Human Pegivirus (HPgV; originally called GB virus C) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are infected with HPgV. No causal association between HPgV and disease has been identified; however, several studies found an association between persistent HPgV infection and prolonged survival of HIV-infected individuals that appears to be related to a reduction in host immune activation. HPgV replicates well in vivo (>10 million genome copies/ml plasma) but grows poorly in vitro and systems to study this virus are limited. Consequently, mechanisms of viral persistence and host immune modulation remain poorly characterized, and the primary permissive cell type(s) has not yet been identified. The overall goals of my thesis were to characterize HPgV tropism, effects of HPgV infection on host immune response and mechanisms of viral persistence. Previous studies found HPgV RNA in T and B lymphocytes and ex vivo infected lymphocytes produce viral particles. To further characterize HPgV tropism, we quantified HPgV RNA in highly purified CD4+ and CD8+ T cells, including naïve, central memory, and effector memory populations, and in B cells (CD19+), NK cells (CD56+) cells and monocytes (CD14+) obtained from persistently infected humans using real time RT-PCR. Single genome sequencing was performed on virus within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4+ and CD8+ T lymphocytes (9 of 9 subjects), B lymphocytes (7 of 9), NK cells and monocytes (both 4 of 5). HPgV RNA levels were higher in naïve (CD45RA+) CD4+ cells than in central memory and effector memory cells (p<0.01). HPgV sequences were highly conserved between patients (0.117 ± 0.02 substitutions per site) and within subjects (0.006 ± 0.003 substitutions per site). The non-synonymous/synonymous substitution ratio was 0.07 suggesting low selective pressure. CFSE-labeled HPgV RNA-positive microvesicles (SEV) from serum delivered CFSE to uninfected monocytes, NK cells, T and B lymphocytes, and HPgV RNA was transferred to peripheral blood mononuclear cells (PBMCs) with evidence of subsequent viral replication. Thus, HPgV RNA-positive SEV may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus. Although HPgV infection reduces NK cell activation in HIV-infected individuals, the mechanism by which this occurs is not characterized. We studied HPgV effects on NK cell non-cytolytic function in HIV-infected people by measuring expression of IL-12 induced interferon gamma (IFNg) and cytolytic function by measuring K562 target-cell induced CD107a and granzyme B. IFNg expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects treated with combination antiretroviral therapy (p=0.02). In contrast, cytolytic NK cell functions were not affected by HPgV. Inhibition of IFNg was due to inhibition of tyrosine kinase (Tyk2) by HPgV envelope protein E2. HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited IL-12-mediated IFNg release by NK cells. Thus HPgV-E2 inhibits NK cell non-cytolytic functions. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV's ability to persist with high viral loads (>10 million genome copies/ml plasma) and reduce immune cell activation. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that have been associated with a myriad of negative human health effects. These man-made compounds were used throughout most of the 20th century and although their intentional production has since been banned and their use limited to closed systems, their prevalence in the environment remains a factor in disease states for exposed populations. The worldwide levels of PCBs has been declining, however, there is evidence for renewed sources of these compounds. The presence of PCBs with lower numbers of chlorine atoms (LC-PCBs) have been verified as unintentional byproducts in paints and pigments, the decomposition of PCB waste, or the recycling or disposal attempts of PCB-laden materials. While exposure to the higher chlorinated congeners (HC-PCBs) is often attributed to the consumption of contaminated water or fatty animal meat, a significant route of exposure to the airborne LC-PCBs is through inhalation. These semi-volatile compounds have been detected in high quantities in both indoor and outdoor air in urban and rural communities, and their presence is pronounced in older buildings (e.g., homes and schools). When compared to HC-PCBs, LC-PCBs are more highly susceptible to metabolic transformations, and recently their sulfated metabolites have gained much interest. Although the sulfation of xenobiotics often is considered a route for their removal from the body, a previous study of Sprague-Dawley rats treated with 4-chlorobiphenyl (PCB 3) resulted