/MS is described. Experiments involving the analysis of gel bands containing multi-Ubiquitin chains with quadrupole time-of-flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching after MS/MS analysis were not sufficiently stringent. False positive and false negative rates were also found to vary significantly depending on the Cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard ion score cutoffs were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high false positive rates were observed. Finally, false positive rates for ubiquitinated proteins were found to be affected by the size of the protein database that is searched. In chapters five and six of my thesis, two biological applications employing the methods I developed for identifying ubiquitinated proteins are described. The first application involves the identification of specific lysine residues within the membrane-anchored transcription factor Mga2p that are targets of the E3 ubiquitin ligase Rsp5p in Saccharomyces cerevisiae. Ubiquitination of these lysines were found to mediate mobilization of the Mga2p transcription factor complex and ist subsequent disassembly. The second application involves the identification of proteins that are ubiquitinated upon mitotic exit in Xenopus laevis embryos. Sequence analysis of these proteins revealed that the majority of poly-ubiquitination occurs through lysine-11 chain polymerization, which is an atypical form of ubiquitination.

A detailed mechanism for heme uptake in pathogenic Neisseriae has not yet been elucidated. Once heme is deposited in the periplasmic space, a heme dedicated ABC transporter has been hypothesized to convey heme into the cytoplasm. To address this hypothesis, we used several molecular techniques to identify and characterize a putative heme ABC transporter in Neisseria meningitidis. Using 2-D gel electrophoresis, we identified six proteins with increasing expression under heme-limiting conditions. Although these proteins appear to be iron-regulated, the genes encoding these proteins are not associated with periplasmic components of ABC transporters. Using DNA microarray analysis, we identified eleven ABC transporter components. A mutation in one of these genes, the ATPase NMB1993, was constructed. No difference in the ability of various iron sources to support the growth of the mutant and wild-type strains was observed under iron-restrictive conditions. No difference in growth rates between the two strains was discerned, suggesting no role in heme metabolism. Another uncharacterized putative periplasmic component, NMB0586, exhibited 27% amino acid identity to a recently characterized periplasmic heme-binding protein in the Haemophilus ducreyi transporter. To address its role in heme metabolism, NMB0586 was disrupted by insertional inactivation. The ability of various iron sources to support the growth of the mutant and wild-type strains was compared under iron-restrictive conditions. The mutant failed to display a heme-deficient phenotype. However, in view of the recognized degenerate binding specificity of the permease component of some ABC transporters, the heme-binding property of NMB0586 was studied. Purified NMB0586 bound to heme at a concentration of 10-4 M, using enhanced chemiluminescence. Furthermore, the binding of purified NMB0586 protein to hemin-agarose demonstrated concentration dependent binding. However, increasing concentrations of competing heme ligand did not result in a decrease in NMB0586 protein binding to hemin-agarose. NMB0586 was also unable to functionally complement a heme-deficient mutant of Escherichia coli. These findings suggest that NMB0586 is unlikely involved in heme acquisition in meningococci. In conclusion, this study describes the difficulties in identifying a meningococcal heme ABC transporter, but highlights several techniques for the future identification and characterization of a heme ABC transporter in Neisseriae.

