Molecular Characterization of Dopamine Transporter Phosphorylation and Regulation

The dopamine transporter (DAT) is a presynaptic membrane phosphoprotein that terminates dopaminergic synaptic transmission by clearing dopamine (DA) back into presynaptic neuron by a reuptake process. DAT is also a molecular target for psychostimulants such as cocaine, amphetamine (AMPH) and methamphetamine (METH) that increase DA synaptic levels either by blocking reuptake or by inducing DA efflux. DAT phosphorylation and transport down regulation are best demonstrated with the treatment of Protein kinase C (PKC) activators such as (phorbol 12-myristate 13-acetate) PMA. Serine(s) and threonine(s) are known to be involved in DAT phosphorylation. Pretreatment with psychostimulant substrates such as AMPH and METH have been shown to regulate DAT phosphorylation and DA transport activity. Here we have examined the effects of DA, cocaine and various other psychoactive transport blockers on DAT phosphorylation and regulation in rDAT expressing LLC-PK1 cells by using 32PO4 metabolic labeling and [3H] DA uptake assays. Pretreatment with cocaine or methylphenidate (MPH) had no effect on basal or PMA stimulated DAT phosphorylation and DA transport. GBR 12909 suppressed PMA-induced DAT phosphorylation and internalization. Treatment with DA did not affect DAT phosphorylation while inducing PKC mediated DA transport down regulation. These results provide information on the potential for endogenous and psychoactive compounds to modulate DAT phosphorylation-mediated regulatory mechanisms that may contribute to drug behavioral or therapeutic properties. N-terminal phosphorylation of DAT is involved in DA efflux, but the DAT amino acid residues, protein kinases and protein phosphatases involved in this process are not known. In this study, we showed that a variety of protein kinases including PKCα, PKA, CaMKII, and MAPKs phosphorylate a recombinantly expressed N-terminal tail of DAT (NDAT) in vitro. Phosphoaminoacid analysis specifically showed that PKCα phosphorylates on serine(s) and ERK1 phosphorylates on threonine(s) of NDAT. Protein phosphatases (PP1 and PP2B) were able to dephosphorylate the PKCα phosphorylated NDAT; however, none of the tested protein phosphatases were able to dephosphorylate the ERK1 phosphorylated NDAT. Further, we have identified T53 as a MAPK phosphorylation site on NDAT in vitroand localized threonine phosphorylation on the N-terminal portion of striatal DAT in vivo. These studies implicate the role of multiple kinases or phosphatases in differential phosphorylation or dephosphorylation of DAT. Further these studies may increase the current understanding of N-terminal phosphorylation ofDAT that has been shown to regulate the DA efflux

A protocol reconfiguration and optimization system for MPI

Modern high performance computing (HPC) applications, for example adaptive mesh refinement and multi-physics codes, have dynamic communication characteristics which result in poor performance on current Message Passing Interface (MPI) implementations. The degraded application performance can be attributed to a mismatch between changing application requirements and static communication library functionality. To improve the performance of these applications, MPI libraries should change their protocol functionality in response to changing application requirements, and tailor their functionality to take advantage of hardware capabilities. This dissertation describes Protocol Reconfiguration and Optimization system for MPI (PRO-MPI), a framework for constructing profile-driven reconfigurable MPI libraries; these libraries use past application characteristics (profiles) to dynamically change their functionality to match the changing application requirements. The framework addresses the challenges of designing and implementing the reconfigurable MPI libraries, which include collecting and reasoning about application characteristics to drive the protocol reconfiguration and defining abstractions required for implementing these reconfigurations. Two prototype reconfigurable MPI implementations based on the framework - Open PRO-MPI and Cactus PRO-MPI - are also presented to demonstrate the utility of the framework. To demonstrate the effectiveness of reconfigurable MPI libraries, this dissertation presents experimental results to show the impact of using these libraries on the application performance. The results show that PRO-MPI improves the performance of important HPC applications and benchmarks. They also show that HyperCLaw performance improves by approximately 22% when exact profiles are available, and HyperCLaw performance improves by approximately 18% when only approximate profiles are available

Pulmonary Delivery of Nanoparticle-Bound Toll-like Receptor 9 Agonist for the Treatment of Metastatic Lung Cancer

CpG oligodeoxynucleotides are potent toll-like receptor (TLR) 9 agonists and have shown promise as anticancer agents in preclinical studies and clinical trials. Binding of CpG to TLR9 initiates a cascade of innate and adaptive immune responses, beginning with activation of dendritic cells and resulting in a range of secondary effects that include the secretion of pro-inflammatory cytokines, activation of natural killer cells, and expansion of T cell populations. Recent literature suggests that local delivery of CpG in tumors results in superior antitumor effects as compared to systemic delivery. In this study, we utilized PRINT (particle replication in nonwetting templates) nanoparticles as a vehicle to deliver CpG into murine lungs through orotracheal instillations. In two murine orthotopic metastasis models of non-small-cell lung cancer-344SQ (lung adenocarcinoma) and KAL-LN2E1 (lung squamous carcinoma), local delivery of PRINT-CpG into the lungs effectively promoted substantial tumor regression and also limited systemic toxicities associated with soluble CpG. Furthermore, cured mice were completely resistant to tumor rechallenge. Additionally, nanodelivery showed extended retention of CpG within the lungs as well as prolonged elevation of antitumor cytokines in the lungs, but no elevated levels of proinflammatory cytokines in the serum. These results demonstrate that PRINT-CpG is a potent nanoplatform for local treatment of lung cancer that has collateral therapeutic effects on systemic disease and an encouraging toxicity profile and may have the potential to treat lung metastasis of other cancer types

Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival

A novel asymmetric synthesis of cinacalcet hydrochloride

A novel route to asymmetric synthesis of cinacalcet hydrochloride by the application of (R)-tert-butanesulfinamide and regioselective N-alkylation of the naphthyl ethyl sulfinamide intermediate is described

Thermal rearrangement of tert-butylsulfinamide

tert-Butylsulfinamides are unstable above room temperature, and in chlorinated solvents they undergo rearrangement to form the more stable N-(tert-butylthio)-tert-butylsulfonamide

Using Application Communication Characteristics to Drive Dynamic MPI Reconfiguration

Modern HPC applications, for example adaptive mesh refinement and multi-physics codes, have dynamic communication characteristics which result in poor performance on current MPI implementations. Current MPI implementations do not change transport protocols or allocate resources based on runtime state of the system or the application characteristics, resulting in degraded application performance. In this paper, we describe PRO-MPI, a Protocol Reconfiguration and Optimization system for MPI that we are developing to meet the needs of dynamic modern HPC applications. PRO-MPI uses profiles of past application communication characteristics to dynamically reconfigure MPI protocol choices. We show that such dynamic reconfiguration can improve the performance of important MPI applications significantly when exact communication profiles are known. We also present preliminary data showing that profiles from past application runs with different (but related) inputs can be used to optimize the performance of later application runs.