Abstract The proglucagon‐derived peptides (PGDP) are expressed in islet alpha and gut enteroendocrine L cells. Although glucagon, glucagon‐like peptide‐1 (GLP‐1), and glucagon like peptide‐2 (GLP‐2) are derived from the same proglucagon gene, energy ingestion and nutrient assimilation represses proglucagon biosynthesis in the α‐cell, but stimulates the synthesis and secretion of GLP‐1 and GLP‐2 from the gut L cell. In the work presented in this thesis, I have identified novel G protein‐coupled receptors that stimulate GLP‐1 secretion and improve glucose homeostasis. G protein‐coupled receptor 119 (GPR119) is expressed in enteroendocrine cells and islets and is activated by nutrients (fatty acid derivatives) and small specific synthetic agonists. Activation of GPR119 enhances glucosestimulated insulin secretion from islet β‐cells and promotes incretin release from enteroendocrine cells in a cyclic AMP (cAMP)‐dependent manner. To determine the importance of gut hormones for the glucoregulatory actions of GPR119, I examined GPR119 activation in normal mice, isolated islets, and in mice with inactivation of gut hormone receptors. GPR119 activation directly stimulates insulin secretion from islets in vitro, yet requires intact incretin receptor signaling and enteral glucose exposure for optimal improvement of glucose tolerance in vivo. In contrast, activation of GPR119 inhibits gastric emptying independent of incretin receptors through GPR119‐dependent pathways. Another important feature of β‐cell GPCRs coupled to cAMP generation is their ability to protect the β‐cell from external injury. I have shown that mice lacking GPR119 (GPR119‐/‐) are more susceptible to streptozotocin (STZ)‐induced apoptosis while pharmacological activation of GPR119 failed to protect the β‐cell from STZ‐induced injury. Furthermore, GPR119‐/‐ mice iv display impaired incretin secretion and beta cell function when chronically fed a high fat (HF) diet. Conversely, abrogation of GPR119 signaling does not affect the beta‐cell adaptation (increased islet number and size) to the metabolic demand of high‐fat feeding. Mechanisms to increase β‐cell mass and function may be useful tools for the treatment of type 2 diabetes. GLP‐1 stimulates insulin biosynthesis, β‐cell proliferation and exerts antiapoptotic actions on β‐cells. To delineate novel mechanisms, important for the regulation of proglucagon gene expression and GLP‐1 secretion in the enteroendocrine L‐cell, I carried out a microarray‐based gene expression profiling and transcriptional networks analysis using RNA from murine gut GLUTag cells. To identify mechanisms unique to enteroendocrine L‐cells, I used the islet αTC1 cell line for comparative purposes. I identified a novel progesterone mediated signaling pathway involving activation of membrane GPCRs for the control of GLP‐1 secretion. In summary, these studies establish that GPR119 engages multiple complementary pathways for control of glucose homeostasis and suggest that endogenous GPR119 signaling plays a critical role in β‐cell adaptation to cytotoxic injury and nutrient excess. The studies provide evidence for a novel role for progesterone, regulating GLP‐1 secretion and controllingglucose homeostasis.

Abstract The proglucagon‐derived peptides (PGDP) are expressed in islet alpha and gut enteroendocrine L cells. Although glucagon, glucagon‐like peptide‐1 (GLP‐1), and glucagon like peptide‐2 (GLP‐2) are derived from the same proglucagon gene, energy ingestion and nutrient assimilation represses proglucagon biosynthesis in the α‐cell, but stimulates the synthesis and secretion of GLP‐1 and GLP‐2 from the gut L cell. In the work presented in this thesis, I have identified novel G protein‐coupled receptors that stimulate GLP‐1 secretion and improve glucose homeostasis. G protein‐coupled receptor 119 (GPR119) is expressed in enteroendocrine cells and islets and is activated by nutrients (fatty acid derivatives) and small specific synthetic agonists. Activation of GPR119 enhances glucosestimulated insulin secretion from islet β‐cells and promotes incretin release from enteroendocrine cells in a cyclic AMP (cAMP)‐dependent manner. To determine the importance of gut hormones for the glucoregulatory actions of GPR119, I examined GPR119 activation in normal mice, isolated islets, and in mice with inactivation of gut hormone receptors. GPR119 activation directly stimulates insulin secretion from islets in vitro, yet requires intact incretin receptor signaling and enteral glucose exposure for optimal improvement of glucose tolerance in vivo. In contrast, activation of GPR119 inhibits gastric emptying independent of incretin receptors through GPR119‐dependent pathways. Another important feature of β‐cell GPCRs coupled to cAMP generation is their ability to protect the β‐cell from external injury. I have shown that mice lacking GPR119 (GPR119‐/‐) are more susceptible to streptozotocin (STZ)‐induced apoptosis while pharmacological activation of GPR119 failed to protect the β‐cell from STZ‐induced injury. Furthermore, GPR119‐/‐ mice iv display impaired incretin secretion and beta cell function when chronically fed a high fat (HF) diet. Conversely, abrogation of GPR119 signaling does not affect the beta‐cell adaptation (increased islet number and size) to the metabolic demand of high‐fat feeding. Mechanisms to increase β‐cell mass and function may be useful tools for the treatment of type 2 diabetes. GLP‐1 stimulates insulin biosynthesis, β‐cell proliferation and exerts antiapoptotic actions on β‐cells. To delineate novel mechanisms, important for the regulation of proglucagon gene expression and GLP‐1 secretion in the enteroendocrine L‐cell, I carried out a microarray‐based gene expression profiling and transcriptional networks analysis using RNA from murine gut GLUTag cells. To identify mechanisms unique to enteroendocrine L‐cells, I used the islet αTC1 cell line for comparative purposes. I identified a novel progesterone mediated signaling pathway involving activation of membrane GPCRs for the control of GLP‐1 secretion. In summary, these studies establish that GPR119 engages multiple complementary pathways for control of glucose homeostasis and suggest that endogenous GPR119 signaling plays a critical role in β‐cell adaptation to cytotoxic injury and nutrient excess. The studies provide evidence for a novel role for progesterone, regulating GLP‐1 secretion and controllingglucose homeostasis.

