Results continued to accumulate that are consistent with the hypothesis that disfacilitation of wake-promoting systems by the hypocretin (Hcrt) receptor antagonist almorexant (ALM) results in less functional impairment than the inhibition of neural activity produced by the benzodiazepine receptor agonist zolpidem (ZOL). In Year 4, the previously- reported results on spatial reference memory (Task 2a) and spatial working memory (Task 2b) were complemented by Psychomotor Vigilance Test studies (Task 2c). The wake-active histaminergic and Hcrt neurons, but neither the cholinergic basal forebrain (BF) neurons nor the serotonergic dorsal raphe neurons, could be activated after sleep deprivation in the presence of ALM; however, none of these four cell types could be activated in the presence of ZOL (Task 3a). Lesions of the locus coeruleus (LC), a wakefulness-promoting area, abolished the ALM-induced decrease in NREM sleep latency without affecting the ZOL-induced decrease (Task 3b). High sleep pressure, rather than the actions of ALM or ZOL per se, is critical for activation of sleep-active cortical neurons (Task 4a). ALM promoted adenosine release in the BF (Task 4b) and cortex (Task 4c), particularly during waking. Hcrt neurons expressing channelrhodopsin-2 can be excited by blue light pulses in vitro and preliminary in vivo experiments indicate that optogenetic activation of Hcrt cells can cause changes in sleep architecture (Tasks 6a).

关键词：剥夺睡眠；腺苷；胆碱能神经

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While the molecular basis of TSC is well established, far less is known about the pathogenetic mechanisms of epileptogensis in TSC, shich is found in the vast majority of patients. We introduce the novel concept that this follows from a disruption of normal synaptic pruning that is due to TSC mutation in astrocytes, the major non-neuronal cell type of the brain. Previous studies mostly focused on focal epileptogenisis in cortical tubers. In this study, we propose that nontuber cortex may present an abnormally excitable neuronal network that could underlie seizure generation. We will concentrate on a non-neuronal mechanism may exist in regulating synaptic function and thus epileptogenisis in TSC. To explore this aim, we will use a wide variety of experimental approaches, including electrophysiological (patch-clamping in culture cells and slice preparations to in vivo video-EEG monitoring), histological (conventional and fluorescent assays), molecular biological (Western blotting, biotinylation), and modern cellular imaging (confocal and two-photon microscopy of tissue preparations) techniques. We will measure biomarkers for astrocyte and neuronal functions and provide an index of glial transmission in the cortex of TSC deficient mouse models and peri-tuber or non- tuber tissue from TSC human brain. We will determine whether astrocyte dysfunction will lead to a failure in pruning excessive excitatory synapses during development, which underlies epilepsy in young TSC patients.

关键词：医学研究；神经细胞；含量测定；星形胶质细胞

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Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin- 6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.

关键词：药品；卵巢癌；铂化合物；脱氧核糖核酸

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In the C3(1)/SV40 T-antigen (Tag) FVB/N mouse model of human estrogen and progesterone receptor-negative breast cancer, the stress response elicited by social isolation is associated with increased expression of metabolic genes in the mammary gland. To further understand accelerated tumor growth associated with social isolation, we separated mammary gland adipocytes from ductal epithelium and stroma and then analyzed individual fractions for changes in metabolic gene expression and function. The increased expression of the key metabolic genes Acaca, Hk2 and Acly was found to be significantly elevated in the adipocytes of the mammary gland, and surprisingly, was not significantly increased in visceral adipose depots of socially isolated female mice. Increased metabolic gene expression in the mammary gland of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin expression. Furthermore, culture media from isolated versus group-housed mouse mammary adipose tissue resulted in relatively increased proliferation of mammary cancer cells. These results suggest that exposure to chronic social isolation results in metabolic changes in mammary gland adipocytes that contribute to increased growth of adjacent epithelial cell tumors. We propose a model in which environmental stress affects estrogen-independent mammary tumor growth, at least in part, through changes in mammary adipocyte biology.

关键词：乳腺癌；代谢；应激（生理）；脂肪组织细胞

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Breast cancer develops from epithelial lesions in breast ducts and lobules that become invasive and can be metastatic. Breast cancer is a disease of uncontrolled cell division. Cell division normally creates two genetically identical daughter cells through severing of a cytoplasmic bridge that interconnects them. The midbody is an organelle involved in severing. Previously midbodies were thought to be lost from cells after division, but we show they can be retained. Here we show that MBs exhibit stem cell properties and are in breast cancer stem cells (Task 1). They are scaffolds for anchoring breast cancer oncogenes, tumor suppressors and breast cancer stem cell proteins. Increasing MB+ cells increases in vitro tumor potential ( Task 2). We activated a MB-degradation pathway that decreased MBs and tumorigenic properties of breast cancer cells (Task 3). Because MBs are in breast cancer stem cells, we believe this MB targeting strategy may be an innovative strategy for therapies focused on the most difficult of all tumors, those thought to be caused by cancer stem cells, namely recurrent, resistant and metastatic cancers.