in the substantial formation of sulfated metabolites (i.e., hydroxylation followed by sulfation of the LC-PCB). This metabolic route accounted for more than half of the treatment dose. Furthermore, LC-PCB sulfates have been shown to bind to the human serum protein, transthyretin, in vitro. Of the health effects associated with PCB exposure, neurotoxicity has been well established through various laboratory and epidemiological studies. It is proposed that the dopaminergic system lies at the core of the observed cognitive, motor, and intellectual dysfunction observed in exposed populations, especially in children exposed perinatally. Interestingly, PCB exposure has been linked to Parkinson's disease (PD) etiology, which is marked by a substantial loss of dopaminergic neurons. This thesis describes studies on the binding of selected LC-PCBs and their hydroxylated and sulfated metabolites to human serum albumin (HSA), the most abundant protein in human serum. The displacement of fluorescent probes, selective for the two major drug binding sites of HSA, indicates that LC-PCB sulfates generally bind to HSA with such affinity that is equal to or greater than that for the LC-PCBs or OH-LC-PCBs This work also included a study of the selective toxicity of these compounds to dopaminergic neuronal cells. The selective toxicity of these compounds was studied in a series of immortalized cell lines (i.e., two neuronal cell lines: the rat midbrain-derived N27 cell line, the human neuroblastoma-derived SH-SY5Y cell line, and the human liver-derived HepG2 cell line). The assessment of toxicity by MTT reduction and LDH release in these cellular models indicated that hydroxylated and sulfated metabolites of LC-PCBs exhibited toxicity that was selective to neuronal cells and, in most cases, selective for the dopaminergic neuronal cells. Furthermore, HPLC analysis of the distribution of the compounds from the extracellular medium into the cellular milieu indicated that the observed toxicity may be due in some cases to selective transport and further metabolism. This work contributes to understanding the neurotoxicity of LC-PCB hydroxylated and sulfated metabolites and the role that binding to serum proteins may play in it. Furthermore, it emphasizes the need for future studies on the effects that metabolism, particularly sulfation, may play in the disposition of LC-PCB congeners as it pertains to their metabolism, retention, and toxic effects.

Ketoconazole is a broad-spectrum antifungal with oral activity and is documented to block steroid biosynthesis in fungi and mammals. Estradiol-primed ovariectomized rats lose the estradiol-induced, cornified layer of the vaginal epithelium as a result of ketoconazole treatment. This effect is not explained by current knowledge of ketoconazole's mechanism of action. Ketoconazole's effect on estradiol's binding to either receptors in target tissues (target-cell receptors) or serum-estradiol-carrying proteins in the aforementioned rats was investigated. The uterus is responsive to estradiol treatment, which causes an increase in uterine size and weight. This tissue is a sensitive means of studying the effects of ketoconazole on target-cell estradiol receptors. Tamoxifen, documented to block the effects of estradiol by binding to target-cell estradiol receptors, was used as a control drug. Uterine weight and horn diameter were measured in estradiol-treated, ovariectomized rats after 7 days of oral, twice-a-day, drug treatment. Ketoconazole had no effect on mean uterine weights and uterine horn diameters at any of the doses tested; however, tamoxifen at a dose of 10 mg/kg caused a significant reduction in uterine weight and horn diameter. Vaginal histopathology showed that ketoconazole and tamoxifen caused decornification. The percentage of unbound estradiol in the serum was measured in estradiol-treated, ovariectomized rats treated with ketoconazole orally, twice-a-day for 3 days to determine the effects of ketoconazole on the serum binding of estradiol. The results of vaginal cytology showed that ketoconazole caused normal decornification. In comparison with control animals, the results of the serum percent-unbound estradiol assays showed that ketoconazole caused a dose-related increase in the percent-unbound estradiol in the blood. The dose required to cause a 5 percent increase in serum unbound estradiol was approximately 6 times greater than the minimum dose required to cause decornification. Ketoconazole's effects on target-cell estradiol receptors and serum estradiol-carrying proteins do not appear to have any relationship to its effects in causing vaginal decornification in this model.

