Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.

A rising problem in agriculture is the increase of antibiotic-resistant Salmonella cases associated with chicken eggs, which transmit infection to humans. To counter this, new approaches to combat Salmonella in chickens and desiccated on eggshells are vital in the prevention of human foodborne illness. Disrupting signaling pathways with naturally occurring compounds provides a potential novel avenue for prevention of Salmonella infections, as this would disrupt sensing of these environments and inhibit subsequent optimal gene expression. Starting with signals identified in previous studies, salicylic acid was found to inhibit Salmonella desiccation survival on both eggshells and plastic. To expand upon this, a desiccation inhibition screen of 285 signals resulted in 9 additional potential desiccation inhibitors, including deoxyribose and guanine. By using natural signals to disrupt bacterial communication pathways, novel therapeutics that serve as viable antibacterial alternatives could be developed to prevent Salmonella contamination at a major source.

Homeodynamic space (HDS) shrinks as vulnerability increases with aging and repeated damage to the cells. HDS is lost in alcoholic pancreatitis patients due to overconsumption of alcohol, smoking, and high fat diets. Etiologically relevant animal models for study of chronic pancreatitis (CP) are needed. In order to begin filling this gap a central purpose of this dissertation research was to examine relationships between the alcohol and high fat diet (AHF) and pancreatitis with attention to hypersensitivity and anxiety-like behaviors. The AHF diet induced pancreatitis described here etiologically mimics human risk factors of AHF consumption for advancement to alcoholic CP. In this study one group of mice was fed long term with a diet of high fat and alcohol for comparison with a group fed normal chow. Mice consumed a liquid diet containing 6% alcohol and a high fat supplement ad libitum over a period of five months. Each group was evaluated for heat and mechanical hypersensitivity, and histology indicative of CP. The association of pancreatitis pathology with anxiety has been understudied. Anxiety, like pain, is useful as a transient state but when anxiety is prolonged it is termed a disorder. Anxiety is often comorbid with pain and depression. Therefore, it is important to determine anxiety in mice with CP histology. This model was characterized for the interaction of pancreatitis histology, as well as persisting pain-, anxiety-, and fear-like behaviors. The AHF diet mice developed hypersensitivity, demonstrated anxiety-like behaviors, and showed concurrent histology consistent with CP. Nontransgenic mouse models where pancreatitis is induced only by a combination of ad libitum liquid food with added alcohol and lard supplementation do not currently exist, nor has an in-depth study of anxiety-like behaviors been conducted in this mouse model. This dissertation research addresses this knowledge gap.

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Autism spectrum disorders (ASD) encompass neurodevelopment disorders characterized by atypical patterns of development that impact multiple areas of functioning beginning in early childhood. The etiology of ASD is unknown and there are currently no preventative treatment options. Gastrointestinal symptoms are commonly associated comorbidities. The microbiota-gut-brain axis is a multidirectional communication chain that connects the central and enteric nervous system that relates brain function to peripheral intestinal functions. Changes within this axis have been postulated in ASD. For example, the “leaky gut theory” proposes that chronic inflammation is linked to alterations in the bacterial profiles of the gut microbiome and subsequent shifts in the amount and type of short-chain fatty acids produced can affect downstream neuronal development. Short-chain fatty acids are signaling molecules produced by bacteria that can trigger nerve afferents in the gut. Dysbiosis causes altered signaling patterns that can be identified by altered intestinal morphology. In this study, C57BL/6J control mice and three mouse models of social behavioral deficits were used to investigate markers of intestinal pathophysiology. Fecal and intestinal samples were collected from adult wild type control mice and the social deficit groups of BTBR genetic knockout mice, C57BL/6J mice injected with valproic acid, and C57BL/6J mice injected with polyinosinic–polycytidylic acid. Short-chain fatty acid profiles that included acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids were obtained from fecal samples to determine differences between the models and control mice. The profiles of the BTBR genetic knockout and valproic acid models were found to be significantly different from control mice. Additionally, postmortem intestinal ileum samples underwent hematoxylin and eosin identification procedures to determine the thickness of the tunica muscularis and tunica mucosa. The thickness of the tunica muscularis was reduced in the valproic acid group compared to the wild type control mice in early stages of development (p=0.0279). This research may illuminate developmental cues that attribute to autism spectrum disorders and may provide markers to assess future therapeutic treatments.

