Remote ischemic preconditioning (rIPC) through transient limb ischemia induces potent cardioprotection against ischemia reperfusion (IR) injury. I examined the delayed phase of protection that appears 24 hours after the initial rIPC stimulus. The primary objective of this study was to establish a mode of sedation and control treatment for delayed rIPC experiments. I used an ex-vivo, Langendorff isolated-mouse heart preparation of IR injury to examine the delayed effects of an intra-peritoneal (IP) injection, sodium-pentobarbital (SP), halothane and nitrous oxide (N2O) anesthesia on post-ischemic cardiac function. Each anesthetic method improved left-ventricular function after IR injury. SP and halothane anesthesia also reduced LV infarct size. Delayed cardioprotection after IP injections was associated with an increase in phosphorylated-Akt levels. The present study shows that IP injections and inhalational anesthesia invoke cardioprotection and, therefore, indicates that these modes of sedation should not be used as control treatments for studies examining the delayed rIPC phenotype.

In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model,MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion(UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS.

Von Hippel Lindau (VHL) is a tumour suppressor protein classically known to target the α subunit of hypoxia inducible factor (HIF) for proteasomal degradation. Emerging evidence has underscored a novel role for VHL in both cell cycle regulation and extracellular matrix assembly. Herein, we provide evidence of VHL multitasking in normal and pathological placentation. Using ex vivo, first trimester human placental tissue and in vitro, JEG-3 choriocarcinoma cell line model we demonstrate that VHL plays a role in regulating the expression of cell cycle modulator CCND1 via a mechanism involving its inhibitor, p15 and HIF-2α. In addition, using a similar experimental strategy we provide evidence supporting a role for VHL in regulating the expression of fibronectin and its receptor integrin α5. Moreover, altered VHL expression observed in preeclampsia is associated with altered expression of cell cycle regulators and contributes to altered FN protein levels which are characteristic of this pathology.

Methyl-CpG-binding proteins (MeCP2, MBD1-MBD3) recruit transcriptional co-repressor molecules to methylated regions and silence transcription. The role of MBD2 in regulating brain function and behavior remains largely unexamined. To begin elucidating whether MBD2 influences neural function, I assessed the behavioral performance of Mbd2 null mice, compared their hippocampal electroencephalographic activity during exploration, and performed protein and mRNA expression assessments. The results indicate that mutant mice display a heightened anxiety-like behavior, diminished explorative activity and reduced sociability compared to wild-type mice. However, these behavioral differences were not paralleled by neurophysiological impairments. Mutant hippocampal and cortical samples display significantly elevated MeCP2 mRNA levels. Yet, MeCP2 protein expression did not mirror the mRNA profile and instead was significantly reduced. Glucocorticoid Receptor mRNA levels were significantly reduced in the hippocampus and cortex regions of Mbd2 null brains. The loss of MBD2 is sufficient to induce behavioral impairments in mice without introducing gross deficits in hippocampal neurophysiology.

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

The contribution of left ventricular (LV) torsion and recoil in augmenting stroke volume during exercise is poorly understood. This study examined the effects of aging on LV torsion and recoil at rest and during sub-maximal exercise in 11 young (YU) and 9 older, untrained males (OU), and 12 age-matched older, endurance-trained males (OT) in upright and supine body positions. LV torsion increased from rest to exercise in YU in upright and supine body positions (9.9±2.3 to 13.2±5.2 degrees, p=.03, and 8.8±3.8 to 12.8±6.6 degrees, p=.02, respectively), but not in OU. LV torsion increased with exercise in the supine body position only in OT (p=.046). There were no differences in EDV or change in ESV with supine exercise across groups suggesting that once the Frank-Starling mechanism is fully recruited, the young heart, and that of older, endurance-trained subjects may augment SV by increasing LV torsion and contractility rather than contractility alone.

