Protein folding in the endoplasmic reticulum (ER) is essential for proper cellular function. However, the sensitive environment in the ER can be perturbed by both pathological processes as well as by physiological processes such as a large biosynthetic load placed on the ER. ER stress is a specific type of intracellular stress caused by the accumulation of immature or abnormal misfolded or unfolded proteins in the ER. Simply defined, ER stress is a disequilibrium between ER load and folding capacity. Cells have an adaptive response that counteracts ER stress called the "Unfolded Protein Response” (UPR). The ability to adapt to physiological levels of ER stress is especially important for maintaining ER homeostasis in secretory cells. This also holds true for pancreatic β-cells, which must fold and process large amounts of the hormone insulin. Pancreatic β-cells minimize abnormal levels of glycemia through adaptive changes in the production and regulated secretion of insulin. This process is highly sensitive, so that small degrees of hypo- or hyperglycemia result in altered insulin release. The frequent fluctuation of blood glucose levels in humans requires that β-cells control proinsulin folding in the ER with exquisite sensitivity. Any imbalance between the load of insulin translation into the ER and the actual capacity of the ER to properly fold and process the insulin negatively affects the homeostasis of β-cells and causes ER stress. In this dissertation, we show that Inositol Requiring 1 (IRE1), an ER-resident kinase/endoribonuclease and a central regulator of ER stress signaling, is essential for maintaining ER homeostasis in pancreatic β-cells. Importantly, IRE1 has a crucial function in the body’s normal production of insulin in response to high glucose. Phosphorylation and subsequent activation of IRE1 by transient exposure to high glucose is coupled to insulin biosynthesis, while inactivation of IRE1 by siRNA or inhibition of IRE1 phosphorylation abolishes insulin biosynthesis. IRE1 signaling under these physiological ER stress conditions utilizes a unique subset of downstream components of IRE1 and has a beneficial effect on pancreatic β-cell homeostasis. In contrast, we show that chronic exposure of β-cells to high glucose causes pathological levels of ER stress and hyperactivation of IRE1, leading to the degradation of insulin mRNA. The term “glucose toxicity” refers to impaired insulin secretion by β-cells in response to chronic stimulation by glucose and is characterized by a sharp decline in insulin gene expression. However, the molecular mechanisms of glucose toxicity are not well understood. We show that hyperactivation of IRE1 caused by chronic high glucose treatment or IRE1 overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1 signaling using a dominant negative form of the protein prevents insulin mRNA degradation in β-cells. Additionally, islets from mice heterozygous for IRE1 retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates. This work suggests that the rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism may represent an essential element in the adaptation of β-cells to chronic hyperglycemia. This adaptation is crucial for the maintenance of β-cell homeostasis and may explain in part why the β-cells of diabetic patients with chronic hyperglycemia stop producing insulin without simply undergoing apoptosis. This work implies that prolonged activation of IRE1 signaling is involved in the molecular mechanisms underlying glucose toxicity. This work therefore reveals two distinct activities elicited by IRE1 in pancreatic β-cells. IRE1 signaling activated by transient exposure to high glucose enhances proinsulin biosynthesis, while chronic exposure of β-cells to high glucose causes hyperactivation of IRE1, leading to the degradation of insulin mRNA. Physiological IRE1 activation by transient high glucose levels in pancreatic β cells has a beneficial effect on insulin biosynthesis. However, pathological IRE1 activation by chronic high glucose or experimental drugs negatively affects insulin gene expression. In the future, a system to induce a physiological level of IRE1 activation, and/or reduce the pathological level of IRE1 activation could be used to enhance insulin biosynthesis and secretion in people with diabetes, and may lead to the development of new and more effective clinical approaches to the treatment of this disorder.

