Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.

The recombinant Hepatitis B surface antigen vaccine inadequately protects those living with HIV from Hepatitis B virus infection. This study utilized saved serum samples from a retrospective cohort of persons with HIV and documented vaccine-induced HBsAb seroconversion to determine factors associated with persistence of protective levels of HBsAb (≥10mIU/ml). HBsAb levels fell below 10mIU/ml in 27% of the cohort after a median follow-up of 43 months. HIV viral load suppression (<50copies/ml) at the time of vaccination was the major factor associated with persistence of protective levels of HBsAb (OR 3.83, p <0.01). Among individuals who lost protective levels of HBsAb, booster doses of vaccine re-instated the development of protective levels of HBsAb. Delaying or repeating HBV vaccination until after suppression of HIV viral load is achieved should be considered HBV antibody levels should be followed over time and boosters given with loss of protective levels of HBsAb.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

The recombinant Hepatitis B surface antigen vaccine inadequately protects those living with HIV from Hepatitis B virus infection. This study utilized saved serum samples from a retrospective cohort of persons with HIV and documented vaccine-induced HBsAb seroconversion to determine factors associated with persistence of protective levels of HBsAb (≥10mIU/ml). HBsAb levels fell below 10mIU/ml in 27% of the cohort after a median follow-up of 43 months. HIV viral load suppression (<50copies/ml) at the time of vaccination was the major factor associated with persistence of protective levels of HBsAb (OR 3.83, p <0.01). Among individuals who lost protective levels of HBsAb, booster doses of vaccine re-instated the development of protective levels of HBsAb. Delaying or repeating HBV vaccination until after suppression of HIV viral load is achieved should be considered HBV antibody levels should be followed over time and boosters given with loss of protective levels of HBsAb.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

B virus (Macacine herpesvirus 1), subfamily Alphaherpesvirinae, causes a fatal, neurovirulent infection in zoonotically infected humans. Macaques (Macaca sp.) serve as the natural host for B virus and they are frequently seropositive for B virus antibodies without showing any overt signs of disease. The global hypothesis of these studies is that B virus, a highly cytopathic virus in macaques, subverts the innate immune responses in the host (macaques) that has co-evolved with it (the virus) differently than it does the foreign host (humans). The foreign host, frequently fails to produce neutralizing antibodies early after infection and this may be due to a dysregulation or inhibition of pathways known to play a role in the innate immune response which directs the adaptive defense responses. Current knowledge is that at least five major signaling pathways can be activated after a pathogen such as B virus enters a host cell (REF). These include the IRF3 pathway, the NFkB pathway, the NFAT pathway, and the MAPK pathway. Early stimulation of one or more of these pathways leads to the induction of the proinflammatory response and subsequent induction of cytokines such as IL6, IL8 and IL10, and apoptosis. Cytokine induction and apoptosis play important roles in host-pathogen interactions, innate defense induction and subsequent adaptive immune responses. Using a primary cell model that is representative of the first target cells of B virus in the natural and foreign host, we investigated one of the key signaling pathways, the MAPK pathway, induced by B virus early after infection (Farah-Abraham and Hilliard, unpublished data). My data suggest that macaque and human cells differ in the induction kinetics of MAPK (JNK and p38) activation. These data reveal differences between foreign and natural host cells in how each controls apoptosis, and demonstrate that inhibition of p38 activation reduced and with high dose inhibition terminated B virus replication in human cells, and played a role in reduction of apoptosis-associated mediators. The importance of each component in the MAPK pathway is investigated with respect to virus replication in macaque and human cells that represent the primary target cells in acute infection. Knowledge of these events provides an understanding of how the innate immune responses can be modulated by B virus to shape the adaptive immune response to limit how the virus replicates and spreads. Further, these data may provide insight into a novel target for the design of new antivirals to inhibit this deadly zoonotic virus. This research will help us understand how the early molecular mechanisms of host-pathogen interactions result in modulation of the innate immune responses and how certain aspects of a normally defensive (protective) host response can be re-directed or modified depending on the nature of the virus:host relationship.

We present a measurement of the top anti-top quark (ttbar)production cross section in the dilepton final states from proton-proton collisions at a center of mass energy at 7 TeV at the LHC. A b-tagging algorithm based on tracks displaced from the event interaction vertex is applied to identify bottom quark jets from top quark decay and reject background events. Given the relatively pure sample of bottom quark jets in ttbar dilepton final states, a new technique to measure in-situ the b-tagging efficiency is introduced that uses the distribution of the number of observed b-tagged jets. We present results with data collected at the ATLAS detector in 2010 with an integrated luminosity of 35 pb-1. The measured ttbar cross section is 176 +22/-21 (stat.) ± 20 (syst.) ± 6 (lum.) pb in the dilepton channel. We will also discuss the future prospects of this measurement.

Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.

Hepatitis B is a serious global health problem with over 2 billion people infected worldwide and 350 million suffering from chronic hepatitis B (CHB) infection. Infection can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) accounting for 320,000 deaths per year. Numerous treatments are available, but with a growing number of therapies each with considerable trade-offs, the optimal treatment strategy is not transparent. This dissertation investigates the relative efficacy of treatments for CHB and estimates the health related quality of life (HRQOL) and health utilities of mild to advanced CHB patients. A systematic review of published randomized controlled trials comparing surrogate outcomes for the first year of treatment was performed. Bayesian mixed treatment comparison meta-analysis was used to synthesize odds ratios, including 95% credible intervals and predicted probabilities of each outcome comparing all currently available treatments in HBeAg-positive and/or HBeAg-negative CHB patients. Among HBeAg-positive patients, tenofovir and entecavir were most effective, while in HBeAg-negative patients, tenofovir was the treatment of choice. Health state utilities and HRQOL for patients with CHB stratified by disease stage were elicited from patients attending tertiary care clinics at the University Health Network in Toronto. Respondents completed the standard gamble, EQ5D, Health Utilities Index Mark 3 (HUI3), Short-Form 36 version-2 and a demographics survey in their preferred language of English, Cantonese or Mandarin. Patient charts were accessed to determine disease stage and co-morbidities. The study included 433 patients of which: 294 had no cirrhosis, 79 had compensated cirrhosis, 7 had decompensated cirrhosis, 23 had HCC and 30 had received liver transplants. Mean standard gamble utilities were 0.89, 0.87, 0.82, 0.84 and 0.86 for the respective disease stages. HRQOL in CHB patients was only impaired at later stages of disease. Neither chronic infection nor antiviral treatment lowered HRQOL. Patients with CHB do not experience lower HRQOL as seen in patients with hepatitis C. The next step in this area of research is to incorporate the estimates synthesized by the current studies into a decision model evaluating the cost-effectiveness of treatment to provide guidance on the optimal therapy for patients with HBeAg-positive and HBeAg-negative CHB.

We present a measurement of the top anti-top quark (ttbar)production cross section in the dilepton final states from proton-proton collisions at a center of mass energy at 7 TeV at the LHC. A b-tagging algorithm based on tracks displaced from the event interaction vertex is applied to identify bottom quark jets from top quark decay and reject background events. Given the relatively pure sample of bottom quark jets in ttbar dilepton final states, a new technique to measure in-situ the b-tagging efficiency is introduced that uses the distribution of the number of observed b-tagged jets. We present results with data collected at the ATLAS detector in 2010 with an integrated luminosity of 35 pb-1. The measured ttbar cross section is 176 +22/-21 (stat.) ± 20 (syst.) ± 6 (lum.) pb in the dilepton channel. We will also discuss the future prospects of this measurement.