HIV infection results in progressive loss of general and HIV-specific cellular immunity. HIV employs a variety of mechanisms to undermine the effectiveness of the host immune system including dysregulation of IL-12 family of Th1 cytokines such as IL-12, IL-23 and IL-27. These cytokines are abundantly produced by monocytic cells following stimulation with various TLR ligands that are components of viruses, bacteria, or microbial products such as LPS and cytokines such as IFNgamma. Monocytic cells in addition to being key sources of Th1 cytokines, act as a bridge between innate and adoptive immune responses and playa crucial role in mv immunopathogenesis. It is well established that IL-12 production by monocytic cells is decreased in HIV-infected individuals and following in vitro infection of monocytic cells with HIV. However, the role played by IL-23 and IL-27 in HIV immunopathogenesis is not clear. Moreover, whether expression of IL-23 and IL-27 is altered in monocytic cells from HIV-infected individuals and following in vitro infection of monocytic cells remains unknown. To understand the molecular mechanisms underlying the loss of protective Th1 responses in HIV infection, it is imperative to investigate the role and regulation of IL-23 and IL-27. Therefore, I have investigated the molecular mechanisms by which LPS alone or in combination with IFNgamma regulate the expression of IL-23 and IL-27 in human monocytic cells and human monocytic cell lines as model systems. I show that LPS-induced IL-23 proteins production is regulated through the activation of JNK, p38 MAPKs and PI3K signalling pathways in THP-1 cells, but following stimulation with IFNgamma and LPS, IL-23 is regulated primarily by the PI3K pathway. In normal monocytes, IFNgamma- or IFNgamma/LPS- induced IL-23 protein production is regulated through the activation of PI3K and calcium pathways. In terms of IL-27 regulation in normal monocytes, I have demonstrated that p38 MAPK and PI3K are the essential signalling pathways that regulate IFNgamma/LPS-induced IL-27 production. Moreover, LPS- and IFNgamma/LPS-induced IL-27 expression is regulated by JNK, p38 MAPKs and PI3K pathways in THP-1 cells. Subsequently, I have elucidated the effects of HIV infection of human monocytes, and monocytic cell line (THP-1 cells) on spontaneous as well as LPS-induced production of1L-23 and IL-27. My results suggest that HIV-infection transiently increases IL-23p19, IL-12/23p40 mRNA and IL-23 proteins, but in terms of IL-27 it only increases IL27EBI3 mRNA expression and does not affect on IL-27 proteins production. In addition, I show that HIV-infection does not affect LPS-induced IL-27 production, but it has a remarkable inhibitory effect on LPS-induced IL-23 production in THP-1 cells and PBMCs. Taken together, this study provides basic understanding of the major signaling pathways involved in the regulation of IL-23 and IL-27 in activated monocytic cells as well as the effects of in vitro HIV infection on the expression of these cytokines. These results may suggest potential strategies aimed at enhancing immune responsiveness.

Cytokine responsiveness in monocytes / macrophages (M/M) is critical in their phagocytic, antimicrobial and antigen presenting function. It has been suggested that HIV can impair cell function by disrupting signal transduction induced in response to antigen and cytokines in different immune cell types including CD4+ T cells, CD8+ T cells, and monocytes. A study by Kryworuchko et al. showed that Interleukin (IL)-2-induced Signal Transducer and Activator of Transcription (STAT) 5 activation was inhibited in CD8+ T cells from a subset of HIV-infected patient's naive to therapy, but was restored, at least in part, after antiretroviral therapy (ART). In view of the important biological role of cytokine signaling via the STAT pathway in the regulation of M/M phagocytosis, proliferation, and cell survival, I hypothesized that disruption of the JAK/STAT pathway may lead to dysregulation of M/M effector functions and programmed cell death (PCD) during the course of chronic HIV infection. Therefore, my objectives were first to evaluate cytokine-induced JAK/STAT signaling in monocytes obtained from HIV- individuals and compare it to that found in two groups of HIV+ patients (ART-treated patients for >1 year and patients off therapy for >6 months). Second, I sought to determine the biological impact and the molecular mechanisms responsible for the alterations observed. Analysis of patient monocytes showed no clear difference in responsiveness among the three groups in the case of Interferon (IFN)-alpha, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 stimulation. However, in the case of IFN-gamma stimulation, the STAT1 transcription factor was significantly upregulated in HIV+ patients off therapy compared to HIV- controls and HIV+ patients on ART. Upregulation of STAT1 activation was not due to alterations in IFN-gamma receptor expression but rather the result of increased total STAT1 expression levels. Treatment of monocytic cells with HIV proteins Gp120 and Vpr was able to induce STAT1 expression in these cells. Investigations into the significance of the STAT1 hyperactivation in patient monocytes showed that, surprisingly, among the STAT1 responsive genes (HLA-DR, IRF-1, CXCL9, and CXCL10) studied, only chemokine CXCL9 expression was elevated in HIV+ patients on ART compared to the other groups. Since IFN-gamma-induced STAT1 activation is associated with PCD and IL-10 has inhibitory effects on IFN-gamma-induced signaling and its downstream effects, I evaluated monocyte cell death in response to IFN-gamma and IL-10 in all groups. Interestingly, spontaneous and IFN-gammainduced monocyte PCD were elevated in HIV+ patients compared to HIV- controls. Spontaneous PCD was significantly correlated with increased total STAT1 expression but not plasma levels of TRAIL. Interestingly, pretreatment with IL-10 could rescue monocytes from this fate. Further investigation of how PCD occurs in normal monocytes and how it is regulated by IFN-gamma and IL-10, showed that IFN-gamma enhanced spontaneous TRAIL secretion and caspase 8 activation. In contrast, IL-10 inhibited spontaneous and IFN-gamma-induced TRAIL secretion and caspase 8 activation. Surprisingly and despite this, spontaneous and IFN-gamma-induced monocyte PCD appeared to be independent of caspase activation but rather depended on autophagy. LC3-II expression, an autophagy marker, was upregulated spontaneously in cultured monocytes, enhanced further upon IFN-gamma stimulation but surprisingly, was also upregulated by IL-10. At the level of the molecular mechanism, I observed that blocking the STAT or Phosphatidyl inositol-3 kinase (PI3K) signaling pathways inhibited PCD in response to IFN-gamma and could also thwart the cytoprotective effects of IL-10. Concordantly, blocking STAT or PI3K activation reduced LC3-II expression III response to IFN-gamma or IL-10 stimulation in monocytes. In conclusion, these results suggest that dysregulation of IFN-gamma signaling may contribute to monocyte dysfunction during HIV infection. Furthermore, IL-10 may have a role in enhancing monocyte survival during chronic HIV infection. Thus, understanding the regulation of monocyte PCD via autophagy pathway may have important implications concerning the elimination of this important cellular reservoir for HIV.