INTRODUÇÃO: Obesidade é, hoje, considerada uma pandemia, com cerca de 500 milhões de obesos no mundo, com cerca de 2,8 milhões de mortes por ano. A cirurgia de bypass gástrico é um importante tratamento para obesidade, porém, não é isenta de riscos. O dispositivo temporário de exclusão duodeno jejunal - DTED (EndoBarrier Gastrointestinal Liner® GIDynamics, Inc. Lexington, MA), apresenta-se como uma nova forma de tratamento endoscópico da obesidade. Apesar dos bons resultados, os mecanismos de ação do DTED ainda não foram estudados, podendo as alterações humorais e do esvaziamento gástrico promovidas, ser os principais responsáveis pelos resultados obtidos. OBJETIVO: Estudar as alterações promovidas pelo DTED no esvaziamento gástrico, e a relação destas alterações com os resultados clínicos de perda de peso e controle do diabetes tipo 2. MÉTODOS: Vinte e cinco obesos e com diabetes tipo 2, que fizeram uso do DTED por período mínimo de 16 semanas e máximo de 24 semanas, realizaram teste de esvaziamento gástrico cintilográfico, antes, durante a 16ª semana de uso e após 4 semanas de retirada do DTED. Foram obtidas medidas de peso e hemoglobina glicada. As médias e desvio-padrão de retenção gástricas foram obtidas e comparadas entre os três exames realizados, e, após, comparados entre os pacientes que obtiveram e os que não obtiveram melhora no parâmetro clínico selecionado (perda de peso maior que 10%, e hemoglobina glicada menor que 7%). Também se avaliou subjetivamente a sensação de saciedade e quantidade de alimento ingerido durante a 16ª semana de uso do dispositivo. RESULTADOS: Quando avaliadas médias de retenção, nota-se que, na 16ª semana de uso, há maior retenção para a primeira, segunda e quarta horas quando comparados ao baseline (1ª h 74 ± 16,3 % p=0,001, 2ª h 45 ± 25% p < 0,001; 4ª 15 ± 15,8% p < 0,001). Não há diferença estatística entre as retenções na 16ª semanas entre os pacientes que atingiram e os que não atingiram o controle do diabetes (p=0,73), entre os que perderam mais de 10% de peso e os que não perderam (p=0,275). Durante a 16ª semana de uso, 23 pacientes (92%) referiram maior sensação de saciedade precoce e maior saciação, e todos referiram comer em menor volume de em relação ao período prévio à colocação do dispositivo. CONCLUSÕES: O DTED causa lentificação no esvaziamento gástrico, reversível após sua retirada, porém esta alteração no esvaziamento gástrico, mesmo sendo sintomática, com aumento de saciedade e saciação, e com diminuição do volume de alimento ingerido, não tem relação com a perda de peso e melhora do diabetes / INTRODUCTION: Obesity is now considered a pandemic, with about 500 million obese worldwide, with about 2.8 million deaths per year. The gastric bypass surgery is an important treatment for obesity, however, not without risks. The temporary duodenal jejunal exclusion device - DTED (EndoBarrier ® Gastrointestinal Liner GIDynamics, Inc. Lexington, MA), presents itself as a new form of endoscopic treatment of obesity. Despite the good results, the mechanisms of action of DTED have not been studied, and the humoral changes and changes in gastric emptying promoted by the device maybe are the main mechanisms of action of the device. OBJECTIVE: To study the changes introduced by DTED in gastric emptying, and the relationship of these changes with clinical outcomes of weight loss and control of type 2 diabetes. METHODS: Twenty five obese patients with type 2 diabetes who used the DTED for a minimum of 16 weeks and maximum 24 weeks underwent a scintigraphic gastric emptying test, before, during the 16th week of treatment and after 4 weeks of withdrawal the DTED. Measurements of weight, glycated hemoglobin were obtained. The mean and standard deviation of gastric retention were obtained and compared between the three tests, and after, compared between patients who were and those who showed no improvement in selected clinical parameters (weight loss greater than 10%, and lower glycated hemoglobin 7%). Also, a subjective evaluation of the feeling of satiety and amount of food ingested during the 16 weeks of device use was done. RESULTS: When evaluated average retention , we note that in the 16th week of use there is greater retention for the first, second and fourth hour compared to baseline (1st h 74 ± 16.3 % p = 0.001, 2nd h 45 ± 25 % p < 0.001 4th 15.8 ± 15 %, p < 0.001). There is no statistical difference among patients who achieved and those who have not reached the control of diabetes (p = 0.73) or among those who lost more than 10 % by weight and not lost (p = . 