Helicobacter pylori is a gram-negative bacterium that infects half the world population and is the etiological cause of numerous gastric pathologies. H. pylori possess numerous mechanisms to promote its survival and modulate host immunity. We propose that H. pylori can modulate intercellular communication by manipulating the host exosome pathway. Exosomes are secreted nanovesicles that contain different proteins and microRNAs that can be transferred between cells to alter cell signaling and gene expression. We demonstrate that H. pylori infection increases host exosome secretion. Furthermore, infection can alter exosome composition as VacA, a bacterial virulence factor, can be exported in exosomes and Argonaute 5, a miRNA effector protein, is upregulated in exosomes during infection. Lastly, we show preliminary evidence that infection-modulated exosomes can modulate immune-regulatory signaling in dendritic cells by activating STAT3. Together, these studies elucidate a novel mechanism by which H. pylori can modulate the host environment and promote its continued survival.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.

G-protein coupled receptors (GPCRs) have been shown to interact with an array of accessory proteins that modulate their function. I hypothesize that the GLP-1R, a B-class GPCR, similarly has interacting proteins that regulate its signaling. An unliganded human GLP-1R was screened using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay and a human fetal brain cDNA prey library to reveal 38 novel interactor proteins. These interactions were confirmed by co-immunoprecipitation and immunofluorescence. When co-expressed with the GLP-1R in cell lines, 15 interactors significantly attenuated GLP-1-induced cAMP accumulation. Interestingly, SiRNA-mediated knock down of three selected novel interactors, SLC15A4, APLP1 and AP2M1, significantly enhanced GLP-1-stimulated insulin secretion from the MIN6 beta cells. In conclusion, this present work generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling; and the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and transmigration across inflamed primary vascular endothelial cells. To determine the response of Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle clamping in mice. This led to significant decreases in both renal tubular necrosis score and neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively, these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia reperfusion injury without compromising protective host innate immune functions.

Kawasaki disease (KD) is an acute inflammatory disease characterized by persistent inflammation of the coronary arteries. KD is characterized by the release of cytokines such as tumor necrosis factor alpha (TNFα) and is thought to be initiated by a superantigen (SAg). The Lactobacillus casei cell wall extract model of KD demonstrates a critical requirement for TNFα and its receptor during pathogenesis, although the precise effect of TNFα is unknown. A persistent T cell infiltrate in the coronary artery disagrees with established fates of SAg activated cells, which undergo apoptosis. In this work, TNFα was found to promote the survival of SAg-reactive T cells. The results demonstrate that TNFα regulates B7.2 molecule expression on antigen presenting cells, and that TNFα indirectly promotes the survival of SEB-stimulated T cells by driving costimulation. These observations demonstrate how TNFα prevents T cell apoptosis and lend support to KD therapies which target TNFα and B7.

G-protein coupled receptors (GPCRs) have been shown to interact with an array of accessory proteins that modulate their function. I hypothesize that the GLP-1R, a B-class GPCR, similarly has interacting proteins that regulate its signaling. An unliganded human GLP-1R was screened using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay and a human fetal brain cDNA prey library to reveal 38 novel interactor proteins. These interactions were confirmed by co-immunoprecipitation and immunofluorescence. When co-expressed with the GLP-1R in cell lines, 15 interactors significantly attenuated GLP-1-induced cAMP accumulation. Interestingly, SiRNA-mediated knock down of three selected novel interactors, SLC15A4, APLP1 and AP2M1, significantly enhanced GLP-1-stimulated insulin secretion from the MIN6 beta cells. In conclusion, this present work generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling; and the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and transmigration across inflamed primary vascular endothelial cells. To determine the response of Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle clamping in mice. This led to significant decreases in both renal tubular necrosis score and neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively, these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia reperfusion injury without compromising protective host innate immune functions.

