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.

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause loss function of the CFTR channel on the apical surface of epithelial cells. ΔF508-CFTR, the major mutation in patients, is misfolded, retained in the endoplasmic reticulum (ER) and degraded. Small molecule corrector compounds partially rescue the trafficking defect of ΔF508-CFTR by allowing escape from the ER and trafficking to the plasma membrane where it exhibits partial function. These compounds may bind directly to the mutant protein and rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis by evaluating the consequence of corrector compound on the conformation of each nucleotide-binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. We found that VRT-325 was capable in partially restoring compactness only in NBD1. In comparison, ablation of the arginine framed peptide sequence: R553XR555 (ΔF508-KXK-CFTR) modified the protease resistance of NBD1, NBD2 and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together these interventions restored processing of ΔF508-CFTR to near wild-type levels. Importantly however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. This defect in channel activation can be fully corrected by addition of the potentiator: VX-770. The experiments performed partly elucidated ii the molecular mechanism of action for drug therapy and suppressor mutation. It is important to understand these basic concepts in hopes to layout a blue print for future drug design.

The effect of decompression stress on circulating microparticles (MPs) from leukocytes (LMP), platelets (PMP), and endothelial cells (EMP) was investigated in fifteen male naval clearance divers. Venous blood samples were obtained 30 min before and 75 min after exposure to 81msw for 20 min. MPs were isolated by differential centrifugation and immunophenotyped using multiparameter flow cytometry. Venous gas emboli (VGE) were assessed using Doppler ultrasound every 40 min post-dive and subsequently graded using the Kisman Integrated Severity Score (mean KISS=21.92, indicating moderate level of VGE). Following the dive there was increased expression of CD41a, CD106, CD62P and CD31 on MPs, while CD45 and CD141 expression decreased. A positive correlation was found between KISS and CD41a expression post-dive. These results indicate that decompression stress activated platelets, producing PMPs and resulting in potential vascular disruption or injury. The inclusion of MP measures in future DCS-related research may help identify biomarkers of DCS.

Craniofacial nerve injury occasionally causes spread of mechanical hypersensitivity in humans. We modeled this abnormality by transecting the infraorbital nerve (IONX) in male and female mice of the 23 AXB-BXA recombinant inbred lines and their progenitor strains, comparing their responsivity to 7 applications of a 0.2 gram Von Frey filament to the ears, paws and tail. When normalizing their mechano-responsivity on postoperative days 14 and 21 by the preoperative values, subtracting data of sham-operated from IONX mice, highly contrasting line/strain-specific differences were demonstrated. Similar line/strain-specific variability in the spread of mechano-allodynia to the paws post-IONX was demonstrated in our novel 3 minute place-avoidance paradigm, assessing parameters of mobility on a smooth surface versus a pro-allodynic granular surface. These genetically-controlled, widespread changes in mechano-sensitivity caused by IONX were minimally sexually dimorphic and mapped to intervals on chromosomes 5, 9, and 13. Further analysis is needed to identify the causative genes.