0.275) during the 16th week of treatment , 23 patients (92%) reported greater sense of early satiety and satiation greater, and all reported eating less volume of food in relation to the period prior to devide placement. CONCLUSIONS: The DTED cause delay in gastric emptying, reversible after withdrawal, though this change in gastric emptying, even being symptomatic with increased satiety and satiation , decreasing the volume of food ingested , has no relation to weight loss and improved diabetes

O objetivo deste trabalho foi avaliar a aplicação clínica e a efetividade da coagulação com plasma de argônio(CPA) usada para realizar a ablação do esôfago de Barrett. A presença desta moléstia é considerada uma condição pré maligna com potencial para o desenvolvimento do adenocarcinoma de esôfago. Um estudo clínico prospectivo foi realizado com um grupo de 30 pacientes portadores de esôfago de Barrett, diagnosticado por meio de endoscopia digestiva alta e histologia. 25 pacientes eram do sexo masculino com idade variando entre 12 e 72 anos (média = 47,1) e 5 pacientes eram do sexo feminino cuja idade variou entre 45 e 60( média=49.9). Os pacientes eram submetidos à cirurgia antirefluxo e depois encaminhados para o tratamento com a CPA. O tratamento era realizado sob sedação em regime ambulatorial. Os pacientes eram submetidos a sessões com intervalos de 30 dias até obter o desaparecimento completo da lesão à endoscopia. Após três meses eram realizadas biópsias para o controle de cura. O tamanho médio da área aplicada mostrou correlação com a sintomatologia e o aparecimento de complicações.Uma área maior que quatro cm correlacionou-se positivamente com estas variáveis. O número médio de sessões foi de 1 a 6 (média=2,1). O tempo médio de seguimento variou entre 29 dias a 4 anos com média de 1 ano e 4 meses. Em dois casos houve presença de epitélio de Barrett sob o neo revestimento que foi tratado novamente com bom resultado. As complicações ocorreram em 2 casos (6,6%) de estenose de esôfago e um caso(3,3%) de pneumo mediastino todas tratadas com bons resultados. O índice de sucesso com ablação total foi de 93,4%. Não houve mortalidade neste estudo. / The aim of this study is to determine the effectiveness of the Argon Plasma Coagulation (APC) in ablation therapy of specialized columnar epithelium in Barrett´s esophagus. The presence of Barrett´s epithelium is considered a premalignant condition with potential development of adenocarcinoma. The incidence of esophageal adenocarcinoma has been rising for the past 3 decades A prospective study performed with a group of 30 patients(25 men/5 women) with 47 years mean age (range 12 to72) for the men and 49,9 years mean age ( range 45 to 60) for the women presented with Barrett´s esophagus demonstrated by endoscopy and histology.They were referred to us after antireflux surgery and were assimptomatic at the beginning of the study. Application of APC was carried out under sedation (midazolan and meperidine) with a gas flow of 2,5 l/min & 70 w. The Barrett\'s epithelium was coagulated from the most proximal to the gastrointestinal junction to the most distal limit. The maximum size treated each time was 4 cm long. Special care was taken on coagulation of the visible small islands of remaining intestinal metaplasia tissue. The treatment performed in large areas, bigger than 4 cm led to the increasing rate of complications and transient symptoms. The patients returned monthly to new session until complete ablation of the Barrett´s esophagus was showed by endoscopy. Then, 3 months later after that, a extensive random biopsies were taken to histology search for Barrett\'s. The mean number of APC sessions was 2,1 (range 1 to 6). In two cases, endoscopy showed absence of intestinal metaplasia but histology shows small islands of intestinal metaplasia under the neosquamous epithelium. Two(6,6%) esophageal stenosis and one (3,3%) pneumomediastin case have been occurred. The follow up ranged to 27days to 4 years (mean one year and four months). Reepitelialization with squamous epithelium indistinguishable of the esophageal mucosa was demonstrated by endoscopy in 93,4%. There has been no relapse or evidence of the development of dysplasia. No deaths or major complication occurred in this study.