关键词：乳腺癌；细胞分裂；细胞质；疾病

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This project is intended to develop the tools and principles necessary to engineer subtilisin proteases which specifically target and deactivate biological warfare agent (BWA) toxins. We are engineering and evolving subtilisin proteases that specifically target and deactivate BoNT, SEB, ricin, and B. anthracis lethal factor (LF), representing four functionally distinct families of toxins. The centerpiece of our design effort is a phage- display selection method for creating tightly-regulated proteases of high specificity. In this system the protease, substrate sequence, and regulatory co- factor are co-evolved. The key accomplishments this past year were: 1. Determined the structure of an evolved variant pT2077 in complex with the substrate sequence used to select it. 2. Design/evolution of a highly active enzyme that can cut P4 = I (pT2050). 3. Computational design of specificity for an ionic P4 amino acid (P4 = E, pT2121 and P4 = K, pT2114); 4. Engineered protease chain reactions that can reliably measure concentrations of 250 fM range in a 20 hour assay.

关键词：毒素和抗毒素；氨基酸；炭疽病；生物制剂

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This is a progress report (year 2) for the development of a novel therapeutic drug (salubrinal) for skeletal diseases, focusing on potential treatment of osteoporosis and bone fracture. The study in the second year using animal models and in vitro cell cultures strongly supported salubrinal s action on prevention of bone loss. In animal experiments, we employed three procedures to induce bone loss. They were ovariectomy, hindlimb suspension, and administration of glucocorticoid (prednisolone). In all three models, salubrinal was able to suppress reduction of bone mineral density. In those experiments, salubrinal was administered via subcutaneous injection as well as oral gavage. In in vitro experiments, salubrinal reduced the development of bone-resorbing osteoclasts and promoted the development of boneforming osteoblasts. A provisional patent was submitted for salubrinal s administration for treatment of bone diseases, and the peer-reviewed articles as well as conference abstract were published. In the third year, we will start evaluating the effects of salubrinal on the healing of bone fracture as well as the regulatory mechanism of salubrinal's action.

关键词：骨折；骨质疏松症；骨细胞（生物学）

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Computer simulation is a valuable tool for the research of physics. These simulations can be especially valuable when there is experimental data available that can be used to validate the model. The main objective of this thesis is to determine whether a computer simulation model can accurately depict the experimentally determined fluid flow for a channel with a series of unique individual constrictions. The experimental data are derived from a scaled-up model of coronary blood flow with localized axisymmetric constrictions (or stenoses), representing an ideal case of atherosclerotic disease. This thesis provides the foundation for future study and simulation to develop a microelectromechanical device mounted on a stent capable of sensing and transmitting changes in blood flow characteristics and properties to an outside receiver for improved treatment of patients with atherosclerotic coronary artery disease.

关键词：计算机模拟；流体流动；血液循环

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The communities of Hartford, Connecticut, and Syracuse, New York, implemented year-long campaigns to test whether NHTSAs high-visibility enforcement (HVE) model could be applied to reduce two specific forms of distracted driving driving while talking on a hand-held cell phone or texting. The HVE model applies strong laws, vigorous targeted law enforcement, extensive media that emphasizes the enforcement, and evaluation. Both sites conducted 4 waves of enforcement between April 2010 and April 2011. NHTSA developed and bought TV and radio spots featuring the tag line Phone in One Hand, Ticket in the Other. Both sites generated ample earned media. Police wrote 100 to 200 citations per 10,000 population for each wave in each site. Driver surveys showed an increase in awareness that cell phone laws were being enforced and recognition of the new slogan. Observed hand-held driver cell phone use dropped from 6.6% to 2.9% in Hartford, and from 3.7% to 2.5% in Syracuse. Connecticuts control area also showed a decrease in use (from 6.6% to 5.6%) but not to the same extent as Hartford. New Yorks control area had similar decreases (5% to 3%), perhaps a result of separate enforcement campaigns running simultaneously in the control site. The results show that high-visibility enforcement campaigns can reduce the number of people who use hand-held cell phones while driving.

关键词：康涅狄格；分心驾驶；驾驶行为

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In this research, twenty batches of concrete with six different mixture proportions were tested with VERIFI to evaluate 1) accuracy and repeatability of VERIFI measurements, 2) ability of VERIFI to adjust slump automatically with water and admixture, and 3) effects on concrete properties when water and admixture are added continuously during transit instead of adding whole at the plant or jobsite. For each batch, concrete was sampled every 30 minutes up to 90 minutes and tested for slump, temperature, air content, unit weight, water content, bleeding (select batches), and rheology (select batches). Cylindrical concrete specimens were cast at 90 minutes and were tested for compressive strength at 3 and 28 d (or 3 and 14 d for IDOT mixes).

关键词：土木工程；混凝土；混凝土行业；建筑

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