Down syndrome (DS), caused by an extra chromosome 21, affects 1 in 750 live births, and is characterized by cognitive impairment as well as several congenital defects. Currently, little is known about the molecular pathogenesis of DS and no direct genotype-phenotype relationship has yet been confirmed. The current screening test for DS subjects many women to undergo invasive procedures such as amniocentesis due to suboptimal sensitivity and specificity. Therefore, this study aimed to discover novel biomarkers to improve screening tests, and to discovery dysregulated molecular pathways in DS-affected fetus to better understand pathogenesis. To achieve this objective, proteomic analyses of amniotic fluid (AF) and amniotic fluid cells (amniocytes) were performed using mass spectrometry (MS), which allows discovery of a large number of proteins in complex biological samples. Since AF contains the most information of the developing fetus, we first generated the most comprehensive list of proteins present in AF by using high resolution MS. We then performed quantitative analyses of proteins from AF as well as amniocytes to reveal novel biomarkers and clues to altered molecular mechanisms of DS. Comparison between the proteome of AF from unaffected and DS-affected pregnancies allowed selection of 60 candidate biomarkers based on spectral counting. Two candidates, APP and TNC-C, were verified by immunoassays to show two-fold increase in AF from DS-pregnancies. Additionally, CPA4, MUC13, CEL, DPP4 and MMP2 were verified to be differentially expressed in trisomy 21-AF via selected reaction monitoring assays using triple-quadruple mass spectrometer. Amniocytes from DS-affected and unaffected fetuses were also quantitatively analyzed by using Stable Isotope Labelling of Amino acids in Cell culture technique. Over 4900 proteins were identified from amniocyte lysate and supernatant by LTQ-Orbitrap mass spectrometer, and 85% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labelled amino acids. Proteins that consistently showed aberrant expression from affected amniocytes have been selected for further verification and molecular network analyses since they may play a role in DS pathogenesis.

Background: There is strong level I evidence that in most patients, mechanical bowel preparation (MBP) is not required. Despite this, physician behaviour has been slow to change in favour of omitting preoperative MBP. Methods: A knowledge translation strategy including: guideline development, consensus, education by opinion leaders, audit and feedback and reminder cards, was used in this study. Results: Overall, 81.1% of patients in the “before” arm and 88.4% in the “after” arm received MBP in compliance with the guideline (p=0.038). Normal diet use was compliant with the guideline in 45.6% of the patients in the “before” arm and 55.8% in the “after” arm (p=0.080). The use of enemas was compliant with the guideline in 88.5% of “before” patients and 94.2% of “after” patients (p<0.001). Conclusions: The results of this study reveal that a tailored, multi-faceted knowledge translation strategy can be used to change surgeon behavior in this clinical scenario.

Introduction: This thesis prospectively evaluated the cognitive and functional outcomes and health-related quality of life of children admitted urgently to a Pediatric Intensive Care Unit (PICU) at the Hospital for Sick Children. Methods: The primary outcome was the Vineland Adaptive Behavioural Scale (VABS-2) measured at 1-month and secondary outcomes were health-related quality of life, daily functioning, and caregiver perceptions. Results: 56 children and 66 caregivers were enrolled; 42 (75%) patients and 49 (74%) caregivers completed the 1-month assessment. Children in the PICU had a mean VABS-2 score of 85(+25). Daily functioning outcomes did not significantly change from baseline to 1-month. In comparison to baseline, children had significantly reduced health-related quality of life at 1-week but no significant change was found at 1-month. Discussion: Children surviving PICU have significant cognitive morbidity and reduced health-related quality of life that is exacerbated by more intense modes of resuscitation and increasing severity of illness.