Helicobacter pylori causes chronic gastritis, peptic ulceration and gastric cancer. This bacterium is one of the most prevalent in the world, but affects mostly the populations with a lower socioeconomical status. While it causes gastric and duodenal ulcers in only 20% of infected patients, less then 1% will develop gastric adenocarcinoma. In fact, H. pylori is the most important risk factor in developing gastric cancer. Epidemiological studies have shown that 80% of gastric cancer patients are H. pylori positive. The outcome of the infection with this bacterium depends on bacterial factors, diet, genetic background of the host, and coinfection with other microorganisms. The most important cofactor in H. pylori induced disease is the host immune response, even though the exact mechanism of how the bacterium is causing disease is unknown. The structural complexity of Helicobacter bacteria makes us believe that different bacterial factors interact with different components of the innate immunity. However, as a whole bacterium it may need mainly the TLR2 receptor to trigger an immune response. The type of adaptive immunity developed in response to Helicobacter is crucial in determining the consequences of infection. It is now known for decades that a susceptible host will follow the infection with a strong Th1 immune response. IFNγ, IL-12, IL-1β and TNF-α are the key components of a strong adaptive Th1 response. This is further supported by our work, where deficient T-bet (a master regulator for Th1 response) mice were protected against gastric cancer, despite maintaining an infection at similar levels to wild type mice. On the other hand, a host that is resistant to Helicobacter develops an infection that is followed by a Th2 response sparing the mucosa from severe inflammation. Human studies looking at single nucleotide polymorphism of cytokines, like IL-1β, IL-10 and TNF-α have clearly demonstrated how genotypes that result in high levels of IL-1β and TNF-α, but low IL-10 expression may confer a 50-fold higher risk in developing gastric cancer. The outcome of Helicobacter infection clearly relies on the immune response and genetic background, however the coinfection of the host with other pathogens should not be ignored as this may result in modulation of the adaptive immunity. In studying this, we took advantage of the Balb/C mice that are known to be protected against Helicobacter induced inflammation by mounting a strong Th2 polarization. We were able to switch their adaptive immunity to Th1 by coinfected them with a T. gondii infection (a well known Th1 infection in mice). The dual infected mice developed severe gastritis, parietal cell loss and metaplastic changes. These experiments have clearly shown how unrelated pathogens may interact and result in different clinical outcomes of the infected host. A strong immune response that results in severe inflammation will also cause a cascade of apoptotic changes in the mucosa. A strict balance between proliferation and apoptosis is needed, as its disruption may result in uncontrolled proliferation, transformation and metaplasia. The Fas Ag pathway is the leading cause of apoptosis in the Helicobacter-induced inflammation. One mechanism for escaping Fas mediating apoptosis is upregulation of MHCII receptor. Fas Ag and MHCII receptor interaction inhibits Fas mediated apoptosis by an impairment of the Fas Ag receptor aggregation when stimulated by Fas ligand. Because H. pylori infection is associated with an upregulation of the MHCII levels on gastric epithelial cells, this indeed may be one mechanism by which cells escape apoptosis. The link between chronic inflammation and cancer is well known since the past century. Helicobacter infection is a prime example how a chronic inflammatory state is causing uncontrolled cell proliferation that results in cancer. The cell biology of “cancer” is regarded not as an accumulation of cells that divide without any control, but rather as an organ formed of cancer stem cells, tumor stromal support cells, myofibroblasts and endothelial cells, which function as a group. The properties of the cancer stem cells are to self-renew and differentiate into tumor cells thus maintaining the tumor grow, emphasizing that a striking similarity exists between cancer stem cells and tissue stem cells. We looked into what role would BMDCs play in chronic inflammation that causes cancer. Using the mouse model of Helicobacter induced adenocarcinoma we discovered that gastric cancer originates from a mesenchymal stem cell coming from bone marrow. We believe that chronic inflammation, in our case of the stomach, sets up the perfect stage for bone marrow stem cells to migrate to the stomach where they are exposed to inflammatory stimuli and transform into cancer stem cells. One of the mechanism by which the MSC migrate to the inflammation site is the CXCR4/SDF-1 axis. Our work sheds new light on Helicobacter induced gastric cancer pathogenesis. I hope that our findings will promote the development of new therapies in the fight against this deadly disease.

