Increasing evidence suggests that normal pulmonary vascular morphogenesis is critical for the formation and maintenance of the lung parenchyma, both pre- and postnatally. Indeed, the disruption of angiogenic pathways, whether through inherent genetic predisposition or as a consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). To understand the etiology of – and advance treatments for – such diseases, we must first identify the fundamental genetic regulators that orchestrate normal parenchymal development. Neuropilin-1 (NRP1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development, and binds two distinct ligand families: Vascular endothelial growth factor (VEGF) and Class 3 Semaphorins (SEMA3). Although VEGF-NRP1 interactions in systemic vascular development have been described, the importance of SEMA3-NRP1 signalling in systemic or pulmonary vascular morphogenesis is uncertain. We hypothesized that SEMA3-NRP1 and VEGF-NRP1 interactions are fundamental pathways in the orchestration of pulmonary vascular development in both the embryo and neonate. Disruption of these pathways would therefore lead to significant interruption of normal angiogenic and vascular maturation processes that are relevant to the pathogenesis of pulmonary diseases. Using extensive histopathological analyses of NRP1 loss-of-function mice, we show evidence of a significant role for SEMA3-NRP1 signalling in fetal microvascular development: congenital loss of SEMA3-NRP1 signalling resulted in severely attenuated development of the distal vasculature and alveolar-capillary interface, leading to fatal respiratory distress at birth that is reminiscent of clinical ACD. By contrast, VEGF-NRP1 and SEMA3-NRP1 signalling appear unessential for normal alveolar and vascular development in the postnatal period, per se, despite increased mortality. Our results demonstrate the critical involvement of SEMA3-NRP1 signalling in endothelial development and substantiate the idea that NRP1 mediates opposing and cooperative functions between SEMA3 and VEGF ligands.

Aggressive fibromatosis is a fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in FAP patients. Tumours are characterized by the stabilization of beta-catenin. Current therapies have yet to offer complete success for primary and recurrent tumours, and there remains a need for more effective therapeutic strategies. In this work, we demonstrate the anti-neoplastic and beta-catenin modulating capacities of Nefopam, a currently approved analgesic agent. We found that Nefopam was able to decrease cell viability and proliferation as well as total beta-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, Nefopam reduced the number of tumours formed in the Apc+/Apc1638N aggressive fibromatosis mouse model. We also demonstrated that androgens contribute to the development of tumours and could also modulate beta-catenin levels as indicated in Testosterone-treated orchidectomized Apc+/Apc1638N mice. Together, this work suggests that Nefopam and androgen signaling-blocking agents are potential candidates to effectively manage aggressive fibromatosis.

Aggressive fibromatosis is a fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in FAP patients. Tumours are characterized by the stabilization of beta-catenin. Current therapies have yet to offer complete success for primary and recurrent tumours, and there remains a need for more effective therapeutic strategies. In this work, we demonstrate the anti-neoplastic and beta-catenin modulating capacities of Nefopam, a currently approved analgesic agent. We found that Nefopam was able to decrease cell viability and proliferation as well as total beta-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, Nefopam reduced the number of tumours formed in the Apc+/Apc1638N aggressive fibromatosis mouse model. We also demonstrated that androgens contribute to the development of tumours and could also modulate beta-catenin levels as indicated in Testosterone-treated orchidectomized Apc+/Apc1638N mice. Together, this work suggests that Nefopam and androgen signaling-blocking agents are potential candidates to effectively manage aggressive fibromatosis.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Bowen-Conradi syndrome is a lethal autosomal recessive disorder affecting Hutterite infants, characterized by severe growth and psychomotor retardation, and leading to death at an average age of thirteen months. Linkage analysis and sequencing identified an A>G mutation in EMG1 as the probable cause of the disease. This gene is implicated in ribosome biogenesis, and the mutation results in an unstable EMG1 protein. The reduction in available EMG1 causes a transient delay in processing of the ribosomal small subunit 18S rRNA, leading to cell cycle delay at G2/M and a subsequent reduction in cell proliferation rates in patient lymphoblasts. A mouse model of Bowen-Conradi syndrome also displayed severe developmental delay, with prominent effects in the cranial central nervous system. Embryos died prematurely during development, probably due to decreased proliferation rates accompanied by apoptosis. These results shed light on the etiology of Bowen-Conradi syndrome, and open the door for development of treatments.