Dementia is the top global public health threat of the twenty first century. Within the dementia spectrum, Alzheimer’s disease (AD) is the most common type of dementia that occurs with aging and accounts for about 60% - 80% of diagnosed cases. But currently available discoveries failed to develop disease-modifying therapies for all patients living with AD. Recent discoveries can only partially slow down cognitive decline in a small subset of patients with limited effectiveness. The heterogeneity and complexity of the pathophysiology of AD indicate that a single drug approach may not be sufficient to prevent disease onset and progression. Human umbilical cord blood cells (HUCBC) and lithium treatment have shown promise against numerous neurological conditions, including AD. Yet, they also show significant unwanted, adverse effects. To address this barrier to yield successful treatments, we employed two key modifications to these two treatment strategy. We used human umbilical cord blood derived serum (HUCBS, also labeled as CBS) rather than HUCBC. We also utilized ionic cocrystal of lithium salicylate l-proline (LISPRO, also labeled as LP) instead of usual lithium salt. Both HUCBS and LISPRO have been shown to have strong neuroprotective, anti-inflammatory properties in separate studies conducted in transgenic AD mouse models. The studies detailed herein independently investigated the effectiveness of biological (HUCBS) and pharmacological (LISPRO) approaches in modulating the pathology and cognitive impairments in AD mouse models (e.g., 5XFAD, 3XTg-AD, APPswe/PS1dE9, and Tg2576). While administration of HUCBC stimulate anti-inflammatory pathways shown in previous studies, we found that HUCBS markedly promoted neurotrophic soluble amyloid precursor protein alpha (sAPPα) through non-amyloidogenic amyloid precursor protein (APP) processing pathway compared to adult (ABS) and aged blood serum (AgBS) in Chinese hamster ovary cells expressing wild type APP (CHO/APPwt). Using chromatographic fractionation, mass-spectrometry, and targeting complement proteins in cord blood serum fraction (αCBSF), we discovered the source of sAPPα in HUCBS as C1 complement protein. Further, intraperitoneal administration of αCBSF via osmotic minipump for 6 weeks showed prevention of cognitive impairment in 5XFAD mice assessed by novel object recognition, and Y-maze test. A series of recent studies have shown that lithium can prevent both AD- and age-associated cognitive decline. But, current United States Food and Drug Administration-approved lithium pharmaceutics (carbonate and citrate forms) have a narrow therapeutic window and unstable pharmaceutics that can cause toxicity without monitoring. Here we investigated the safety, pharmacokinetics, and therapeutic efficacy of LISPRO (LP), lithium salicylate (LS), and lithium carbonate (LC) in cell culture and mouse (B6129SF2/J, Tg2576, and 3xTg-AD) models. Cytokine profiles from the brain, plasma and splenocytes demonstrated that 8-week oral treatment with LISPRO downregulates pro-inflammatory cytokines, upregulates anti-inflammatory cytokines and suppress renal cyclooxygenase 2 (COX2) expression in Tg2576 mice. Pharmacokinetic studies indicated that LISPRO provides significantly higher brain and more steady plasma lithium levels in both B6129SF2/J and transgenic Tg2576 mice compared with lithium carbonate. Oral administration of LISPRO for 28 weeks significantly reduced β-amyloid plaques and tau phosphorylation. In addition, LISPRO significantly elevated pre-synaptic (synaptophysin) and post-synaptic protein (post synaptic density protein 95) expression in brains from transgenic 3XTg-AD mice. Moreover, female APPswe/PS1dE9 mice at 4 months of age were orally treated with LP, LS, or LC for 8- to 9- months at 2.25 mmol lithium/kg/day followed by measuring body weight, internal organs’ growth, and cognitive and non-cognitive function. LISPRO treatment prevented cognitive decline compared with transgenic APPswe/PS1dE9 cohort, as shown by shorter escape latency during training and probe trials in the Morris water maze and longer contextual freezing time during fear conditioning. As expected, LISPRO treatment also reduced depression assayed by tail suspension test and irritability assessed with the touch escape test. But, lithium treatment did not alter anxiety or locomotor activity as assessed by open field, elevated plus maze or accelerated rotarod tests. Taken together, these data indicate that both biological HUCBS and pharmacological LISPRO treatment may prove to be viable effective strategy for ameliorating Alzheimer’s like pathology and cognitive impairment in preclinical models.