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Spontaneous cases of chronic poisoning by Pteridium aquilinum in cattle were studied. The clinical forms of the disease were squamous cell carcinoma (SCCs) of the upper digestive tract (UDT) and bovine enzootic hematuria (BEH). The cases were from the midland Region of the Midwest of Rio Grande do Sul State, Brazil. For the epidemiological study, the profile of the farms was analyzed about herd purpose, main activity of the farm, altitude, and area of plant infestation. No differences were observed among the clinical forms, according to these criteria. Analysis of the ager, gender, and breed of the affected cattle revealed that, in both clinical forms of disease, mixed breed cows (more common in that region) were more affected. In BEH, adult cattle (3-to-7-years-old) were more frequently affected. In the form of UDT SCCs, aged cattle (more than 8- years-old) were more affected. For the clinical study, clinical signs and blood work were evaluated at terminal phase of disease. Cattle with UDT SCCs, had progressive weigth loss, ruminal atony, cough, dysphagia, bloating, and regurgitation. Hematuria was clinically observed in one case of this disease form. In cattle with BEH, hematuria was observed in all cases, followed by progresive weigth loss. Non-regenerative anemia was detected in 33.33% in UDT SCCs form and in 66.66% of BEH form. Changes in white blood count occurred in some cases but drop in lymphocytes was uncommon in both forms of disease. For the morfological study, urinary bladders of 46 UDT SCCs cases and 11 BEH cases were analyzed. Grossly, 16/46 bladders of the UDT SCCs form had gross lesions (vesical red or pale nodules, hemorrhages, and papilomas; red urine in the three cases). In BEH form, the bladder had nodules, large neoplastic masses, red urine, papilomas, and hemorrhages. Pielonephritis and hidronephosis were seen in a few cases. Microscopically, in the UDT SCCs form, 44/46 (95.65%) bladders had 22 different types of morphological changes, caracterized by neoplastic lesions (5/22) and non-neoplastic lesions (17/22), which were subdivided in non-neoplastic epithelial changes (6/17), general changes of the lamina propria (6/17), and inflammatory changes (5/17). The bladder changes in BEH form were of 19 different types, caracterized by neoplastic lesions (5/19) and non-neoplastic lesions (14/19), which were subdivided in non-neoplastic epitelial changes (9/14), general changes of the lamina propria (3/14), and inflammatory changes (2/14). In BEH, mesenchymal neoplasms were more observed than epithelial ones, and most of them were malignant. Immunohistochemistry was utilized to characterize the histogenesis of poorly differentiated neoplasms. In conclusion, the morfological study showed that urinary bladder lesions are very often in cattle with the UDT SCCs form, and were identical to the ones seen in cattle with BEH. / Foram estudados casos espontâneos de intoxicação crônica por samambaia em bovinos nas formas clinicopatológicas de carcinoma de células escamosas (CCE) no trato alimentar superior (TAS) e de hematúria enzoótica bovina (HEB), provenientes da Mesorregião Centro Ocidental do Rio-Grandense e encaminhados ao Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM). Para o estudo epidemiológico, foi avaliado o perfil das propriedades quanto à finalidade da criação, principal atividade da propriedade, altitude e área de infestação pela planta. Quanto a esses parâmetros, as propriedades com uma ou outra forma de intoxicação crônica não apresentaram diferenças. Foi