Introduction: Hypertension is the number one risk factor for cardiovascular diseases. Total peripheral resistance (TPR) is strongly involved in blood pressure homeostasis. TPR is primarily determined by resistance arteries (RAs). Pathogenic factors which change RA structure are associated with cardiovascular disease. Despite this, methods employed in the study of RAs lack efficiency. Methods: A polymer microfluidic device (Artery-on-a-Chip Device, AoC) made from polydimethylsiloxane (PDMS) was developed. RAs from CD1 mice were measured on the device. Their responses to phenylephrine (PE), acetylcholine (Ach), FURA-2 imaging, and 24-h culture were assessed. Results: Following several modifications, vessel function on the AoC device was successfully measured. Robust PE constriction and Ach-induced vasodilation were observed. AoC arteries were viable after 24-hour culture, and FURA-2 was successfully imaged. Conclusions: The AoC device is a viable alternative to cannulation myography. The AoC can greatly increase the efficiency of RA studies, while also decreasing training time and difficulty.

Partitioning-defective protein 6 (Par6), a regulator of cell polarity, is emerging as a mediator of cell differentiation. Herein I sought to assess the contribution of Par6 to trophoblast fusion in normal and pathological human placentae. I hypothesized that Par6 regulates fusion in response to oxygen and transforming growth factor 3 (TGF3) and that this process is altered in preeclampsia (PE). Using silencing and overexpression strategies in choriocarcinoma BeWo cells, my results demonstrate Par6 negatively regulates trophoblast fusion via its roles on tight junctions and cytoskeleton dynamics. Additionally, Par6 expression is elevated in PE, a pathology characterized by placentalhypoxia, increased TGF3, and altered trophoblast fusion. Using low O2 conditions to model PE in BeWo and primary trophoblast cells, Par6 levels increased, and thisassociated with maintenance of tight junctions at cell boundaries and decreased fusion. Overall, my data provides insight into the mechanisms involving Par6 in contributing to the pathogenesis of PE.

Several genes thought to confer susceptibility to dyslexia have been identified, and the purpose of this study is to 1) determine the expression pattern of one of these gene products and 2) characterize the function of the product of one of these genes, namely KIAA0319-Like (KIAA0319L), using the developing chick visual system as a model. Whole mount in situ hybridization was performed for KIAA0319L on embryonic day (E)3 – E5 and in situ hybridization on sections was performed at later stages. Engineered microRNA (miRNA) constructs targeting KIAA0319L were prepared and their specificity and efficiency for knocking down KIAA0319L were tested. miRNAs were electroporated in E5 optic tecta (OT). Embryos were sacrificed at E12. OT were removed, sectioned and analyzed. Results demonstrate that KIAA0319L is expressed in the developing chick visual system. Knockdown of KIAA0319L in the OT results in abnormal migration indicating that KIAA0319L is necessary for this process.

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic and intestinal anti-inflammatory hormone. Hence, I hypothesized that treatment with degradation-resistant hGly2GLP-2 increases, while blocking endogenous GLP-2 decreases colorectal cancer (CRC) in rodents. In mice, treatment with dextran sodium sulphate (DSS) and azoxymethane (AOM) induced colitis-associated CRC, which was further increased by treatment with hGly2GLP-2 and reduced by blocking endogenous GLP-2 with the antagonist hGLP-23-33. Moreover, while colonic damage score (CDS) was not altered by hGly2GLP-2 or hGLP-23-33 treatment, hGly2GLP-2 increased small intestinal growth and hGLP-23-33 reduced jejunal crypt cell proliferation. In rats fed with of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and high fat (HF) diet for aberrant crypt foci (ACF) induction, treatment with hGly2GLP-2 increased small intestinal growth and ACF occurrence. Moreover, in rats fed with PhIP-HF diet for tumour induction, early treatment with hGly2GLP-2 appears to increase the occurrence of intestinal tumours. Collectively, these findings indicate a pro-carcinogenic role for both exogenous and endogenous GLP-2.