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

<p>Brominated flame retardants (BFRs) are a diverse group of chemicals, which are used to slow down or inhibit the development of fires. BFRs are incorporated into a wide range of consumer products that are considered as potential fire hazards, such as TV-sets, household appliances, computers, and textiles. The production and use of BFRs is extensive and consists of mainly tetrabromobisphenol A (TBBPA), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecan (HBCD). BFRs in general, but in particular the PBDEs, have led to both scientific and public concern since they have been found to bioaccumulate in humans and wildlife. The general population is targeted by the PBDEs due to their applications and via the food web. Occupational exposure occurs not only during direct handling of BFRs, but also during handling, repair and dismantling of flame retarded goods.</p><p>This thesis is aimed to assess occupational exposure to BFRs. It is mainly focused PBDEs and especially the PBDEs with high bromine content, such as decabromodiphenyl ether (BDE-209). The work has been accomplished by analysis of BFRs in indoor air at industries handling BFRs or flame retarded goods, and by analysis of blood drawn from workers with potential exposure to BFRs. A referent group, abattoir workers with no occupational exposure to PBDEs, was also investigated. Data from these cross-sectional investigations and from serum sampling during vacation in PBDE-exposed workers have been used for calculation of apparent halflives of hepta- to decaBDE in serum.</p><p>The results clearly show that the workers were exposed to PBDEs when handling PBDE containing products or goods. The serum PBDE levels in computer technicians were found to correlate to the estimated cumulative work hours with computers. Exceptionally high concentrations of BDE-209, almost up to 300 pmol/g lipid weight (l.w.) were observed in serum from rubber workers manufacturing or handling rubber compound that was flame retarded with a technical mixture of decabromodiphenyl ether (DecaBDE). Elevated concentrations of PBDEs with eight or nine bromine substituents were also observed. In an electronics dismantling plant, where elevated levels of PBDEs previously had been observed, reduced serum levels of some, but not all PBDE congeners were observed after industrial hygiene improvements. Notably, it was observed that the BDE-209 concentrations in referents with no occupational exposure were similar to the concentrations of 2,2’,4,4’- tetrabromodiphenyl ether (BDE-47), often referred to as the most abundant PBDE congener in humans and wildlife. Additionally, PBDEs with high bromine content were found to have a fast rate of elimination or transformation in humans, based on serum analysis. BDE-209 had an apparent half-life in serum of only 15 days.</p><p>The possibility of quantifying BFRs, such as PBDEs, in human serum at low levels of detection has been achieved by reducing the contamination of the samples and procedural blanks. Major efforts have been done to develop routines and clean up methodology to enable an almost contamination-free environment at the laboratory. The use of a clean room has decreased PBDE levels in the blanks to acceptable limits. The modifications of the original analytical method have made it possible to quantify almost all PBDE congeners of interest in one GC/MS run.</p><p>Occupational and general background exposure of BFRs to humans will continue as long as these chemicals are a part of our daily life and present as environmental contaminants. The present scientific knowledge of the potential health risks of these BFRs still needs to be further developed. It should be stressed that health effects to PBDEs have not been assessed in this work. It is the author's wish that this thesis should add another piece of knowledge to the puzzle of BFRs and BFR exposure to humans and that these data will be used in future risk assessments of PBDEs in particular.</p>

Serum-free mouse embryo (SFME) cells, which were derived from 16-day-old Balb/c mouse embryo brain, grow in absence of serum without losing genomic normality or proliferative potential, and require epidermal growth factor (EGF) for normal growth. EGF is a well studied mitogen that binds to a specific receptor on the cell surface membrane to activate the proliferative signal transduction pathways. The activated receptor is a tyrosine specific protein kinase, and tyrosine phosphorylation is one of the important mediators of EGF receptor (EGFR) signal transduction. Using anti-phosphotyrosine Western immunoblotting, we detected a 100 kDa protein which is tyrosine-phosphorylated in response to EGF in SFME cells. This protein is constitutively phosphorylated in an SFME cell line which expresses the neu oncogene. The neu oncogene encodes an analog protein of EGFR which does not require a ligand for activation, and neu-transformed SFME cells are tumorgenic in mice.This protein, p100 was not a fragment of EGFR, and was not antigenically related to other signal transduction phosphoproteins of about 100 kDa. We attempted to purify p100 from neu SFME tumor cells for amino acid sequencing. / Graduation date: 1997

Graduation date: 1996

Advances in recent nanoscience technologies have generated a new compilation of biocompatible, fluorescent nanoparticles derived from semiconductor quantum dots (QDs). QDs are extremely small in size and possess very large surface areas, which gives them unique physical properties and applications distinct from those of bulk systems. When exposed to biological fluid, these QDs may become coated with proteins and other biomolecules given their dynamic nature. These protein-QD systems may affect or enhance the changes in protein structure and stability, leading to the destruction of biological function. It is believed that these QDs can act as nucleation centers and subsequently promote protein fibril formation. Protein fibrillation is closely associated with many fatal human diseases, including neurodegenerative diseases and a variety of systemic amyloidoses. This topic of protein-QD interaction brings about many key issues and concerns, especially with respect to the potential risks to human health and the environment. Herein, the behavioral effects of dihydrolipoic acid (DHLA)-capped CdSe/ZnS (core/shell) QDs in hen egg-white lysozyme (HEWL) and human serum albumin (HSA) protein systems were systematically analyzed. This study gives rise to a better understanding of the potentially useful application of these protein-QD systems in nanobiotechnology and nanomedicine as a bioimaging tool and/or as a reference for controlled biological self-assembly processes.