determinado o perfil dos bovinos afetados quanto à idade, sexo e raça. Os mais afetados por ambas as formas clínicas foram fêmeas de raça mista (predominante na região). Na HEB houve predomínio de bovinos adultos (três a sete anos) e a forma de CCEs no TAS afetou mais bovinos idosos (mais de oito anos). Para o estudo clínico foram avaliados os sinais clínicos de bovinos com CCEs no TAS e com HEB e realizados hemogramas na fase terminal da doença. Os principais sinais clínicos nos bovinos com CCEs no TAS foram emagrecimento progressivo, atonia ruminal, tosse, disfagia, timpanismo, regurgitação e hematúria, vista em um caso. Nos bovinos com HEB, hematúria foi o principal sinal, observado em todos os casos, seguido de emagrecimento progressivo. No exame hematológico, 33,33% dos bovinos com CCEs no TAS e 66,67% dos bovinos com HEB apresentaram anemia arregenerativa. Alterações no leucograma ocorreram em alguns casos, mas linfopenia foi um achado infreqüente em ambas as formas de intoxicação. Para o estudo morfológico, foram avaliadas as bexigas de 46 casos de CCEs no TAS e de 11 casos de HEB. Macroscopicamente, 16/46 bexigas dos casos de CCEs no TAS apresentaram alterações macroscópicas, que foram nódulos vermelhos ou pálidos, hemorragia e papilomas; urina vermelha foi observada em três casos. Nos casos de HEB, os achados macroscópicos vesicais foram nódulos vermelhos, massas neoplásicas focalmente extensas, urina vermelha, papilomas, hemorragias e ruptura de bexiga; pielonefrite e hidronefrose foram observados em poucos casos. Histologicamente, 44/46 (95,65%) bexigas de bovinos com CCEs no TAS apresentaram 22 tipos diferentes de alterações morfológicas, que foram classificadas em alterações neoplásicas (5/22) e alterações não-neoplásicas (17/22), as quais foram divididas em alterações epiteliais não-neoplásicas (6/17), alterações gerais na lâmina própria (6/22) e alterações inflamatórias (5/17). Os achados histológicos das bexigas dos casos de HEB foram classificados da mesma forma, resultando em 19 tipos diferentes de alterações morfológicas. Dessas, 5/19 eram alterações neoplásicas e 14/19, alterações não-neoplásicas (9/14 alterações epiteliais não neoplásicas, 3/14 alterações gerais na lâmina própria e 2/14 alterações inflamatórias). Na HEB, os neoplasmas mesenquimais foram mais observados que os epiteliais, e a maior parte era maligno. A técnica de imuno-histoquímica foi utilizada para caracterizar os aspectos morfológicos, principalmente dos neoplasmas. Através do estudo morfológico concluiu-se que é muito freqüente a ocorrência de lesões vesicais em bovinos com a forma crônica de CCEs no TAS e que essas são idênticas às encontradas nos bovinos com HEB.

Tissue homeostasis in the adult Drosophila melanogaster intestine is maintained by controlling the proper balance of stem cell self-renewal and differentiation. In the adult fly midgut, intestinal stem cells (ISCs) are the only dividing cells and their identity maintenance is crucial to the proper functioning of the fly gut. Various pathways such as Notch, JAK-STAT and Wingless are known to regulate ISC division and differentiation. Here I used a pathogen feeding model to study conditions that accelerate ISC division and guide intestinal cell differentiation favoring enterocyte development. I also examined the role of Tumor Suppressor Gene 101 (TSG101) in ISC maintenance and function. TSG101, a part of the ESCRT1 complex. It is known to stimulate the Notch pathway and to play a role in endocytic trafficking. TSG101 loss-of-function mutants show developmental defects in various fly and mammalian tissues. The protein also plays a role in virus abscission from host cells. In my experiments I have observed that TSG101 is required for ISC maintenance. TSG101 knockdown and loss of function mutant clones have defects in ISC proliferation that hinder the normal intestinal responses to oral pathogen ingestion. Based on these results I conclude that TSG101 is needed in the adult fly intestine for proper ISC maintenance and function, thereby being an important player in intestinal homeostasis.

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.