Central driving forces in the pathogenesis of liver disease are hepatocyte death and immune cell-driven inflammation. The interplay between outcomes, stemming from these two major cell types, is present from the earliest ethanol exposure, and are both determinants in advanced stages of liver disease particularly in alcoholic liver disease (ALD). The complexities associated with advanced ALD are many and therapies are limited. Due to the liver’s role in ethanol metabolism and filtering gut-derived products, it is becoming increasingly clear that innate immunity plays a central role in triggering activation of cell death and inflammatory pathways in ALD. We identified interferon regulatory factor 3 (IRF3) activation as a mediator of hepatocyte death as the first event after ethanol exposure, and the inflammasome as a protein complex responsible for the subsequent inflammatory cascade, driven by the NLRP3 inflammasome. Our novel findings in murine samples and human patients with alcoholic hepatitis demonstrate that ethanol-induced inflammasome activity results in Caspase-1-mediated pyroptosis and extracellular ASC aggregates in the liver and circulation. Pyroptosis can be abrogated by therapeutic inhibition of inflammasome components, NLRP3 or Caspase-1. Taken together, the event leading to mtDNA release into the cytoplasm is the inception of the pathogenesis of ALD, triggering hepatocyte death, culminating in a pro-inflammatory cascade driven by the NLRP3 inflammasome and pyroptotic release of ASC.

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Chronic alcohol use results in accelerated liver injury, leading to alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. However, due to the complex nature of this disease process, a central, druggable mechanism has remained elusive. microRNAs are potent post-transcriptional regulators of gene expression. A single miRNA has the ability to regulate hundreds of pathways simultaneously, defining cellular fate and function. microRNA-122 (miR-122), the most abundant miRNA in hepatocytes, has a demonstrated role as an tumor suppressor, regulator of hepatocyte metabolism, and hepatic differentiation. In this dissertation I demonstrate the role of miR-122 on alcoholic liver disease (ALD) pathogenesis over four parts. In chapter II, I will demonstrate chronic alcoholic patients, free of neoplastic changes, have a reduction of miR-122 and that this miRNA regulates HIF-1α, a determinant of ALD pathogenesis. In chapter III, using hepatocytetropic adeno-associated virus 8 (AAV8) vector, I demonstrate that miR-122 inhibition mimics ALD pathogenesis, and furthermore, using hepatocyte-specific HIF-1α-null (HIF1hepKO) mice that this phenomenon is HIF-1α dependent. Given this finding, in chapter IV, I demonstrate that ectopic expression of miR-122 in vivo can reverse alcoholinduced liver damage, steatosis, and inflammation by directly targeting HIF-1α. Finally, in chapter V, I present evidence that alcohol-induced dysregulation of grainyhead-like proteins 1 and 2 (GRHL2), mediate the inhibition of miR-122 at the transcriptional level. These findings dissect a novel mechanistic regulatory axis of miR-122 and indicate a potential opportunity for restoration of miR-122 as a therapy in early ALD.

HCV afflicts many millions of people globally, and antiviral therapies are often ineffective and intolerable. The Food and Drug Administration approved the HCV protease inhibitors telaprevir and boceprevir in May 2011, marking an important milestone in anti-HCV research over the past two decades. Nevertheless, severe drug side effects of combination therapy – flu-like symptoms, depression and anemia – limit patient adherence to treatment regimens. The acquisition of resistance challenges the long-term efficacy of antiviral therapies, including protease inhibitors, as suboptimal dosing allows for the selection of drug resistant viral variants. A better understanding of the molecular basis of drug resistance is therefore central to developing future generation protease inhibitors that retain potency against a broader spectrum of HCV strains. To this end, my research characterizes the molecular basis of drug resistance against HCV protease inhibitors. Chapter II defines the mode of substrate recognition by the common volume shared by NS3/4A substrate products – the substrate envelope. Chapter III then correlates patterns of drug resistance to regions where drugs protrude from the substrate envelope. Lastly, Chapter IV elucidates the molecular underpinnings of resistance against four leading protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – and provides practical approaches to designing novel drugs that are less susceptible to resistance. I ultimately hope my work appeals to the broader biomedical community of virologists, medicinal chemists and clinicians, who struggle to understand HCV and other human pathogens in the face of rapid disease evolution.