Brominated flame retardants (BFRs) are a diverse group of chemicals, which are used to slow down or inhibit the development of fires. BFRs are incorporated into a wide range of consumer products that are considered as potential fire hazards, such as TV-sets, household appliances, computers, and textiles. The production and use of BFRs is extensive and consists of mainly tetrabromobisphenol A (TBBPA), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecan (HBCD). BFRs in general, but in particular the PBDEs, have led to both scientific and public concern since they have been found to bioaccumulate in humans and wildlife. The general population is targeted by the PBDEs due to their applications and via the food web. Occupational exposure occurs not only during direct handling of BFRs, but also during handling, repair and dismantling of flame retarded goods. This thesis is aimed to assess occupational exposure to BFRs. It is mainly focused PBDEs and especially the PBDEs with high bromine content, such as decabromodiphenyl ether (BDE-209). The work has been accomplished by analysis of BFRs in indoor air at industries handling BFRs or flame retarded goods, and by analysis of blood drawn from workers with potential exposure to BFRs. A referent group, abattoir workers with no occupational exposure to PBDEs, was also investigated. Data from these cross-sectional investigations and from serum sampling during vacation in PBDE-exposed workers have been used for calculation of apparent halflives of hepta- to decaBDE in serum. The results clearly show that the workers were exposed to PBDEs when handling PBDE containing products or goods. The serum PBDE levels in computer technicians were found to correlate to the estimated cumulative work hours with computers. Exceptionally high concentrations of BDE-209, almost up to 300 pmol/g lipid weight (l.w.) were observed in serum from rubber workers manufacturing or handling rubber compound that was flame retarded with a technical mixture of decabromodiphenyl ether (DecaBDE). Elevated concentrations of PBDEs with eight or nine bromine substituents were also observed. In an electronics dismantling plant, where elevated levels of PBDEs previously had been observed, reduced serum levels of some, but not all PBDE congeners were observed after industrial hygiene improvements. Notably, it was observed that the BDE-209 concentrations in referents with no occupational exposure were similar to the concentrations of 2,2’,4,4’- tetrabromodiphenyl ether (BDE-47), often referred to as the most abundant PBDE congener in humans and wildlife. Additionally, PBDEs with high bromine content were found to have a fast rate of elimination or transformation in humans, based on serum analysis. BDE-209 had an apparent half-life in serum of only 15 days. The possibility of quantifying BFRs, such as PBDEs, in human serum at low levels of detection has been achieved by reducing the contamination of the samples and procedural blanks. Major efforts have been done to develop routines and clean up methodology to enable an almost contamination-free environment at the laboratory. The use of a clean room has decreased PBDE levels in the blanks to acceptable limits. The modifications of the original analytical method have made it possible to quantify almost all PBDE congeners of interest in one GC/MS run. Occupational and general background exposure of BFRs to humans will continue as long as these chemicals are a part of our daily life and present as environmental contaminants. The present scientific knowledge of the potential health risks of these BFRs still needs to be further developed. It should be stressed that health effects to PBDEs have not been assessed in this work. It is the author's wish that this thesis should add another piece of knowledge to the puzzle of BFRs and BFR exposure to humans and that these data will be used in future risk assessments of PBDEs in particular.