Introduction: Peritoneal carcinomatosis is an end stage sequela occurring in 10% of patients with colorectal cancer. Palliative approaches have evolved over the past several decades and the role for surgical cytoreduction with hyperthermic intraperitoineal chemotherapy (HIPEC) has proven efficacy in several studies. Optimization of HIPEC therapy includes the addition of adjuncts to the carrier solution of intraperitoneal chemotherapy to improve tumor cell killing. In this study the addition of vitamin C, selenium, and quercetin ("micronutrient combination") to mitomycin C is evaluated in-vitro, and a novel murine model of HIPEC is developed using a hyperthermic chemotherapy infuser device designed de novo and printed on a 3D resin printer. Methods: HCT-116 cells were grown in culture and divided into treatment groups including: control, micronutrient combination, mitomycin C, and mitomycin C + micronutrient combination. Groups were cultured up to 72 hours after treatment and then subjected to MTT assay, crystal violet assay, trypan blue synergy assay, clonogenicity assay, cell cycle assessment by flow cytometry with propidium iodide, and western blotting for cleaved caspase-3. The infuser device was designed in a CAD environment, printed on a 3D resin printer, and underwent fluid temperature stability analysis and flow experiments by infusing methylene blue into live mice followed by necropsy and analysis of dyeing patterns. Results: MCC treated cells proliferated at 32.7%, and tumor cells treated with MCC + MNC carrier solution proliferated at 27.3%. Normothermic MCC and the MNC alone caused a 26.8% and 33.3% reduction in cell survival, and MCC delivered to cells in the micronutrient combination solution decreased cell survival by 53.2%. 95.3% and 99% of cells treated with MCC or MNC alone demonstrated viability, and 85% of cells treated with MCC + MNC demonstrated short term viability, suggesting synergy. HCT-116 clonogenicity is disrupted by MCC and MNC individually, and nonexistent in the MCC + MNC treatment group. Cleaved caspase-3 mediated apoptosis is upregulated by MCC, and by MNC to a lesser extent. Flow cytometry apoptosis demonstrates increased S-phase cell cycle arrest in the MCC + MNC sample. The mouse infuser HIPEC apparatus demonstrated an thorough distribution of blue dye in predictable regions of the abdomen with an acceptable range of hyperthermic regulation.

Hepatitis C virus (HCV) is a serious global health problem with an estimate of 170 million carriers worldwide. Most individuals exposed to this blood-borne pathogen develop chronic infection, which may result in severe liver complications as well as end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options are suboptimal with no effective vaccines available to date. Development of a readily accessible mouse model that is permissive to natural HCV infection is important to facilitate drug and vaccine discovery, and also to better understand the viral pathogenesis. The inherent difficulty is that HCV displays very limited tropism, infecting only livers from humans or chimpanzees. An attempt was made to elucidate the key determinants in rendering the murine intracellular environment permissive to HCV replication. The results revealed that deletion of the interferon regulatory factor-3 and overexpression of microRNA-122 can independently enhance viral subgenomic replication in murine fibroblasts, with microRNA-122 being the stronger determinant. Interestingly, the phenotype established by these genetic manipulations was insufficient to support full-length HCV genome replication. Murine hepatic cell lines, with or without microRNA-122 expression, were also non-permissive to genomic HCV replication, despite the fact that translation of viral RNA was observed. These results suggest that additional host-specific factor(s) are required to support replication of full-length HCV RNA. These studies provide insight on the essential factors capable of influencing permissiveness of rodent cells to HCV replication, and also suggest genetic modifications to be considered when modeling the complete viral life cycle in a rodent animal model.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. D486N/+ female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.