Skeletal muscle damage can lead to cell death and the subsequent release of intracellular molecules. To investigate whether molecules from skeletal muscle can interact with the heart, the Langendorff isolated heart preparation was used to assess cardiac function, while NF-κB and AP-1 activation were assessed by EMSA to observe inflammatory status. Hearts were perfused for 75 min with 1 μg/ml of either soleus, white gastrocnemius (WG), or heat stressed white gastrocnemius (HSWG) skeletal muscle homogenate and a decreased LVDP, +dP/dt and –dP/dt were observed when compared to untreated (control) hearts. Greater early and late decreases in cardiac function were observed in hearts treated with HSWG and soleus muscle homogenates, respectively. No alterations in NF-κB or AP-1 activation were detected. These data suggests the contents of skeletal muscle are capable of interacting with the heart and altering contractile function in a fibre type specific manner, possibly related to muscle HSP content.

Skeletal muscle damage can lead to cell death and the subsequent release of intracellular molecules. To investigate whether molecules from skeletal muscle can interact with the heart, the Langendorff isolated heart preparation was used to assess cardiac function, while NF-κB and AP-1 activation were assessed by EMSA to observe inflammatory status. Hearts were perfused for 75 min with 1 μg/ml of either soleus, white gastrocnemius (WG), or heat stressed white gastrocnemius (HSWG) skeletal muscle homogenate and a decreased LVDP, +dP/dt and –dP/dt were observed when compared to untreated (control) hearts. Greater early and late decreases in cardiac function were observed in hearts treated with HSWG and soleus muscle homogenates, respectively. No alterations in NF-κB or AP-1 activation were detected. These data suggests the contents of skeletal muscle are capable of interacting with the heart and altering contractile function in a fibre type specific manner, possibly related to muscle HSP content.

Introduction: This thesis prospectively evaluated the cognitive and functional outcomes and health-related quality of life of children admitted urgently to a Pediatric Intensive Care Unit (PICU) at the Hospital for Sick Children. Methods: The primary outcome was the Vineland Adaptive Behavioural Scale (VABS-2) measured at 1-month and secondary outcomes were health-related quality of life, daily functioning, and caregiver perceptions. Results: 56 children and 66 caregivers were enrolled; 42 (75%) patients and 49 (74%) caregivers completed the 1-month assessment. Children in the PICU had a mean VABS-2 score of 85(+25). Daily functioning outcomes did not significantly change from baseline to 1-month. In comparison to baseline, children had significantly reduced health-related quality of life at 1-week but no significant change was found at 1-month. Discussion: Children surviving PICU have significant cognitive morbidity and reduced health-related quality of life that is exacerbated by more intense modes of resuscitation and increasing severity of illness.

Background: There is strong level I evidence that in most patients, mechanical bowel preparation (MBP) is not required. Despite this, physician behaviour has been slow to change in favour of omitting preoperative MBP. Methods: A knowledge translation strategy including: guideline development, consensus, education by opinion leaders, audit and feedback and reminder cards, was used in this study. Results: Overall, 81.1% of patients in the “before” arm and 88.4% in the “after” arm received MBP in compliance with the guideline (p=0.038). Normal diet use was compliant with the guideline in 45.6% of the patients in the “before” arm and 55.8% in the “after” arm (p=0.080). The use of enemas was compliant with the guideline in 88.5% of “before” patients and 94.2% of “after” patients (p<0.001). Conclusions: The results of this study reveal that a tailored, multi-faceted knowledge translation strategy can be used to change surgeon behavior in this clinical scenario.

Metformin has been associated with a reduction in breast cancer incidence, however its effect on mortality following cancer has not been adequately examined. The purpose of this study was to evaluate the impact of metformin therapy on mortality in women with breast cancer. Using Ontario health databases, this retrospective cohort examined the impact of metformin on mortality among women aged 66 years or older with diabetes and breast cancer. After a mean follow-up of 4.5 years, there was no association between cumulative metformin use and either all-cause or breast cancer-specific mortality (HR 0.97, 95% CI 0.92-1.07; HR 0.91, 95% CI 0.81-1.03 respectively per additional year of cumulative metformin use). Though metformin was not associated with a reduction in mortality in our study of older women with breast cancer, there is still a need to examine whether metformin has an effect on mortality in other breast cancer populations.