Adult mammopoiesis occurs in close synchronization with reproductive development when the hypothalamic-pituitary-ovarian axis delivers integral systemic hormone cues to propel mammary morphogenesis during puberty, remodeling during reproductive cycles and functional differentiation following pregnancy. While hormones remain the driving force behind normal glandular development, increased life-time hormone exposure is a strong risk factor for breast cancer. Breast cancer heterogeneity has been attributed to different cells of origin and/or different mutation repertoires. Stem/progenitor cells are intensely investigated as cells of origin given their regenerative and self-renewal properties that provide conceivable advantage in cancer. Although hormones have a fundamental influence in breast cancer, their capacity to regulate stem/progenitor cells was unknown, and presents the central directive in this thesis. Employing mouse models, we show that mammary epithelial subpopulations and in particular, stem cells, are highly responsive to ovarian hormones and depend on key molecular events. A progesterone peak during the luteal phase of reproductive cycles results in a significant increase in stem cell-enriched basal cells and an expansion of stem cells measured by in vivo transplantation assays, with rapid development of lobuloalveoli. Progesterone was found to stimulate expression of mitogenic ligands, RANKL and Wnt4, in ER+PR+ luminal epithelial niche cells concomitant with increased expression of their receptors and target genes in the ER-PR- basal stem cell population, suggesting a cross-talk between luminal and basal cells that elicits stem cell expansion within the niche. The requirement of RANKL signaling for hormone-induced mammary stem cell dynamics was further explored utilizing mice deficient for its receptor, RANK, and by pharmacological inhibition of RANKL. Disruption of RANKL/RANK signaling resulted in abrogated activation of the basal stem cell-enriched population and alveolar progenitor cells in response to progesterone. This was accompanied by a marked reduction in cell proliferation, cell cycle regulators, alveolar lineage determinants and notably, in epithelial Wnt responsiveness. Thus, progesterone orchestrates a series of molecular events in the mammary stem cell niche where RANK is effectively positioned to deliver instructive signals to stem cells, culminating in stem cell recruitment and alveolar regeneration, processes which when deregulated have considerable potential to promote breast cancer pathogenesis.

The non-cluster homeobox gene TLX1 was initially identified at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in the malignant cells. Three independent research groups have generated transgenic mouse models expressing TLX1 in various hemopoietic cells resulting in development of B cell lymphoma and T-ALL. T-ALL tumours were characterized as immature Double Negative or/and Double Positive phenotypes. In addition, all TLX1 transgenic mice showed a long latency for leukemia development suggesting requirement of additional genomic abnormalities for the conversion of premalignant cells to full-blown malignancy. We hypothesized that unresolved double strand DNA breaks might act as additional genetic mutation and contribute to T-ALL progression in TLX1 overexpressed thymocytes. To address this hypothesis, we generated double mutant IgHμ-TLX1TgPrkdcScid/Scid mice, which accelerated the development of leukemia relative to PrkdcScid/Scid littermates. We identified that multiple genes associated with chromosome segregation, apoptosis and cell cycle progression were aberrantly expressed in IgHμ-TLX1TgPrkdcScid/Scid premalignant thymocytes. We found that IgHμ-TLX1TgPrkdcScid/Scid thymocytes were prone to chromosome instability suggesting malfunction of the mitotic spindle assembly checkpoint. In addition to T-ALL, 46% of IgHμ-TLX1TgPrkdcScid/Scid mice developed Acute Myeloid Leukemia, suggesting that the cancer initiating effects of TLX1 are not limited to cells of lymphoid origin. Transplantation experiments revealed that T cell acute lymphoblastic leukemia initiating cells (T-ALL-ICs) reside in the thymus of IgHμ-TLX1TgPrkdcScid/Scid mice and T-ALL-ICs were enriched in the c-kit+CD44+CD25- fraction. We showed that T-ALL tumour cells from IgHμ-TLX1TgPrkdcScid/Scid were transplantable and there was a tendency for the latency period of T-ALL development to be reduced with secondary and tertiary transplantations. We demonstrated that premalignant IgHμ-TLX1TgPrkdcScid/Scid myeloid progenitors exhibited deregulated apoptosis and proliferation. Collectively, our studies demonstrate that TLX1 expression in DNA-PK-deficient cells activats multiple oncogenic pathways leading to apoptosis resistance, accelerated proliferation and deregulation of the spindle assembly checkpoint. We propose that activation of the same pathways supporting survival and proliferation in various cells may be indicative of the universal principles driving TLX1-induced tumourogenesis. Our data provide clinically relevant information of the molecular mechanisms involved in the pathogenesis of leukemia that makes IgHμ-TLX1TgPrkdcScid/Scid mouse model a powerful tool to explore potential treatment options directed to delay disease progression.