Herpesviruses have been previously correlated to vascular disease and shown to cause thrombogenic and atherogenic changes to host cells. The work conducted in this thesis showed that even in the absence of cells, purified cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) could initiate thrombin production. Clotting and chromogenic assays showed that purified HSV-1 and HSV-2 provide the procoagulant phospholipid (proPL) necessary for assembly of factors Xa (FXa) and Va (FVa) into prothrombinase, which is responsible for generating thrombin. These findings are consistent with earlier CMV studies. These observations were confirmed by electron microscopy and flow cytometry using FVa and annexin V, respectively, as proPL-specific probes. CMV, HSV-1 and HSV-2 each had the ability to facilitate FXa generation from the inactive precursor factor X (FX), but only when factor VII/VIIa and Ca$\sp{2+}$ were present. Monoclonal antibodies specific for the coagulation initiator, tissue factor (TF), inhibited this FX activation and furthermore enabled identification of TF antigen on each virus type by electron microscopy and flow cytometry. Collectively, these data show that CMV, HSV-1 and HSV-2 can initiate the generation of thrombin by having essential proPL and TF activities on their surface. Unlike the normal cellular source, the viral activity is constitutive and therefore not restricted to sites of vascular injury. Thus cell-independent thrombin production may be the earliest event in vascular pathology mediated by herpesviruses.

Pichinde virus belongs to the arenavirus family, which harbours several serious human pathogens. This work was performed in order to investigate the basis for the mouse-attenuated phenotype of a temperature sensitive mutant of Pichinde, and to identify potential genetic changes responsible. Since arenaviruses demonstrate a tropism for monocytes and macrophages, the ability of the mutant, ts13, to replicate in tissues enriched for these cells was assessed. Compared to the wild type (wt) parental virus, the mutant ts13 displayed markedly reduced replication in resident peritoneal cells (RPC) from infected mice, in both the macrophage-rich adherent population, and the non-adherent population, which is a mixture of monocytes and lymphocytes. This indicated that reduced growth in macrophages may be involved in the reduced growth of ts13 in vivo. This was further supported by the observation that the replication of ts13 was also limited in a macrophage cell line, and in RPC infected in vitro. Investigations such as those described here will provide valuable information regarding mechanisms of arenavirus attenuation, and the genetic changes required to limit growth and virulence. Ultimately, this will be of use in the design of effective arenaviral vaccines, which will become imperative in the midst of new and emerging arenaviral pathogens. (Abstract shortened by UMI.)

This study was designed to: (a) describe the occurrence and distribution of the protozoan parasites Cryptosporidium sp. and Giardia sp. in raw and treated water supplies on the St. Lawrence River; (b) to find a suitable microbial indicator for the presence of Cryptosporidium sp. and Giardia sp. in raw water; (c) to measure the survival capability of Cryptosporidium parvum in both the raw water and the treated water supplies of the St. Lawrence River, and (d) to determine whether C. parvum oocysts aged (or stressed) in raw water are more susceptible than fresh oocysts to chlorine and monochloramine disinfection. Twelve sampling sites were chosen on the St. Lawrence River (between Morrisburg and Lancaster, Ontario), consisting of eight raw surface water sites, and four water treatment plant effluents. Two of the plants practiced full conventional treatment of the water, including alum coagulation, rapid-sand filtration and chlorination, while the other two used a one-step chlorination process to treat the drinking water. Each site was sampled three times between July and November 1995, and a further 5 to 7 times between April and October 1996. (Abstract shortened by UMI.)